RESUMEN
Migraine patients are particularly prone to develop medication overuse headache (MOH). However, the risk factors for the transformation of migraine to MOH are still not clear. We investigated gene polymorphisms, personality traits, and characteristics of headache and lifestyle in 47 migraine patients (aged 36.4 ± 10.3) and 22 MOH patients (aged 39.6 ± 9.9) who progressed from migraine and made a scoring system for a predictive index (PI) of the onset of MOH in patients with migraine. By multivariate logistic stepwise regression analysis, type of migraine, regular and sufficient dietary intake, and methylenetetrahydrofolate reductase (MTHFR) C677T (rs1801133) and dopamine D2 receptor (DRD2) C939T (rs6275) polymorphisms were selected as significant factors that contribute independently to the development from migraine to MOH (P < 0.05). The regression coefficients (ß) of these four selected factors were approximated and scored. The PI score in MOH patients (7.32 ± 1.60) was significantly higher than that in migraine patients (4.62 ± 1.83, P < 0.001). The proposed scoring system should in the future be the object of larger studies to confirm its validity.
Asunto(s)
Cefaleas Secundarias/epidemiología , Trastornos Migrañosos/complicaciones , Adulto , Edad de Inicio , Cartilla de ADN , Femenino , Genotipo , Cefaleas Secundarias/genética , Cefaleas Secundarias/psicología , Humanos , Estilo de Vida , Modelos Logísticos , Masculino , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Persona de Mediana Edad , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/genética , Migraña con Aura/complicaciones , Migraña con Aura/tratamiento farmacológico , Migraña sin Aura/complicaciones , Migraña sin Aura/tratamiento farmacológico , Personalidad , Pruebas de Personalidad , Polimorfismo Genético/genética , Valor Predictivo de las Pruebas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Dopamina D2/genética , Factores de Riesgo , Encuestas y CuestionariosRESUMEN
AIMS/HYPOTHESIS: The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating the expression of the leptin gene, we examined the methylation status and methylation-sensitive transcription factors during 3T3-L1 differentiation. METHODS: DNase I footprinting, electrophoretic mobility-shift assays, and a Southwestern analysis were carried out using nuclear extracts from preadipocytes and adipocytes. Promoter activity was measured by luciferase assays. The CpG methylation pattern was determined. RESULTS: Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough transactivation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt -161, all of which were highly methylated ( > 92 %) in preadipocytes. Seven of these sites showed a varying degree of demethylation during differentiation, while the site at nt -54 remained methylated. In electrophoretic mobility-shift assays, DNA fragments from nt -115 to nt -70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kMr protein that binds strongly to the methylated probes. Promoter activity was reduced by methylation of the CpG sites up to nt -115, but not up to nt -70. CONCLUSION/INTERPRETATION: These results suggest that methylation of specific CpG sites between nt -115 and nt -70 and a methylation-sensitive protein could contribute to leptin gene expression during adipocyte differentiation in 3T3-L1 cells.
Asunto(s)
Adipocitos/citología , Leptina/genética , Regiones Promotoras Genéticas , Células 3T3 , Regiones no Traducidas 5'/genética , Adipocitos/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Metilación de ADN , Cartilla de ADN , Ratones , Datos de Secuencia MolecularRESUMEN
The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2-7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.
