RESUMO
Hand hygiene has the biggest impact and is the least expensive way to prevent and control health care-associated infections. In this study, we assessed the effectiveness of the multimodal strategy of the World Health Organization to improve health care-associated infection rates, hand hygiene compliance, and the related knowledge of health care professionals in a Brazilian university hospital. We observed the necessity for an alternative approach in hospitals with high staff turnover and low attendance of educational sessions.
Assuntos
Higiene das Mãos/normas , Organização Mundial da Saúde/organização & administração , Centros Médicos Acadêmicos/normas , Centros Médicos Acadêmicos/estatística & dados numéricos , Brasil , Fidelidade a Diretrizes/normas , Fidelidade a Diretrizes/estatística & dados numéricos , Higiene das Mãos/estatística & dados numéricos , Pessoal de Saúde/normas , Pessoal de Saúde/estatística & dados numéricos , Humanos , Controle de Infecções/normas , Controle de Infecções/estatística & dados numéricos , Inquéritos e QuestionáriosRESUMO
Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2-/- results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.
Assuntos
Fissura Palatina/embriologia , Proteínas de Ligação a DNA/fisiologia , Modelos Animais de Doenças , Proteínas Repressoras/fisiologia , Glândula Tireoide/embriologia , Fatores de Transcrição/fisiologia , Animais , Fissura Palatina/genética , Proteínas de Ligação a DNA/genética , Endoderma , Fatores de Transcrição Forkhead , Hipotireoidismo/genética , Camundongos , Camundongos Knockout , Morfogênese , Proteínas Repressoras/genética , Glândula Tireoide/patologia , Fatores de Transcrição/genéticaRESUMO
Permanent congenital hypothyroidism (CH) is a common disease that occurs in 1 of 3,000-4,000 newborns. Except in rare cases due to hypothalamic or pituitary defects, CH is characterized by elevated levels of thyroid-stimulating hormone (TSH) resulting from reduced thyroid function. When thyroid hormone therapy is not initiated within the first two months of life, CH can cause severe neurological, mental and motor damage. In 80-85% of cases, CH is associated with and presumably is a consequence of thyroid dysgenesis (TD). In these cases, the thyroid gland can be absent (agenesis, 35-40%), ectopically located (30-45%) and/or severely reduced in size (hypoplasia, 5%). Familial cases of TD are rare, even though ectopic or absent thyroid has been occasionally observed in siblings. The pathogenesis of TD is still largely unknown. Although a genetic component has been suggested, mutations in the gene encoding the receptor for the thyroid-stimulating hormone (TSHR) have been identified in only two cases of TD with hypoplasia. We report mutations in the coding region of PAX8 in two sporadic patients and one familial case of TD. All three point mutations are located in the paired domain of PAX8 and result in severe reduction of the DNA-binding activity of this transcription factor. These genetic alterations implicate PAX8 in the pathogenesis of TD and in normal thyroid development.
Assuntos
Hipotireoidismo Congênito , Proteínas de Ligação a DNA/genética , Mutação , Proteínas Nucleares , Glândula Tireoide/anormalidades , Transativadores/genética , Sequência de Aminoácidos , Sequência de Bases , Feminino , Humanos , Recém-Nascido , Masculino , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , LinhagemRESUMO
We report the characterization of a two-qubit processor implemented with two capacitively coupled tunable superconducting qubits of the transmon type, each qubit having its own nondestructive single-shot readout. The fixed capacitive coupling yields the sqrt[iSWAP] two-qubit gate for a suitable interaction time. We reconstruct by state tomography the coherent dynamics of the two-bit register as a function of the interaction time, observe a violation of the Bell inequality by 22 standard deviations after correcting readout errors, and measure by quantum process tomography a gate fidelity of 90%.
