RESUMO
BACKGROUND: Amyloid-ß peptide (Aß) deposition in Alzheimer's disease (AD) is due to an imbalance in its production/clearance rate. Aß is transported across the blood-brain barrier by LRP1 and P-gp as efflux transporters and RAGE as influx transporter. Vitamin D deficit and polymorphisms of the vitamin D receptor (VDR) gene are associated with high prevalence of mild cognitive impairment (MCI) and AD. Further, vitamin D promotes the expression of LRP1 and P-gp in AD-animal model brains. OBJECTIVE: To associate VDR polymorphisms Apa I (rs7975232), Taq I (rs731236), and Fok I (rs2228570) with the risk of developing MCI in a Chilean population, and to evaluate the relationship of these polymorphisms to the expression of VDR and Aß-transporters in peripheral blood mononuclear cells (PBMCs). METHODS: VDR polymorphisms Apa I, Taq I, and Fok I were determined in 128 healthy controls (HC) and 66 MCI patients. mRNA levels of VDR and Aß-transporters were evaluated in subgroups by qPCR. RESULTS: Alleles A of Apa I and C of Taq I were associated with a lower risk of MCI. HC with the Apa I AA genotype had higher mRNA levels of P-gp and LRP1, while the expression of VDR and RAGE were higher in MCI patients and HC. For Fok I, the TC genotype was associated with lower expression levels of Aß-transporters in both groups. CONCLUSION: We propose that the response to vitamin D treatment will depend on VDR polymorphisms, being more efficient in carriers of protective alleles of Apa I polymorphism.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Receptores de Calcitriol/genética , Idoso , Chile/epidemiologia , Disfunção Cognitiva/epidemiologia , Estudos de Coortes , Feminino , Expressão Gênica , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Fatores de Risco , Taq Polimerase/genética , Taq Polimerase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Background Thermostable DNA polymerase (Taq Pol ?) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30-40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. Results We provided a novel, simplified and low-cost method to purify the Taq Pol ? after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save labor, time, and cost. Conclusions Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol ?, and simplifies the operation, and increases the enzyme recovery rate and quality.
Assuntos
Taq Polimerase/isolamento & purificação , Taq Polimerase/genética , Etanol/química , Precipitação Química , Reação em Cadeia da PolimeraseRESUMO
Schizophrenia is a severe psychotic disorder with recurrent relapse and functional impairment. It results from a poorly understood gene-environment interaction. The Taq1A polymorphism (located in the gene cluster NTAD) is a likely candidate for schizophrenia. Its rs1800497 polymorphism was shown to be associated with DRD2 gene expression. Therefore the present work aims to investigate a possible association between schizophrenia and such polymorphism. The compared distribution of the alleles and genotypes of the studied polymorphism was investigated in a Brazilian sample of 235 patients and 834 controls. Genotypic frequencies were in Hardy-Weinberg equilibrium. There was a trend of allelic association between the Taq1A polymorphism (rs1800497) with schizophrenia in the studied sample. However no statistically differences were found between cases and controls when analyzed by gender or schizophrenia subtypes.
Assuntos
Interação Gene-Ambiente , Polimorfismo Genético/genética , Receptores de Dopamina D2/genética , Esquizofrenia/genética , Taq Polimerase/genética , Brasil , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , MasculinoRESUMO
Schizophrenia is a severe psychotic disorder with recurrent relapse and functional impairment. It results from a poorly understood gene-environment interaction. The Taq1A polymorphism (located in the gene cluster NTAD) is a likely candidate for schizophrenia. Its rs1800497 polymorphism was shown to be associated with DRD2 gene expression. Therefore the present work aims to investigate a possible association between schizophrenia and such polymorphism. The compared distribution of the alleles and genotypes of the studied polymorphism was investigated in a Brazilian sample of 235 patients and 834 controls. Genotypic frequencies were in Hardy-Weinberg equilibrium. There was a trend of allelic association between the Taq1A polymorphism (rs1800497) with schizophrenia in the studied sample. However no statistically differences were found between cases and controls when analyzed by gender or schizophrenia subtypes.
A esquizofrenia é um grave transtorno psicótico que apresenta frequentes recaídas e incapacitação progressiva. Resulta de uma interação gene-ambiente ainda pouco compreendida. O polimorfismo Taq1A (localizado no grupamento genético NTAD) é considerado um possível candidato para esquizofrenia. O polimorfismo genético rs1800497 foi associado com alteração da expressão do gene do DRD2. Assim, o presente trabalho objetivou investigar a possível associação de tal polimorfismo com esquizofrenia. A distribuição de seus alelos e genótipos foi investigada em uma amostra brasileira composta de 235 pacientes e 834 controles. As frequências genotípicas estavam em equilíbrio de Hardy-Weinberg. Houve uma tendência de associação alélica entre o polimorfismo Taq1A (rs1800497) e esquizofrenia na amostra estudada. No entanto, não houve diferenças estatisticamente significantes entre os grupos de casos e controles, quando analisados por gênero e subtipos da esquizofrenia.
