RESUMO
Genomic imprinting is an important epigenetic phenomenon, wherein genes or gene clusters are marked by DNA methylation during gametogenesis. This plays a major role in several functions of normal cells, including cell differentiation, X chromosome inactivation, and the maintenance of chromatin structure, in mammalian development. The aim of this study was to investigate the possible differences in SNRPN gene methylation profiles in non-obese and obese individuals, and in children and adults. Our results did not reveal any statistical correlations between the DNA methylation profiles of the SNRPN gene in children or adult obese and non-obese groups. However, a comparison of the methylation levels with the chronological age revealed statistically significant differences between the means of methylation in adults and children (46.20 ± 5.88 and 39.40 ± 2.87, respectively; P < 0.001). Pearson's correlation analysis indicated a positive association between the level of DNA methylation and the chronological age (R2 = 0.326; P < 0.001). Therefore, we concluded that the methylation profile of the SNRPN promoter (in blood) is not a useful biomarker for determining the predisposition of an individual to obesity. Additionally, we have confirmed that SNRPN methylation increases with age, which raises further questions regarding the role of SNRPN expression during the aging process.
Assuntos
Peso Corporal/genética , Metilação de DNA , Estudos de Associação Genética , Proteínas Centrais de snRNP/genética , Adolescente , Adulto , Fatores Etários , Criança , Humanos , Pessoa de Meia-Idade , Obesidade/genética , Adulto Jovem , Proteínas Centrais de snRNP/químicaRESUMO
High-resolution melting (HRM) is considered an inexpensive, rapid, and attractive methodology for methylation analysis. In the application of the polymerase chain reaction (PCR) to methylation analysis, amplification efficiencies are biased towards unmethylated, rather than methylated, templates: a phenomenon known as PCR bias. To overcome PCR bias, primers that include CpG site(s) and are fully complementary to the methylated sequence have been proposed. However, genes mapped within imprinted regions usually present higher methylation levels, and an unusual PCR bias towards the methylated template can therefore arise. The manipulation of primer affinity attempts to overcome this problem. We attempted to show that mismatches at the primer's methylated binding sites increase the area between the 50 and 100% methylation plots on the melting curves, and may increase HRM accuracy for samples that have high methylation levels. Sets of primers for imprinted genes that included CpG sites at their binding sequences were designed, and were complementary to methylated or unmethylated templates. Primers fully complementary to methylated templates produced a very small area between the 50 and 100% methylation plots. When using primers that were fully complementary to the unmethylated sequence, we were able to increase the area between the 50 and 100% methylation plots. Therefore, when samples are highly methylated, such as targets in genes mapped in imprinted regions, we propose that primers should favor amplification of the rarest, unmethylated sequence. Primers may be designed to include one CpG at its binding site and be fully complementary to the unmethylated template.
Assuntos
Primers do DNA/metabolismo , Impressão Genômica/genética , Desnaturação de Ácido Nucleico/genética , Adulto , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Proteínas/genéticaRESUMO
AIMS: The polymorphisms of pro- and anti-inflammatory cytokines may be involved in type 2 diabetes (T2D) pathogenesis and its complications. METHODS: We investigated in 102 T2D patients the association of the cytokine polymorphisms in the TNF-α, IL-10, IL-6, TGF-ß1, and IFN-γ genes with the T2D microvascular complications and comorbidities (hypertension, dyslipidemia, and obesity). Cytokine genotypes were determined by PCR using Cytokine Genotyping Tray kit. RESULTS: Diabetic retinopathy was associated with GG genotype and G allele in TGF-ß1 codon 25C/G polymorphism (p = 0.004 and p = 0.018) and the nephropathy was associated the lower frequency of GG genotype in IL-10 -1082G/A polymorphism (p = 0.049). Hypertension was associated with the CC genotype and C allele for IL-10 -592C/A polymorphism (p = 0.013 and p = 0.009) and higher frequencies of T (p = 0.047) and C (p = 0.033) alleles of the TGF-ß1 codon 10T/C and IL-10 -819T/C polymorphisms, respectively. The TGF-ß1 codon 10T/C polymorphism was associated with the BMI groups (p = 0.026): the CC genotype was more frequent in the group with BMI < 25 Kg/m(2), while the TC genotype was more frequent in the group with BMI = 30 Kg/m(2). CONCLUSIONS: Our findings suggest that TGF-ß1 and IL-10 polymorphisms are involved in complications and comorbidities in T2D patients.
Assuntos
Complicações do Diabetes/genética , Diabetes Mellitus Tipo 2/genética , Interferon gama/genética , Interleucina-10/genética , Interleucina-6/genética , Polimorfismo de Nucleotídeo Único , Fator de Crescimento Transformador beta1/genética , Fator de Necrose Tumoral alfa/genética , Idoso , Alelos , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
AIMS: Obesity is one of the major concerns in the world currently, its prejudicial effect is exerted by proteins secreted by adipose tissue, among them visfatin was demonstrated to be related with BMI and cardiovascular diseases. The HMG-CoA reductase inhibitors are known to minimize the cardiovascular risk in hyperlipidemic patients and recently the discovery of various pleiotropic effects has made the statins evidencing among others anti-inflammatory effect. Our objective in this study was to determinate if simvastatin treatment may modulate visfatin levels in obese women without any other metabolic disorder. METHODS: We recruited 25 obese women without any other metabolic disorder and treated with simvastatin for 6 weeks 20 mg/day. RESULTS: The levels of plasma visfatin were similar before and after treatment (22 ± 20 versus 27 ± 14 ng/mL, p > 0.05) and correlated with BMI before treatment (p = 0.001). We also found correlations among visfatin and insulin levels (p = 0.015) and HOMA-IR (p = 0.025) only after treatment. CONCLUSION: These findings suggest that visfatin is not modulated by simvastatin treatment in this group but the treatment may interfere on the relation among visfatin, BMI, insulin and HOMA-IR.