RESUMO
BACKGROUND AND AIMS: The American Diabetes Association (ADA) has revised criteria for diagnosis of type 2 diabetes recommending an A1C cut point of ≥6.5% in addition to criteria based on glucose levels. We compared A1C, fasting plasma glucose (FPG) or 2-h post-challenge glucose (2-hPG) criteria for the diagnosis of diabetes in a cohort of Italian Caucasians. METHODS AND RESULTS: A total of 1019 individuals without known diabetes completed an oral glucose tolerance test (OGTT) and had A1C measured. Moderate agreement existed for A1C and FPG criteria for diagnosis of type 2 diabetes (κ coefficient = 0.522), with 85.5% of individuals classified as not having diabetes by both A1C and FPG criteria, and 5.8% classified as having diabetes by both A1C and FPG criteria. Discordant classifications occurred for 5.5% of individuals who had an A1C ≥ 6.5% and FPG <126 mg dl(-1), and for 3.2% who had an A1C <6.5% and FPG ≥126 mg dl(-1). Modest agreement existed for A1C and 2-hPG criteria for diagnosis of type 2 diabetes (κ coefficient = 0.427), with 81.8% of individuals classified as not having diabetes by both A1C and 2-hPG criteria, and 6.0% classified as having diabetes by both A1C and 2-hPG criteria. The area under the receiver operating characteristic curve of A1C for identifying subjects with diabetes according to FPG or 2-hPG criteria was 0.856 and 0.794, respectively. Modest agreement existed for A1C and FPG and/or 2-hPG criteria for diagnosis of type 2 diabetes (κ coefficient = 0.446). CONCLUSIONS: A1C ≥ 6.5% demonstrates a moderate agreement with fasting glucose and 2-hPG for diagnosing diabetes among adult Italian Caucasians subjects.
Assuntos
Glicemia/análise , Diabetes Mellitus Tipo 2/diagnóstico , Hemoglobinas Glicadas/análise , População Branca , Adulto , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Diabetes Mellitus Tipo 2/sangue , Jejum , Feminino , Teste de Tolerância a Glucose , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Curva ROC , Fatores de RiscoRESUMO
PURPOSE: This study was done to investigate the efficacy and safety of percutaneous renal denervation with the Symplicity catheter for reducing blood pressure in patients with essential hypertension resistant to medical therapy (systolic blood pressure >160 mmHg despite the use of three or more antihypertensive drugs, including a diuretic). MATERIALS AND METHODS: In September 2010, five patients affected by essential hypertension resistant to medical therapy were treated. All patients were studied by computed tomography angiography (CTA) of the renal arteries before the procedure and underwent follow-up at 30 and 60 days with colour Doppler ultrasound (CDUS) with evaluation of resistive index, glomerular filtration rate (GFR), 24-h blood pressure and serum catecholamine concentration. Student's t test was used to assess the effectiveness of the procedure in lowering blood pressure. RESULTS: In treated patients, mean blood pressure at baseline was 171/100 mmHg [standard deviation (SD) ± 8/10]; mean GFR was 91.6 ml/min/1.73 m(2) (SD ± 15). Blood pressure after the procedure was reduced by -18/-5 and -13/-10 mmHg at 30 and 60 days, respectively, with a mean medication reduction of 3.6. No complications occurred during the intra- or periprocedural period or during short-term follow-up. CONCLUSIONS: The Symplicity system proved to be efficacious and without serious adverse events in reducing blood pressure and antihypertensive medication use in patients affected by essential hypertension resistant to medical therapy. Although encouraging, our data are preliminary and need to be validated by larger prospective randomised studies.
Assuntos
Ablação por Cateter/métodos , Denervação/métodos , Procedimentos Endovasculares/métodos , Hipertensão/cirurgia , Rim/inervação , Pressão Sanguínea , Resistência a Medicamentos , Humanos , Hipertensão/tratamento farmacológicoRESUMO
Schistosomiasis is a parasitic disease caused by trematode worms of the genus Schistosoma and affects over 200 million people worldwide. The control and treatment of this neglected tropical disease is based on a single drug, praziquantel, which raises concerns about the development of drug resistance. This, and the lack of efficacy of praziquantel against juvenile worms, highlights the urgency for new antischistosomal therapies. In this review we focus on innovative approaches to the identification of antischistosomal drug candidates, including the use of automated assays, fragment-based screening, computer-aided and artificial intelligence-based computational methods. We highlight the current developments that may contribute to optimizing research outputs and lead to more effective drugs for this highly prevalent disease, in a more cost-effective drug discovery endeavor.