Assuntos
Feminino , Humanos , Masculino , Interação Gene-Ambiente , Polimorfismo Genético/genética , /genética , Esquizofrenia/genética , Taq Polimerase/genética , Brasil , Estudos de Casos e Controles , Frequência do Gene , Predisposição Genética para Doença , GenótipoRESUMO
We report a study of TGFA/ Taq I polymorphisms and environmental factors in non-syndromic oral cleft in Southern Brazil. Nonsyndromic cleft case-parent triads were recruited to participate. Clinical data was collected with an emphasis on tobacco and alcohol use during pregnancy. DNA was extracted from peripheral blood and TGFA/ Taq I polymorphisms were analyzed by PCR/RFLP with Taq I restriction enzyme. Association of clefts and TGFA/ Taq I polymorphisms was determined using a transmission disequilibrium test (TDT). Association of environmental factors, clefts, and genotypes was evaluated with Fisher's exact test. The minor allele frequency was 0.064. We found no evidence of association between TGFA/ Taq I polymorphisms and clefting (TDT p = 0.335). We also found no association between TGFA/ TaqI polymorphisms and environmental factors (alcohol and/or tobacco). Therefore, no evidence was found that TGFA/ Taq I polymorphisms play a role in clefting in this population. No evidence was found that tobacco or alcohol exposure during pregnancy was related to clefting, however a larger sample size is needed to confirm these results.
Assuntos
Feminino , Humanos , Masculino , Gravidez , Fenda Labial/genética , Fissura Palatina/genética , Polimorfismo Genético/genética , Taq Polimerase/genética , Fator de Crescimento Transformador alfa/genética , Brasil , Frequência do Gene , Exposição Materna , Reação em Cadeia da Polimerase , Fatores de Risco , FumarRESUMO
We report a study of TGFA/ Taq I polymorphisms and environmental factors in non-syndromic oral cleft in Southern Brazil. Nonsyndromic cleft case-parent triads were recruited to participate. Clinical data was collected with an emphasis on tobacco and alcohol use during pregnancy. DNA was extracted from peripheral blood and TGFA/ Taq I polymorphisms were analyzed by PCR/RFLP with Taq I restriction enzyme. Association of clefts and TGFA/ Taq I polymorphisms was determined using a transmission disequilibrium test (TDT). Association of environmental factors, clefts, and genotypes was evaluated with Fisher's exact test. The minor allele frequency was 0.064. We found no evidence of association between TGFA/ Taq I polymorphisms and clefting (TDT p = 0.335). We also found no association between TGFA/ TaqI polymorphisms and environmental factors (alcohol and/or tobacco). Therefore, no evidence was found that TGFA/ Taq I polymorphisms play a role in clefting in this population. No evidence was found that tobacco or alcohol exposure during pregnancy was related to clefting, however a larger sample size is needed to confirm these results.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Polimorfismo Genético/genética , Taq Polimerase/genética , Fator de Crescimento Transformador alfa/genética , Brasil , Feminino , Frequência do Gene , Humanos , Masculino , Exposição Materna , Reação em Cadeia da Polimerase , Gravidez , Fatores de Risco , FumarRESUMO
OBJECTIVE: To test the TGFA/Taq I polymorphism in the development of nonsyndromic cleft lip and palate. DESIGN AND SETTING: The research was based on a case-control study, including nonsyndromic cleft lip and palate patients (140 individuals) and a control sample of unaffected individuals (142) to ascertain the absence or presence of genic mutation at the TGFA locus. INTERVENTIONS: The DNA of carriers of nonsyndromic cleft lip with or without cleft palate was obtained by buccal swab, and the DNA of the control group was extracted from peripheral blood leucocytes. TGFA/Taq I polymorphism was determined genetically by polymerase chain reaction using specific primers and fragment digestion with Taq I restriction enzyme. RESULTS: No significant association was detected when patients and controls were compared with the genotype for TGFA/Taq I polymorphism. CONCLUSION: Mutations in TGFA gene have no association with nonsyndromic cleft lip and palate in the sample from Rio Grande do Sul. Therefore, based on this study, it is not possible to determine the role played by TGFA in the expression of cleft lip and palate.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Polimorfismo Genético/genética , Fator de Crescimento Transformador alfa/genética , Adenina , Adolescente , Adulto , Alelos , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 2/genética , DNA/genética , Feminino , Genótipo , Humanos , Lactente , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Deleção de Sequência/genética , Taq Polimerase/genética , Timina , Adulto JovemRESUMO
The DRD2 TaqI A and DBH-1021 C/T polymorphisms were genotyped in smoking alcoholics (N = 100), non-alcoholic smokers (N = 120) and nonsmoking controls (N = 112). Alcoholic and non-alcoholic smokers presented a higher frequency of the DRD2 TaqI A1 allele (P = 0.04) than non-smoking controls. Individuals who had at least one DBH-1021 T allele smoked fewer cigarettes per day than CC homozygotes (P = 0.03). These results are coherent with the expected effects of these polymorphisms on dopaminergic function.
Assuntos
Dopamina beta-Hidroxilase/genética , Polimorfismo Genético/genética , Receptores de Dopamina D2/genética , Fumar/genética , Adulto , Alcoolismo/genética , Alelos , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 9/genética , Comorbidade , Frequência do Gene , Genótipo , Homozigoto , Humanos , Masculino , Reação em Cadeia da Polimerase , Taq Polimerase/genéticaRESUMO
The Thermus aquaticus DNA Polymerase I (Taq Pol I) gene was cloned into the pOSEX4 plasmid under the osmo-inducible promoter proU and subsequently expressed into the Escherichia coli MKH13 strain. The suitability of the enzyme in polymerase assays was determined in standard 35S dATP incorporation tests and by PCR. The Taq Pol I expression in this system, which is under the control of the osmotic pressure in the growth medium, was analyzed in different media and in different sodium chloride concentrations. A study of the osmolarity effects in the growth of the strain and in Taq Pol I expression shows that an increase in sodium chloride concentration limits the growth. At 0.25 M of NaCl maximum activity was observed; at higher values of osmolarity, we found an unexpected decline of activity. This is the first report of using the pOSEX vector for the expression of an heterologous protein and it is very advantageous to make a regulated, non toxic, simple and cost-effective manner of induction in a biotechnology process using just NaCl or other non-permeable osmolyte.