Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Marcadores genéticos associados com a resposta inflamatória na meningite bacteriana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA) misamplification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serological investigation and PCR in detection of pathogenic leptospires in snakes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identificação e caracterização de genes vip e cry coleóptero-específicos em isolados de Bacillus thuringiensis

O objetivo deste trabalho foi identificar e caracterizar os genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de 1.078 isolados de Bacillus thuringiensis potencialmente tóxicos para larvas de coleópteros. Foram utilizados pares de oligonucleotídeos iniciadores gerais obtidos a partir de regiões conservadas dos genes e do alinhamento de sequências consenso. Posteriormente, os isolados positivos foram caracterizados por meio da técnica de PCR‑RFLP, tendo-se utilizado enzimas de restrição específicas, para identificar novas subclasses de genes nos isolados. Cento e cinquenta e um isolados foram positivos para os genes avaliados, com maior frequência para o gene vip1/vip2 (139 isolados). Pela técnica de PCR‑RFLP, foram observados 14 perfis polimórficos, o que indica a presença de diferentes alelos e, consequentemente, de distintas subclasses desses genes.

Identification of new isolates of Bacillus thuringiensis using rep-PCR products and delta-endotoxin electron microscopy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização molecular e laboratorial da talassemia beta e da interação hemoglobina s/talassemia beta

Beta thalassemia arises as a consequence of the reduction (β+, β++, βsilent) or absence (β0) of beta globin chain synthesis and results from a number of mechanisms that lead to genetic defects. The inheritance of beta thalassemia is characterized by the existence of heterozygous individuals, compound heterozygotes, homozygotes and those with coinheritance of beta thalassemia allele and other thalassemias and/or hemoglobin variants. The aim of this study was to perform molecular and laboratory characterization of beta thalassemia in heterozygous and homozygous individuals and in those with coinheritance of S beta thalassemia. A total of 48 individuals were included (35 heterozygotes, 4 homozygotes and 9 S beta thalessemia carriers) referred to the Integrated Laboratory of Clinical Analyses of the Federal University of Rio Grande do Norte (UFRN) and the Hematology Ambulatory Facility of the Dalton Barbosa Cunha Hemocenter (Hemonorte Natal, Brazil). Peripheral blood samples form each patient underwent the following laboratory examinations: erythrogram, hemoglobin electrophoresis at alkaline pH, measurements of Hb A2, Fetal Hb and serum ferritin. DNA was extracted using the illustra blood genomicPrep Mini Spin Kit and molecular characterization was performed by the PCR/RFLP technique, which involves digestion with specific restriction enzymes for IVS-1 nt 1 (G®A), IVS-1 nt 6 (T®C) and codon 39 (CAG®TAG) mutations. Of the 35 heterozygotes, 37.1% showed IVS-1 nt 6 mutation, 42.9% IVS-1 nt 1 and 20% were carriers of other mutations not identified by the technique used. The four homozygous patients presented with the IVS-1 nt 6 mutation, while 66.7% of the individuals with S beta thalassemia had the IVS-1 nt 1 mutation. Codon 39 was not detected in any of the patients investigated. Of the thallasemic alleles found, 40.4% were IVS- 1 nt 1, 40.4% IVS-1 nt 6 and 19.2% were not identified. Laboratory data showed that the heterozygotes exhibited microcytosis and hypochromia, evidenced by MCV ranging from 57 to 75fL and MCH from 15.9 to 23.6 pg. Hemoglobin A2 varied between 3.7 and 7.2%. The homogygotes also showed reduced MCV and MCH and elevated HbA2.. Comparison of laboratory data between heterozygous individuals with IVS-1 nt 1 and IVS-1 nt 6 mutations showed that heterozygotes for the IVS1-1 mutation had significantly lower mean MCV and MCH (p = 0.023 and 0.007, respectively) and significantly higher hemoglobin A2 (p < 0.001) when compared to heterozygotes for the IVS-1 nt 6 mutation. PCR/RFLP was useful in identifying the presence or absence of IVS-1 nt 6, IVS-1 nt 1 and codon 39 mutations in most of the patients investigated here. This is the first study conducted in the state of Rio Grande do Norte, Brazil aimed at identifying beta thalassemia mutations and represents an important contribution to the knowledge regarding the molecular profile of beta thalassemia in our country