Asunto(s)
Carcinoma Papilar/genética , Proteínas Portadoras/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas/fisiología , Simportadores , Glándula Tiroides/fisiología , Neoplasias de la Tiroides/genética , Secuencia de Bases/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Genoma , Humanos , Yoduros/metabolismo , Yoduros/farmacocinética , Mutación , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Fragmentos de Péptidos/genética , ARN Mensajero/metabolismo , Valores de Referencia , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Factor Nuclear Tiroideo 1 , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) induce apoptosis and exert antiproliferative effects on several carcinoma cell lines. The present study investigates the expression of PPARgamma and the possibility that agonists for PPARgamma also inhibit the growth of human thyroid carcinoma cells. We examined this hypothesis using six cell lines, designated BHP thyroid carcinoma cells, which originated from patients with papillary thyroid carcinoma. RT-PCR analysis revealed that the thyroid carcinoma cell lines BHP2-7, 7-13, 10-3, and 18-21 express PPARgamma. More PPARgamma was expressed in carcinoma than in adjacent normal thyroid tissue in three of six samples of human papillary carcinoma of the thyroid. PPARgamma-positive thyroid carcinoma cells were treated with agonists of PPARgamma, troglitazone, BRL 49653, and 15-deoxy-12,14-prostaglandin J2. Troglitazone (10 micromol/L), BRL 49653 (10 micromol/L), and 15-deoxy-12,14-prostaglandin J2 (1 microg/mL) decreased [(3)H]thymidine incorporation and reduced cell number, respectively, in BHP carcinoma cell lines that expressed PPARgamma. Under low serum conditions, ligands for PPARgamma induced condensation of the nucleus and fragmentation of chromatin into nucleosome ladders. These findings indicate that the death of thyroid carcinoma cells is a form of apoptosis. To investigate the molecular mechanism of the apoptosis, we assessed expression of the apoptosis-regulatory genes bcl-2, bax, and c-myc. Troglitazone significantly increased the expression of c-myc messenger RNA but had no effect on the expression of bcl-2 and bax in thyroid carcinoma cells. These results suggest that, at least in part, the induction of apoptosis in human papillary thyroid carcinoma cells may be due to an increase of c-myc. Troglitazone (500 mg/kg.day) significantly inhibited tumor growth and prevented distant metastasis of BHP18-21 tumors in nude mice in vivo. Taken together, these results suggest that PPARgamma agonist inhibit cell growth of some types of human thyroid cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Papilar/tratamiento farmacológico , Cromanos/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazoles/farmacología , Tiazolidinedionas , Neoplasias de la Tiroides/tratamiento farmacológico , Factores de Transcripción/agonistas , Animales , Northern Blotting , Carcinoma Papilar/patología , División Celular/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosiglitazona , Timidina/metabolismo , Neoplasias de la Tiroides/patología , Factores de Transcripción/genética , Troglitazona , Células Tumorales CultivadasRESUMEN
Gene therapy with thyroglobulin (TG) promoter and a prodrug/suicide gene combination may prove useful as a treatment for thyroid carcinoma. However, most poorly differentiated and anaplastic thyroid carcinomas have lost the ability to express the TG gene expression accompanied by loss of transcription factors [thyroid transcription factor-1 (TTF-1), TTF-2, or Pax-8] interacting with the TG promoter. In anticipation of developing transcriptionally targeted gene therapy of TG-nonproducing thyroid carcinomas, we investigated the effect of TTF-1 gene transfer on TG promoter activity and the cytotoxic effect obtained by the TG promoter-driven HSV-TK gene along with ganciclovir in thyroid carcinoma and nonthyroidal cells. Using a chimeric construct containing the 5'-flanking region of the rat TG gene between -826 and +39 bp and the luciferase gene, TG promoter activity was detected in a normal rat thyroid cell line (FRTL-5), but not in a dedifferentiated line of thyroid cells (FRT) expressing Pax-8 but not TTF-1, TTF-2, or TG [TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)], or in a human papillary thyroid carcinoma cell line [BHP15-3; TTF-1(-)/TTF-2(-)/Pax-8(-)/TG(-)], a human pulmonary cell line [H441; TTF-1(+)/TTF-2(-)/Pax-8(-)/TG(-)], or a dog kidney epithelial cell line [MDCK; TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. Cotransfection of the TTF-1 expression vector stimulated TG promoter activity in FRT and BHP15-3 dedifferentiated thyroid cells, but not in H441 pulmonary cells. Only weak activation was observed in MDCK kidney cells. We then constructed recombinant adenovirus vectors, AdTTF-1 and ADTGTK: AdTTF-1 contained cytomegalovirus promoter and rat TTF-1 cDNA; AdTGTK carried the TG promoter-driven HSV-TK gene. Infection with AdTGTK and combined with GCV treatment induced a cytotoxic effect in FRTL-5 cells but not in dedifferentiated thyroid or nonthyroid cells. Cotransduction of AdTTF-1 and AdTGTK permitted 90% cytotoxicity for BHP15-3 and >95% cytotoxicity for FRT, as well as for BHP7-13 and BHP18-21v thyroid cancer cell lines [both/TTF1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. In contrast, little cytotoxicity was seen for H441 and MDCK cell lines even with 300 microg/ml of ganciclovir. These results suggest that cotransduction of a TG promoter-controlled suicide gene and the TTF-1 gene by adenoviral vectors confers transcriptionally targeted gene-mediated cytotoxicity in poorly differentiated thyroid carcinoma cells unable to express the TG gene.