Assuntos
Inteligência Artificial , Descoberta de Drogas/métodos , Schistosoma/efeitos dos fármacos , Esquistossomose/tratamento farmacológico , Esquistossomicidas , Animais , HumanosRESUMO
Compared to matched controls, 17 patients with Duchenne muscular dystrophy showed decreased insulin binding to monocytes due to decreased receptor concentration. These patients showed no signs of altered glucose metabolism and retrospective analysis of the clinical records of a further 56 such patients revealed no modification in carbohydrate metabolism. These data suggest that reduced insulin receptor number does not produce overt modifications of glucose metabolism in Duchenne muscular dystrophy.
Assuntos
Glucose/metabolismo , Distrofias Musculares/metabolismo , Receptor de Insulina/metabolismo , Adolescente , Adulto , Membrana Celular/metabolismo , Criança , Humanos , Monócitos/metabolismoRESUMO
BACKGROUND AND AIM: Weight gain is associated with a decline in insulin sensitivity and a compensatory increase in insulin secretion. IGF-1 is a plausible candidate to explain these divergent phenomena. In this cross-sectional study, we analyzed the relationship between IGF-1 levels, insulin sensitivity and secretion in 110 nondiabetic subjects with a wide range of BMI to verify this hypothesis. METHODS AND RESULTS: Subjects underwent OGTT, IVGTT and euglycemic-hyperinsulinemic clamp. HOMA-beta, IVGTT-derived and OGTT-derived indexes for first-phase and second-phase insulin secretion were higher in obese as compared with overweight and normal-weight groups, while glucose disposal was lower. IGF-1 levels were negatively correlated with IVGTT-derived and OGTT-derived indexes first-phase and second-phase insulin secretion, and positively correlated with glucose disposal. These correlations were no longer significant after adjustment for BMI. In a multivariate analysis, the variables associated with glucose disposal were IGF-1, age, triglycerides, and 2-h post-load glucose accounting for 23.4% of its variation. When BMI was entered into the model, the variables associated with glucose disposal were triglycerides, 2-h post-load glucose and BMI accounting for 27.2% of variation. In a multivariate analysis, the only variable associated with IVGTT-derived first-phase and second-phase insulin secretion was IGF-1 accounting for 10.4% and 15.1% of variation, respectively. When BMI was entered into the model, it became the only variable associated with both first-phase and second-phase insulin secretion accounting for 25.7% and 37.6% of variation, respectively. CONCLUSION: These data suggest that progressive reduction in IGF-1 levels may be involved in obesity-related changes in both insulin sensitivity and secretion.
Assuntos
Adiposidade , Resistência à Insulina , Fator de Crescimento Insulin-Like I/análise , Insulina/metabolismo , Adulto , Idoso , Envelhecimento , Glicemia/análise , Índice de Massa Corporal , Estudos Transversais , Feminino , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Secreção de Insulina , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/complicações , Sobrepeso/sangue , Sobrepeso/complicações , Triglicerídeos/sangue , População Branca , Adulto JovemRESUMO
BACKGROUND AND AIMS: Growing evidence suggests that the metabolic syndrome (MetS) has both a genetic and environmental basis. To evaluate the possibility of a further genetic analysis, we estimated prevalence rates and heritabilities for the MetS and its individual traits in the adult population of Linosa, a small and isolated Italian Island in the southern-central part of the Mediterranean Sea. METHODS AND RESULTS: The Linosa Study (LiS) group consisted of 293 Caucasian native subjects from 51 families (123 parents; 170 offsprings). The MetS was defined according to NCEP/ATP III criteria and the following prevalence rates were calculated: hyperglycaemia 20.3%; central obesity 34.9%; hypertension 43.4%; hypertriglyceridaemia 29.9%; "low HDL" 56.6%; MetS 29.9%. Waist circumference was significantly related to all the quantitative parameters included in the NCEP/ATP III MetS definition. The MetS showed a heritability of 27% (p=0.0012) and among its individual components, treated as continuous and discrete traits, heritability ranged from 10% for blood glucose to 54% for HDL-cholesterol. Among MetS subtypes, the clustering of central obesity, hypertriglyceridaemia and "Iow HDL" had the highest heritability (31%; p<0.001). CONCLUSION: These data showed high prevalence rates for the MetS and its related traits in an isolated and small Caucasian population. The appreciable heritability estimates for the MetS and some of its components/clusters in the LiS population might support the observation of genetic factors underlying the pathogenesis of the MetS and encourage further analysis to identify new susceptibility genes.