Assuntos
Citocinas/sangue , Hipolipemiantes/uso terapêutico , Nicotinamida Fosforribosiltransferase/sangue , Obesidade/sangue , Obesidade/tratamento farmacológico , Sinvastatina/uso terapêutico , Adulto , Índice de Massa Corporal , Colesterol/sangue , Feminino , Humanos , Insulina/sangue , Resistência à Insulina , Pessoa de Meia-Idade , Triglicerídeos/sangueRESUMO
OBJECTIVE: To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, on the secretion of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMCs) and on the generation of reactive oxygen species (ROS) by leukocytes. METHODS: PBMCs and leukocytes were obtained from venous blood samples collected from 20 healthy individuals. VEGF secretion was evaluated using a commercial ELISA kit, while ROS production was determined using a luminol-dependent chemiluminescence assay. RESULTS: Rosiglitazone and calphostin C (a protein kinase C inhibitor) inhibited VEGF secretion by PBMCs by 63.7 and 62.3%, respectively. Both agents reduced ROS production in non-stimulated human leukocytes and down-regulated the enhanced generation of ROS in leukocytes that had been stimulated with the PKC activator phorbol 12,13-dibutyrate. CONCLUSION: The results support the involvement of PKC as a direct, and/or NADPH-oxidase as an indirect, target for the action of rosiglitazone on VEGF secretion by PBMCs and ROS production in human leukocytes.
Assuntos
Leucócitos Mononucleares/citologia , PPAR gama/metabolismo , Tiazolidinedionas/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Humanos , Hipoglicemiantes/farmacologia , Leucócitos/citologia , Luminescência , Pessoa de Meia-Idade , Naftalenos/farmacologia , Dibutirato de 12,13-Forbol/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio , RosiglitazonaRESUMO
OBJECTIVE: To compare the role of Akt/PKB signaling pathway in the modulation of reactive oxygen species (ROS) production by autologous plasma in peripheral blood mononuclear cells (PBMNC) from type 2 diabetic patients and healthy subjects. MATERIALS AND METHODS: This study was approved by Santa Casa Ethical Committee and has included patients diagnosed with diabetes type 2 (DM2) and control group (non-diabetic) (ND). PBMNC were purified utilizing Ficoll-hypaque gradient. ROS was quantified by luminol-dependent chemiluminescence. The Akt/PKB phosphorylation was measured using a CASE kit. Statistical analyses were made with t Student test and chi-square (chi(2)). p<0.05 was considered significant. RESULTS: 12, 13-Phorbol dibutyrate (PDB) stimulated the production of higher levels of ROS in PBMNC from type 2 diabetic patients than that from healthy subjects. Autologous plasma, however, inhibited induced or not ROS production in PBMNC in both groups. The inhibition of PBMNC-ROS derived by autologous plasma from healthy subjects was higher than that from type 2 diabetic patients. Plasma phosphorylated (activated) Akt/PKB. The percentage of phosphorylation induced by autologous plasma in PBMNC from patients and healthy control were 14% and 93%, respectively. Inhibition of ROS production in PBMNC from DM2 were similar for PBMNC+plasma; PBMNC+Akti; and PBMNC+plasma+Akti. However, in ND control, plasma showed a higher ROS inhibition than Akti or plasma plus Akti. CONCLUSIONS: Our results suggest that the low antioxidant capacity observed in autologous plasma from DM2 patients in conjunction with the decreased activation of PKB may cause an imbalance in the oxidizing/reducing responses, possible inducing an oxidative stress state, which could be associated with tissular damage.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Leucócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucócitos/efeitos dos fármacos , Pessoa de Meia-Idade , NADPH Oxidases/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidoresRESUMO
AIMS/HYPOTHESIS: Our aim was to compare the therapeutic effect of thalidomide and rosiglitazone on the prevention of diabetic retinopathy in streptozotocin-induced diabetic rats. METHODS: Male Holtzman rats of 6 to 8 weeks of age and weighing 170+/-30 g were randomly divided into four groups: control ( n=13), untreated diabetic ( n=17) and diabetic rats treated with thalidomide (200 mg kg(-1) day(-1)) ( n=8) or rosiglitazone (1 mg kg(-1) day(-1)) ( n=22) for 3 months. Diabetes was induced by streptozotocin with the rats having a body weight of 70 mg/kg. After treatment, vascular endothelial growth factor (VEGF) concentrations in ocular fluid were compared between the different groups, and retinal capillary basement membrane thickness was measured by electron microscopy. RESULTS: Higher VEGF concentrations in ocular fluid and thicker basement membranes were observed in untreated diabetic rats compared to the control rats. Similar VEGF concentrations and basement membrane thickness were observed for the thalidomide-treated group compared with the control group, whereas no difference in these parameters was observed between the rosiglitazone-treated rats and the control or untreated diabetic rats. CONCLUSIONS/INTERPRETATION: Our findings confirm the association between VEGF concentrations and diabetic retinopathy as suggested by other investigators. Thalidomide, but not rosiglitazone, was associated with the inhibition of basement membrane thickening and the blockade of the increase of VEGF in ocular fluid, thus representing a potential therapeutic drug for the prevention of diabetic retinopathy.