Asunto(s)
Carcinoma Papilar/genética , Terapia Genética/métodos , Proteínas Nucleares/genética , Tiroglobulina/genética , Neoplasias de la Tiroides/genética , Factores de Transcripción/genética , Activación Transcripcional , Adenoviridae/genética , Animales , Carcinoma Papilar/tratamiento farmacológico , Carcinoma Papilar/terapia , Ganciclovir/farmacocinética , Ganciclovir/farmacología , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/genética , Células HeLa , Humanos , Regiones Promotoras Genéticas , Ratas , Simplexvirus/enzimología , Simplexvirus/genética , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Tiroglobulina/biosíntesis , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/terapia , Factor Nuclear Tiroideo 1 , Transducción Genética , Células Tumorales CultivadasRESUMEN
Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that is thought to play a major role in the regulation of growth and differentiation of thyroid cells. However, little is known of its detailed mechanisms of action in thyrocytes. We have therefore studied the molecular mechanisms of TGF-beta1 action on thyroglobulin (TG) gene expression by focusing our attention on TGF-beta1 regulation of thyroid-specific transcription factors. TGF-beta1 decreased TG messenger RNA (mRNA) expression both in the presence and in the absence of TSH in rat thyroid FRTL-5 cells. Transfected into FRTL-5 cells, the activity of reporter plasmids containing the rat TG promoter ligated to a luciferase gene was significantly suppressed by the addition of TGF-beta1. When the nuclear extracts prepared from TGF-beta1-treated FRTL-5 cells were used in gel mobility shift assays, the amount of protein-DNA complex formed by Pax-8 was reduced, both in the presence and in the absence of TSH, but protein-DNA complexes formed by thyroid transcription factor-1 (TTF-1) and TTF-2 were not. The suppressive effect of TGF-beta1 on Pax-8/DNA complex formation is in part due to the suppression of Pax-8 mRNA and protein levels by TGF-beta1. Expressions of Pax-8 mRNA and protein, which were assessed by Northern blot and Western blot analyses, respectively, were decreased by TGF-beta1 treatment of FRTL-5 cells in a concentration-dependent manner. In a transfection experiment, mutation of the Pax-8-binding site caused a loss of both TGF-beta1- and TSH-responsiveness in TG promoter activity. Overexpression of Pax-8 abolished the TGF-beta1 suppression of TG promoter activity. These results indicate that TGF-beta1 decreases Pax-8 mRNA levels as well as Pax-8 DNA-binding activity, which, at least in part, seems to be involved in the TGF-beta1-induced suppression of TG gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Nucleares , Tiroglobulina/genética , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Bovinos , Línea Celular , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Luciferasas/genética , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Proteínas Recombinantes/farmacología , Glándula Tiroides , Tirotropina/farmacología , TransfecciónRESUMEN
A 44-year-old female with familial hypocalciuric hypercalcemia (FHH) due to a homozygous missense mutation (Pro40Ala) in calcium sensing receptor (CaSR) gene has type 2 diabetes mellitus. The identical heterozygous mutation of CaSR gene was observed in consanguineous parents and all other family members examined except her two sisters. Many subjects with abnormal glucose tolerance were observed in this family, which is compatible with maternal inheritance. Mitochondrial function of complex I (NADH-coenzyme Q reductase) activity in cybrid cells between mitochondrial DNA (mtDNA)-deleted (rho(0)) HeLa cells and mtDNA from the proband was decreased by 35%. The proband has eight substitutions and among these 4833 A/G is a missense substitution in NADH dehydrogenase 2 gene and may probably be a major pathogenic mutation of impaired complex I activity. These results suggest that coexistence of nuclear gene and mtDNA mutations may have caused or modified the development of abnormal glucose tolerance in this family.