Assuntos
Síndrome Metabólica/epidemiologia , Síndrome Metabólica/genética , Adolescente , Adulto , Fatores Etários , Idoso , Glicemia/genética , Glicemia/metabolismo , HDL-Colesterol/sangue , HDL-Colesterol/genética , Feminino , Ligação Genética/genética , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/epidemiologia , Hipertrigliceridemia/genética , Resistência à Insulina/genética , Itália , Masculino , Síndrome Metabólica/diagnóstico , Pessoa de Meia-Idade , Fatores Sexuais , Fumar/epidemiologia , População Branca , Adulto JovemRESUMO
Endothelial damage, with loss of the vascular protective effects of nitric oxide (NO), is an important early step in the development of microvascular and macrovascular complications of diabetes. Endothelial dysfunction is closely associated with diabetic nephropathy in type 1 and 2 diabetes. In this review we will discuss the mechanisms by which hyperglycemia may cause kidney damage and endothelial dysfunction. Hyperglycemia causes microvascular dysfunction, which contributes to the development of end stage renal disease. Determining the role of endothelial abnormalities in the development of diabetic nephropathy is critical to understanding the etiology and pathogenesis of the microvascular complications of diabetes. Endothelial function can be assessed by invasive and noninvasive techniques both in the coronary and peripheral circulation. Endothelial dysfunction is considered a reversible phenomenon; pharmacological intervention with hypolipidemic agents, insulin sensitizers, ACE inhibitors and angiotensin II receptor blockers (ARB) as well as dietary and lifestyle modifications have been shown to reverse it.
Assuntos
Nefropatias Diabéticas/etiologia , Endotélio Vascular/fisiopatologia , Humanos , Hiperglicemia/complicações , Óxido Nítrico/fisiologia , Fatores de RiscoRESUMO
Numerous studies have shown a marked increase in the incidence of diabetes mellitus worldwide. Diabetes mellitus is currently considered equivalent to coronary artery disease in terms of prognostic risk stratification, and its high prevalence makes this clinical condition the first cause of end-stage renal disease requiring chronic hemodialysis or kidney transplant. Even if chronic kidney disease remains the ''Cinderella of the cardiovascular profile'', the presence of microalbuminuria is closely related to a high risk of development of coronary artery disease. The same risk factors that impair heart function are also harmful to the kidney, and the common pathophysiological features of the two systems are at the origin of a new subspecialty, cardionephrology. A crucial task of cardiologists and nephrologists is the early identification of high risk patients with concurrent cardiovascular and kidney disease. The utilization of simple screening methods such as assessment of microalbuminuria and glomerular filtration rate by family doctors may help in establishing prevention strategies directed towards cardiovascular risk and progression of kidney disease. In conclusion, early stratification of cardiovascular risk, coupled with primary prevention strategies aimed at the general population, is warranted to obtain a significant reduction of kidney and cardiovascular disease and of the need for chronic hemodialysis treatment. This strategy is safe and cost-effective in comparison with the costs of chronic dialysis of patients affected by chronic kidney disease.
Assuntos
Doenças Cardiovasculares/complicações , Complicações do Diabetes/complicações , Nefropatias/complicações , Albuminúria/complicações , Humanos , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológico , Prognóstico , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: to compare carbon dioxide and saline solution distension in diagnostic hysteroscopies with regards to patient discomfort and procedural time and in accordance with the instrument diameter (5 mm vs 3.5 mm). The secondary outcome was to evaluate the role of the patient age and the obstetrical history on perception of pain and procedural time. STUDY DESIGN: This is a prospective multicenter randomized study including 1982 patients that underwent office diagnostic hysteroscopy in: Policlinico Abano Terme, Università Cattolica del Sacro Cuore in Rome and Ente Ecclesiastico Ospedale Generale Regionale "F. Miulli" in Acquaviva delle Fonti. They were firstly randomized according to distension medium and secondly according to instrument diameter. Pain perception after the procedure was assessed by VAS (Visual Analogue Scale) score and procedural time was registered. Mann-Whitney U test was used to compare data. RESULTS: Lower pain score and procedural time were recorded with the employment of Carbon Dioxide (p < 001). Patient discomfort and procedural time were significantly influenced by the instrument diameter independent of the distension medium used, though in the subgroup where gas was employed VAS score after 1 min (VAS1) resulted lower compared to saline solution in both the traditional and mini-hysteroscopy procedures (p < 001). CONCLUSION: Carbon dioxide and saline solution are both suitable distension media for outpatient diagnostic hysteroscopy; nonetheless, carbon dioxide confers advantages in terms of pain perception and procedural time.