Asunto(s)
ADN Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Mutación Missense , Receptores de Superficie Celular/genética , Adulto , Animales , Calcio/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Femenino , Homocigoto , Humanos , Hipercalcemia/complicaciones , Hipercalcemia/genética , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Linaje , Ratas , Receptores Sensibles al Calcio , Receptores de Superficie Celular/análisisRESUMEN
Aldose reductase (AKR1B1) is the first enzyme in the polyol pathway through which glucose is converted to sorbitol, and has been implicated in the etiology of diabetic complications. However, its physiological role is still not well understood. In the kidney, AKR1B1 is quite abundant in the collecting tubule cells and thought to provide protection against hypertonic environment. We report here that the mice lacking AKR1B1 showed hypercalciuria, hypercalcemia, hypermagnesemia, and reduced ability to concentrate urine, suggesting a new physiological role of AKR1B1 in divalent cation homeostasis.
Asunto(s)
Aldehído Reductasa/genética , Aldehído Reductasa/fisiología , Calcio/orina , Magnesio/orina , Aldehído Reductasa/biosíntesis , Aldehído Reductasa/metabolismo , Animales , Glucemia/metabolismo , Southern Blotting , Western Blotting , Peso Corporal , Cationes , Cloruros/sangre , Cloruros/orina , Creatinina/sangre , Creatinina/orina , Desamino Arginina Vasopresina/farmacología , Eliminación de Gen , Biblioteca de Genes , Genotipo , Glucosa/metabolismo , Inmunohistoquímica , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Fenotipo , Fosfatos/sangre , Fosfatos/orina , Potasio/sangre , Potasio/orina , Fármacos Renales/farmacología , Sodio/sangre , Sodio/orina , Agua/metabolismo , Privación de AguaRESUMEN
From a family of 16 diabetic patients with typical maternal inheritance, we investigated a 69-year-old woman with type 2 diabetes. The proband showed no major deletions in the mitochondrial DNA (mtDNA). Direct sequencing revealed 7 missense and 5 ribosomal RNA homoplasmic nucleotide substitutions when compared with the Cambridge Sequence and its recent revision. When compared with the control cybrid cells, the proband cybrid cells showed 6 nucleotide substitutions. Among these, 14577 T/C, which turned out to be 98.9% heteroplasmic, is a new missense substitution in the NADH dehydrogenase 6 gene. We also observed 2 other patients with 14577 T/C substitution from another group of 252 unrelated diabetic patients, whereas no individual from a group of 529 control subjects had 14577 T/C substitution. Furthermore, these 6 substitutions were in linkage disequilibrium. Mitochondrial respiratory chain complex I activity and O2 consumption rates of the proband cybrid cells, which were obtained by the fusion of mtDNA-deleted (rho0) HeLa cells and mtDNA from the proband, showed 64.5 and 61.5% reductions, respectively, compared with control cybrid cells. The present study strongly indicates that the new mtDNA mutation at 14577 T/C is probably a major pathogenic mutation for type 2 diabetes in this family.
Asunto(s)
ADN Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Impresión Genómica , Mutación Missense , NADH Deshidrogenasa/genética , Anciano , Grupo Citocromo b/genética , Diabetes Mellitus Tipo 2/enzimología , Femenino , Células HeLa , Humanos , Células Híbridas , Masculino , Mitocondrias/metabolismo , Consumo de Oxígeno , Linaje , ARN Ribosómico/genética , ARN Ribosómico 16S/genéticaRESUMEN
The sodium iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease (ATD). A recent study has described the development of a bioassay for human (h) NIS antibody detection, but this will not detect antibodies that bind the symporter without modulating its activity. Therefore, the establishment of a binding assay is of importance to determine the overall prevalence of hNIS antibodies in ATD patients. An in vitro transcription and translation system was used to produce [35S]-labeled hNIS. The radiolabeled ligand reacted specifically in immunoprecipitation experiments with rabbit antiserum raised against a peptide fragment of hNIS. Subsequently, the reactivity of control and ATD sera to translated [35S]hNIS was determined using RIAs. A significant difference in the frequency of hNIS antibody-positive sera was found when patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH) were compared with normal controls (P = 0.01 and P = 0.03, respectively). Of 49 GD and 29 AH sera tested, 11 (22%) and 7 (24%), respectively, were found to contain hNIS antibodies. Differences were also significant when the antibody-binding indices of the control group of sera were compared with those of both the GD and the AH patient sera (P < 0.0001 and P = 0.001, respectively). In contrast, sera from 10 patients with Addison's disease and 10 patients with vitiligo (without associated ATD) were all negative for antibody reactivity to the symporter. No differences were detected when the antibody binding indices of either the Addison's disease or the vitiligo sera were compared with those of the normal sera group (P = 0.9 and P = 0.6, respectively). Eight of the 11 (73%) GD and 3 of the 7 (43%) AH sera, which were positive for hNIS antibodies in the immunoprecipitation assay, were also found to inhibit iodide uptake in hNIS-transfected CHO-K1 cells, suggesting the existence of antibodies in some serum samples that bind to the symporter without modulating its function. Overall, a significant correlation was found between the iodide uptake inhibition and the binding assays for hNIS antibody detection (r = 0.49, P < 0.0001). In summary, we have developed a specific and quantitative assay for the detection of hNIS binding antibodies in sera of patients with ATD. This system offers the advantage of studying antibody reactivity against conformational epitopes and will be useful in understanding the role of NIS autoreactivity in the pathogenesis of ATD.
Asunto(s)
Autoanticuerpos/sangre , Proteínas Portadoras/inmunología , Enfermedad de Graves/inmunología , Proteínas de la Membrana/inmunología , Simportadores , Tiroiditis Autoinmune/inmunología , Enfermedad de Addison/sangre , Enfermedad de Addison/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cricetinae , Femenino , Enfermedad de Graves/sangre , Humanos , Hipotiroidismo/sangre , Hipotiroidismo/inmunología , Yoduros/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Conformación Proteica , Conejos , Radioinmunoensayo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Tiroiditis Autoinmune/sangre , TransfecciónRESUMEN
A 45-year-old man had been complaining of thirst and polydypsia for the last 3 months and was diagnosed as having type 2 diabetes mellitus because his fasting blood glucose showed 221 mg/dl with positive urinary ketone. He was hospitalized to a private hospital and Penfil 30R was started. However, serum gamma-GTP and aminotransferases began to elevate after insulin treatment and exceeded 1000 IU/l. Insulin was discontinued and serum gamma-GTP and aminotransferases returned close to the normal range. Since his glycemic control became poor again, Penfil 30R was restarted and serum gamma-GTP and aminotransferases elevated again. Therefore, insulin was discontinued and the patient was referred to the Third Department of Internal Medicine, Yamanashi Medical University Hospital because of liver dysfunction. His plasma glucose decreased by diet therapy, and improved further by the administration of glibenclamide. After obtaining informed consent, Humalin R was challenged. Seven days after insulin injection, serum aminotransferases began to elevate again. Lymphocyte stimulation test was negative against three preparations (Penfil R, Penfil N and Humalin R). The present case suggests that human insulin itself can cause liver dysfunction and we need to pay more attention to liver function tests when we start insulin treatment.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Insulina/efectos adversos , Hepatopatías/etiología , Alanina Transaminasa/sangre , Fosfatasa Alcalina/sangre , Anticuerpos Antivirales/sangre , Aspartato Aminotransferasas/sangre , Bilirrubina/sangre , Péptido C/orina , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/dietoterapia , Gliburida/uso terapéutico , Antígenos de Superficie de la Hepatitis B/sangre , Humanos , Hipoglucemiantes/uso terapéutico , Inmunoglobulina M/sangre , Insulina/uso terapéutico , Anticuerpos Insulínicos/sangre , Cuerpos Cetónicos/orina , L-Lactato Deshidrogenasa/sangre , Hepatopatías/diagnóstico , Hepatopatías/patología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Sed , gamma-Glutamiltransferasa/sangreRESUMEN