Assuntos
Dióxido de Carbono/efeitos adversos , Histeroscopia/efeitos adversos , Percepção da Dor/efeitos dos fármacos , Dor Processual/etiologia , Solução Salina/efeitos adversos , Adulto , Procedimentos Cirúrgicos Ambulatórios/efeitos adversos , Procedimentos Cirúrgicos Ambulatórios/métodos , Feminino , Doenças dos Genitais Femininos/diagnóstico , Humanos , Histeroscopia/métodos , Pessoa de Meia-Idade , Duração da Cirurgia , Medição da Dor , Dor Processual/psicologia , Satisfação do Paciente , Estudos Prospectivos , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
A novel mouse homeobox-containing gene, Nkx-2.2, has been isolated. Nkx-2.2 is a member of a family of genes whose homeodomains are homologous to that of the Drosophila NK-2 gene. Nkx-2.2 transcripts are found in localized domains of the brain during mouse embryogenesis. Nkx-2.2 expression in the brain abuts and partially overlaps with the expression domains of two other related homeobox-containing genes, TTF-1 and Dlx. The expression domains of the three genes in the developing prosencephalon coincide with anatomical boundaries, particularly apparent in the diencephalon. This result raises the possibility that these genes may specify regional differentiation of the developing diencephalon into its anatomically and functionally defined subregions. Nkx-2.2 may be involved in specifying diencephalic neuromeric boundaries.
Assuntos
Regulação da Expressão Gênica/genética , Genes Homeobox/genética , Prosencéfalo/embriologia , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , DNA/genética , Diencéfalo/embriologia , Diencéfalo/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes Homeobox/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Morfogênese/fisiologia , Hibridização de Ácido Nucleico , Prosencéfalo/fisiologia , Transcrição Gênica/genéticaRESUMO
Congenital thyroid gland defects - resulting in reduced production of the hormones triiodothyronine (T3) and thyroxine (T4) - can be a consequence of either reduced or absent thyroid tissue (thyroid dysgenesis) or, less frequently, of impairment in the biochemical mechanisms responsible for hormone biosynthesis (thyroid dyshormonogenesis). Recent studies have revealed how mutations in the genes encoding either transcription factors or the thyroid stimulating hormone receptor cause, in humans or in mouse models, thyroid dysgenesis. This demonstrates, for the first time, the heritability of this condition. New genes responsible for thyroid dyshormonogenesis have also been discovered.
Assuntos
Hipotireoidismo Congênito , Hipotireoidismo/genética , Animais , Humanos , Camundongos , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Hormônios Tireóideos/biossínteseRESUMO
OBJECTIVES: To evaluate the effect of hydroalcoholic crude extract (HCE) from Chenopodium ambrosioides leaves on the development of type II collagen-induced arthritis (CIA) and on pro-inflammatory cytokine balance. METHODS: Collagen-induced arthritis was induced in DBA1/J mice. On the 21st day, the mice were treated orally with HCE or methotrexate, daily. Six weeks after beginning the treatment, the following measures were determined: lymphoid organs cell numbers, percentage of blood cells, IL-6, IFN-γ, TNF-α and IL-17 serum concentrations, activity of hepatic and kidney glutathione S-transferase, hepatic 7-ethoxyresorufin-O-deethylase activity, bone density and histopathology. KEY FINDINGS: Treatment of CIA mice with HCE 5 mg/kg (HCE5) reduced the percentage of neutrophils and macrophages and the number of bone marrow cells and increased the lymphocyte numbers and the inguinal lymph node cellularity. This treatment inhibited the serum concentration of IL-6 and TNF-α, which may be related to the preservation of bone density and to the slight thickening of periarticular tissues, with minimal fibrosis and fibroblast proliferation in the joints. The CIA group presented advanced articular erosion and synovial hyperplasia. Phytochemical analysis showed mainly flavonols. CONCLUSIONS: HCE5 presented anti-arthritic potential and reduced IL-6 and TNF-α, which participate directly in the development and maintenance of the inflammatory process in rheumatoid arthritis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Chenopodium ambrosioides/química , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Animais , Anti-Inflamatórios/isolamento & purificação , Artrite Experimental/imunologia , Artrite Experimental/patologia , Densidade Óssea/efeitos dos fármacos , Interleucina-6/sangue , Masculino , Camundongos Endogâmicos DBA , Patela/efeitos dos fármacos , Patela/patologia , Extratos Vegetais/isolamento & purificação , Fator de Necrose Tumoral alfa/sangueRESUMO