A point mutation of a nucleotide within a single gene can have a profound effect on a specific organ and/or the entire human body. DNA sequences associated with human diseases may differ from the corresponding normal sequences by single nucleotide mutations or by large alterations such as deletions, insertions, duplications, or translocations of DNA segments or entire chromosomes. As a result of the heterogeneity of DNA alterations and genetic mutations, various screening approaches are required to detect these alterations. However, methods which facilitate the detection of large mutations in the genome are typically insensitive to point mutations, whereas methods which detect point mutations are not appropriate to detect large alterations within the genome. Since there is no single perfect method to screen for unknown mutations, combinations of these methods may be necessary for accurate genetic diagnosis. The applications of polymerase chain reaction (PCR) technology to genomic screening have made rapid and accurate genetical diagnosis possible. Furthermore, recent developments in the technology of DNA microarrays have opened the way for high throughput sequence analysis by hybridization, which shows great potential in both molecular biology and medicine in the near future.
Asunto(s)
Pruebas Genéticas/métodos , Mutación , HumanosRESUMEN
We determined by PCR the number of (A-C)n repeats in the 2. 1 kb upstream of the aldose reductase (AR2) gene in healthy control subjects and in patients with NIDDM in Japanese. Sixty-one patients were recruited based on the severity of retinopathy and subdivided into two groups with proliferative retinopathy and without retinopathy. Japanese exhibited 10 different alleles in this region. The most prevalent allele was designated Z ((A-C)24 repeats) allele. The Z-4 allele was significantly associated with patients with proliferative retinopathy, whereas the Z+2 allele was significantly associated with patients without retinopathy. Erythrocyte AR2 protein levels were significantly elevated in patients exhibiting the Z-4 allele compared to those exhibiting other alleles. When Z-4 allele was ligated in transfection experiments to luciferase vector containing the promoter region of the AR2 gene, the construct showed significantly higher transcription of the reporter gene compared to constructs without (A-C) repeat or with Z-2, Z or Z+2 alleles. Our results suggest that the Z-4 allele in the 2. 1 kb upstream of the AR2 gene may enhance gene transcription and may be a genetic risk factor, which determines the predisposition to retinopathy in Japanese patients with NIDDM.
Asunto(s)
Aldehído Reductasa/genética , Alelos , Diabetes Mellitus Tipo 2/genética , Retinopatía Diabética/genética , Luciferasas/genética , Transcripción Genética , Aldehído Reductasa/metabolismo , Células Cultivadas , ADN/análisis , Cartilla de ADN/química , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/enzimología , Retinopatía Diabética/sangre , Retinopatía Diabética/enzimología , Repeticiones de Dinucleótido , Eritrocitos/enzimología , Femenino , Genes Reporteros , Humanos , Japón , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , TransfecciónRESUMEN
GLUT4, the major glucose transporter in adipose tissue, is expressed during the differentiation of 3T3-L1 cells from preadipocytes to adipocytes. We previously examined the mouse GLUT4 promoter activity up to -590 bp, and demonstrated that the 5'-flanking region of the GLUT4 gene between -200 and -100 bp contains sequences that act as a repressor in preadipocytes, but not in adipocytes. Here we examine in detail the activity of this repressor in 3T3-L1 cells. Transient transfections indicated that the region extending from -125 to -112 bp functions as a repressor element only in preadipocytes. In electrophoretic mobility shift assay (EMSA), this GLUT4 repressor element (G4RE) generated specific bands with nuclear extracts from preadipocytes, but not from adipocytes. Southwestern blot analysis identified a protein of approximately 96 kDa from preadipocytes that bound to the G4RE site. Mutation of the G4RE site, which abolished the protein/DNA complex formation by EMSA, increased GLUT4 promoter activity only in preadipocytes. These results suggest that the G4RE site and its binding protein may regulate GLUT4 gene transcription during adipocyte differentiation.
Asunto(s)
Adipocitos/citología , Adipocitos/fisiología , Regulación de la Expresión Génica , Proteínas de Transporte de Monosacáridos/genética , Proteínas Musculares , Proteínas Represoras/metabolismo , Transcripción Genética , Células 3T3 , Animales , Diferenciación Celular , Núcleo Celular/metabolismo , Transportador de Glucosa de Tipo 4 , Ratones , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , TransfecciónRESUMEN
Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.
Asunto(s)
Óxido Nítrico/biosíntesis , Inhibidores de Fosfodiesterasa/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sulfonamidas , Tetrazoles/farmacología , Cilostazol , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dexametasona/farmacología , Humanos , Inositol/análisis , Isoquinolinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Sorbitol/análisis , Células Tumorales CultivadasRESUMEN
Graves' hyperthyroidism is thought to be caused by thyroid-stimulating antibodies, which interact with the thyrotropin receptors in the thyroid. In this sense there is no ideal treatment for Graves' hyperthyroidism, yet, according to the pathogenesis of the disease. Therefore, drugs of thionamide which inhibit thyroid hormone synthesis have still been used as the antithyroid drugs widely. However, there are many problems left for antithyroid drug therapy. The author described update considerations to solve these problems faced during the therapy of Grave's hyperthyroidism. Major issues discussed are as follows; 1) Thionamide therapy depending on the severity of Graves' hyperthyroidism. 2) Evaluation of drug effectiveness. 3) Possibility and choice of therapy. 4) Change of therapy from antithyroid drugs. 5) Explanation of antithyroid drug to patients. 6) Side effects. 7) When antithyroid drug should be stopped?
Asunto(s)
Antitiroideos/uso terapéutico , Enfermedad de Graves/tratamiento farmacológico , Antitiroideos/efectos adversos , HumanosRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) is known to modulate the expression of thyroid-specific genes, such as thyroglobulin (TG), contributing to the pathogenesis of autoimmune thyroid disease. In the present study, we show that TNF-alpha suppresses DNA-binding activity of thyroid transcription factors, Pax-8 and thyroid transcription factor-1 (TTF-1), which is, in part, involved in TNF-alpha-induced decrease in TG gene expression. Transfected into rat thyroid FRTL-5 cells, the activity of reporter plasmid containing the rat TG promoter ligated to a luciferase gene was significantly suppressed in the presence of TNF-alpha. In gel mobility shift analyses, protein-DNA complexes formed by TTF-1 and Pax-8 were reduced when the nuclear extracts prepared from TNF-alpha-treated FRTL-5 cells were used. The suppressive effect of TNF-alpha on TTF-1-DNA complex formation is, in part, caused by suppression of TTF-1 gene transcription by TNF-alpha. Expressions of TTF-1 messenger RNA and protein, which were assessed by Northern blot and Western blot analyses, respectively, were decreased by TNF-alpha treatment of FRTL-5 cells. In contrast, TNF-alpha did not affect the expression of Pax-8 messenger RNA. Treatment of FRTL-5 cells with TNF-alpha caused a decrease in Pax-8 protein in nuclear extracts and accumulation of the protein in the cytoplasm, as assessed by Western blot analyses. Mutation of the TTF-1/Pax-8-binding site lost the TNF-alpha-induced decrease in TG promoter activity in a transfection experiment. These results indicate that TNF-alpha suppresses the activity of TTF-1 and Pax-8 by different mechanisms, which, in part, seem to be involved in TNF-alpha-induced decrease in TG gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Glándula Tiroides/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Animales , Secuencia de Bases/genética , Sitios de Unión/fisiología , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quimera/genética , Citoplasma/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Mutación/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ratas , Tiroglobulina/genética , Glándula Tiroides/citología , Factor Nuclear Tiroideo 1 , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Histologic examination of sural nerve of a 32-year-old man with an aggressive polyneuropathy associated with autonomic failure demonstrated amyloid deposition, and familial amyloidotic polyneuropathy (FAP) was diagnosed. Immunohistochemical staining showed transthyretin (TTR) staining of the amyloid deposits in nerve. Sequencing revealed G to A transition in the codon 54 causing TTR Lys 54. This is a new variant TTR associated with aggressive FAP.