Recent studies have identified several polymorphisms in the human insulin receptor substrate-1 (IRS-1) gene. The most prevalent IRS-1 variant, a Gly-->Arg change at the codon 972, has been reported to be increased in prevalence among patients with type 2 diabetes. Carriers of the Arg(972) substitution are characterized by lower fasting insulin and C-peptide levels compared with non-carriers, suggesting that the Arg(972) IRS-1 variant may contribute to impairment of insulin secretion. In this study, we stably overexpressed both wild-type IRS-1 (RIN-WT) and Arg(972) IRS-1 variant (RIN-Arg(972)) in RIN beta cells to investigate directly whether the polymorphism in codon 972 of IRS-1 impairs insulin secretion. The Arg(972) IRS-1 variant did not affect expression or function of endogenous IRS-2. RIN-WT showed a marked increase in both glucose- and insulin-stimulated tyrosine phosphorylation of IRS-1 compared with control RIN cells. The Arg(972) IRS-1 variant did not alter the extent of either glucose- or insulin-stimulated tyrosine phosphorylation of recombinant IRS-1. However, RIN-Arg(972) showed a significant decrease in binding of the p85 subunit of phosphatidylinositol-3-kinase (PI 3-kinase) with IRS-1, compared with RIN-WT. Compared with control RIN cells, insulin content was reduced to the same extent in RIN-WT or RIN-Arg(972) at both the protein and mRNA levels. Both glucose- and sulfonylurea-induced insulin secretion was increased in RIN-WT compared with control RIN cells. By contrast, RIN cells expressing Arg(972) IRS-1 exhibited a marked decrease in both glucose- and sulfonylurea-stimulated insulin secretion compared with RIN-WT. These data suggest that the insulin signaling pathway involving the IRS-1/PI 3-kinase may play an important role in the insulin secretory process in pancreatic beta cells. More importantly, the results suggest that the common Arg(972) IRS-1 polymorphism may impair glucose-stimulated insulin secretion, thus contributing to the relative insulin deficiency observed in carriers of this variant.
Assuntos
Substituição de Aminoácidos/genética , Arginina/genética , Glicina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Fosfoproteínas/genética , Polimorfismo Genético , Animais , Glucose/farmacologia , Humanos , Insulina/genética , Proteínas Substratos do Receptor de Insulina , Secreção de Insulina , Insulinoma/enzimologia , Insulinoma/genética , Insulinoma/metabolismo , Líquido Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato/genética , Compostos de Sulfonilureia/farmacologia , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
We used the lung epithelial cell-specific surfactant protein B (SPB) gene promoter as a model with which to investigate mechanisms involved in transcriptional control of lung-specific genes. In a previous study, we showed that the SPB promoter specifically activated expression of a linked reporter gene in the continuous H441 lung cell line and that H441 nuclear proteins specifically protected a region of this promoter from bp -111 to -73. In this study, we further show that this region is a complex binding site for thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3 (HNF-3). Whereas TTF-1 bound two highly degenerate and closely spaced sites, HNF-3 proteins bound a TGT3 motif (TGTTTGT) that is also found in several liver-specific gene regulatory regions, where it appears to be a weak affinity site for HNF-3. Point mutations of these binding sites eliminated factor binding and resulted in significant decreases in transfected SPB promoter activity. In addition, we developed a cotransfection assay and showed that a family of lung-specific gene promoters that included the SPB, SPC, SPA, and Clara cell secretory protein (CCSP) gene promoters were specifically activated by cotransfected TTF-1. We conclude that TTF-1 and HNF-3 are major activators of lung-specific genes and propose that these factors are involved in a general mechanism of lung-specific gene transcription. Importantly, these data also show that common factors are involved in organ-specific gene expression along the mammalian foregut axis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Pulmão/fisiologia , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Proteolipídeos/genética , Surfactantes Pulmonares/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Fator 3-alfa Nuclear de Hepatócito , Fator 3-beta Nuclear de Hepatócito , Técnicas In Vitro , Fígado/fisiologia , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Glândula Tireoide/fisiologia , Fator Nuclear 1 de Tireoide , Ativação TranscricionalRESUMO