Asunto(s)
Neuropatías Amiloides/genética , Adulto , Edad de Inicio , Neuropatías Amiloides/patología , Mapeo Cromosómico , Ácido Glutámico , Humanos , Inmunohistoquímica , Japón , Lisina , Masculino , Reacción en Cadena de la Polimerasa , Prealbúmina/genética , Nervio Sural/patologíaRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) are cytokines that can individually or additively suppress thyroid cell function and the expression of thyroid-specific genes, such as thyroglobulin (TG) and thyroperoxidase (TPO). Thyroid transcription factor-2 (TTF-2) is a DNA-binding protein that modulates the expression of TG and TPO genes. In the present study, we examine the effects of TNF-alpha and IFN-gamma on TTF-2 gene expression, as well as the DNA-binding activity of TTF-2. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with TNF-alpha, IFN-gamma, or TNF-alpha plus IFN-gamma. Total RNA was isolated and Northern blotted. TNF-alpha (50 ng/ml) only slightly suppressed (61+/-2% compared with control), whereas IFN-gamma (100 U/ml) modestly decreased TTF-2 messenger RNA (mRNA) levels (34+/-4%). TNF-alpha and IFN-gamma simultaneously caused a marked decrease in TTF-2 mRNA levels (13+/-2%). The suppressive effects of TNF-alpha and IFN-gamma on TTF-2 mRNA levels were concentration dependent and maximal at 50 ng/ml TNF-alpha with 100 U/ml IFN-gamma. The suppressive effect was also time dependent, reaching a maximum 12 h after exposure. Moreover, the suppressive effects of TNF-alpha and IFN-gamma upon rat TG and TTF-2 mRNA levels were similar. To test whether TNF-alpha and IFN-gamma alter TTF-2-binding to DNA, we performed electrophoretic mobility shift assays using a TTF-2-binding element in the rat TG gene as a probe. Formation of the TTF-2/DNA complex was decreased by TNF-alpha and/or IFN-gamma. Our results demonstrate that TNF-alpha and IFN-gamma additively reduce the gene expression and DNA-binding of TTF-2. These data suggest that TTF-2 is involved in the TNF-alpha and IFN-gamma-induced suppression of thyroid-specific gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Proteínas Represoras/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ratas , Proteínas Represoras/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Thyrotropin receptor (TSHR) is expressed during the differentiation of rat preadipocytes and is highly abundant in mature fat adipocytes, but its physiological role is unknown. In this article, long-term effects of thyrotropin (TSH) on the proliferation and the differentiation were investigated using cultured rat preadipocytes. When TSH was added at the beginning of differentiation, TSH increased the number of preadipocytes and 3H-thymidine uptake. Apoptosis of the cells was not influenced by TSH. Preadipocytes incubated in the presence of TSH had fewer fat droplets, decreased level of mRNA for lipoprotein lipase (LPL), a marker of adipocyte differentiation. Histochemical study showed that the cells that increased their number and expressed lower level of LPL in response to TSH were preadipocytes, and not contaminating fibroblasts. In conclusion, TSH causes the proliferation and inhibits the differentiation of rat preadipocytes. Results suggest that TSH may be a potent regulator of preadipocyte proliferation and differentiation in vivo.