Expression of oncogenic Ras in thyroid cells results in loss of expression of several thyroid-specific genes and inactivation of TTF-1, a homeodomain-containing transcription factor required for normal development of the thyroid gland. In an effort to understand how signal transduction pathways downstream of Ras may be involved in suppression of the differentiated phenotype, we have tested mutants of the Ras effector region for their ability to affect TTF-1 transcriptional activity in a transient-transfection assay. We find that V12S35 Ras, a mutant known to interact specifically with Raf but not with RalGDS or phosphatidylinositol 3-kinase (PI3 kinase) inhibits TTF-1 activity. Expression of an activated form of Raf (Raf-BXB) also inhibits TTF-1 function to a similar extent, while the MEK inhibitors U0126 and PD98059 partially relieve Ras-mediated inactivation of TTF-1, suggesting that the extracellular signal-regulated kinase (ERK) pathway is involved in this process. Indeed, ERK directly phosphorylates TTF-1 at three serine residues, and concomitant mutation of these serines to alanines completely abolishes ERK-mediated phosphorylation both in vitro and in vivo. Since activation of the Raf/MEK/ERK pathway accounts for only part of the activity elicited by oncogenic Ras on TTF-1, other downstream pathways are likely to be involved in this process. We find that activation of PI3 kinase, Rho, Rac, and RalGDS has no effect on TTF-1 transcriptional activity. However, a poorly characterized Ras mutant, V12N38 Ras, can partially repress TTF-1 transcriptional activity through an ERK-independent pathway. Importantly, concomitant expression of constitutive activated Raf and V12N38 Ras results in almost complete loss of TTF-1 activity. Our data indicate that the Raf/MEK/ERK cascade may act in concert with an as-yet-uncharacterized signaling pathway activated by V12N38 Ras to repress TTF-1 function and ultimately to inhibit thyroid cell differentiation.
Assuntos
Regulação da Expressão Gênica , Genes ras , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular , Proteínas de Homeodomínio/genética , Fosforilação , Ratos , Proteínas Repressoras/genética , Transdução de Sinais/genética , Glândula Tireoide/fisiologia , Fator Nuclear 1 de Tireoide , Transcrição GênicaRESUMO
A 420-bp fragment from the 5' end of the rat thyroperoxidase (TPO) gene was fused to a luciferase reporter and shown to direct cell-type-specific expression when transfected into rat thyroid FRTL-5 cells. Analysis of this DNA fragment revealed four regions of the promoter which interact with DNA-binding proteins present in FRTL-5 cells. Mutation of the DNA sequence within any of these regions reduced TPO promoter activity. The trans-acting factors binding to these sequences were compared with thyroid transcription factor 1 (TTF-1) and TTF-2, previously identified as transcriptional activators of another thyroid-specific gene, the thyroglobulin (Tg) gene. Purified TTF-1 binds to three regions of TPO which are protected by FRTL-5 proteins. Two of the binding sites overlap with recognition sites for other DNA-binding proteins. One TTF-1 site can also bind a protein (UFB) present in the nuclei of both expressing and nonexpressing cells. TTF-1 binding to the proximal region overlaps with that for a novel protein present in FRTL-5 cells which can also recognize the promoter-proximal region of Tg. Using a combination of techniques, the factor binding to the fourth TPO promoter site was shown to be TTF-2. We conclude, therefore, that the FRTL-5-specific expression of two thyroid restricted genes, encoding TPO and Tg, relies on a combination of the same trans-acting factors present in thyroid cells.