Characterization and polymorphism screening of IGF-I and prolactin genes in Nelore heifers

Insulin growth factor I (IGF-I) and prolactin (PRL) are peptide hormones that exert complementary effects on reproductive traits by acting on folliculogenesis. In view of the lack of information about the IGF-I and PRL genes in Bos indicus, the objective of this study was to partially characterize the promoter regions of these genes and to screen animals of different ages at first pregnancy for the presence of polymorphisms in these regions. In addition, we determined whether polymorphisms influence the regulation of the two hormone genes, evaluating their association with sexual precocity.The animals were divided into three groups according to age at first pregnancy: 1) 100 heifers considered to be sexually precocious that became pregnant at 15-16 months of age, 2) 100 heifers that became pregnant during the normal breeding season at 24 months of age, and 3) 100 heifers that did not become pregnant until 24 months of age. For the IGF-I gene, PCR-RFLP-SnaBI analysis showed the presence of genotypes AB and BB at frequencies of 0.02 and 0.98, respectively. Sequencing of the IGF-I gene fragment revealed a single nitrogen base change from cytosine to thymine, corresponding to the restriction site of SnaBI. The polymorphisms identified in the 5'-flanking region of the IGF-I gene may serve as a basis for future studies of molecular markers in cattle. For the PRL gene, PCR-RFLP-HaeIII analysis showed the presence of only one migration pattern, a finding characterizing the region studied as monomorphic. The study of other regions in the IGF-I and PRL genes might provide molecular data that can be used in the future for the selection of sexually precocious animals.

Estudo de polimorfismos em genes relacionados ao metabolismo do ácido fólico e sua associação com o desenvolvimento de fendas orais não-sindrômicas

The congenital facial clefts are characterized by incomplete formation of the structures that separate the oral and nasal cavity. It is known that several environmental and genetic factors are involved in its development, among these, polymorphisms associated with folic acid metabolism have been investigated. In this sense, the objective was to observe the frequency of polymorphisms C677T and A1298C methylenetetrahydrofolate reductase gene (MTHFR), methionine synthase A2756G of (MTR), A66G of methionine synthase reductase (MTRR) A80G and the reduced folate carrier (RFC1) in patients with non-syndromic oral clefts, trying to match them with their development. Methods: We studied 140 patients with non-syndromic oral clefts and their mothers and 175 control subjects with their mothers, who underwent a questionnaire to obtain family information. Were collecting blood for DNA extraction from patients and their mothers to identify the genotypes of both by PCRRFLP, in addition to carrying out the determination of glucose, AST, ALT and serum creatinine, folic acid and vitamin B12 Serum and plasma homocysteine, and the hemogram. Results: Most patients have cleft lip and palate (55.8%), followed by isolated cleft palate (24.2%) and cleft lip (20%). Regarding gender, 62% of patients were male and 48% female and, after subdivision of the type of screwdriver according to sex was found a prevalence of males in the cracks of the type lip and palate (69 %) and lip (69.2%) and in the case of cleft palate was a female predominance (59%). The average concentration of serum folate in the group of mothers of cleft patients was significantly lower (13.8 ± 2.4 ng / mL) compared with the group of mothers of control subjects (18.8 ± 3.4 ng / mL) This was also observed for the group of cleft children as compared to controls, the dosage of folic acid had a significant difference with values of 15.6 ± 0.6 (ng / mL) and 17.9 ± 0.6 (ng / mL), respectively. For the biochemical measurements of glucose, AST, ALT and creatinine were not statistically different, nor was observed for haematological parameters performed. In assessing the frequency of polymorphisms C677T and A1298C MTHFR, A2756G MTR, MTRR A66G and A80G of the RFC1 there was no statistically significant difference in genotype distribution between cases and controls both for mothers and in the cleft. Conclusion: Although not observed association of polymorphisms with the development of cracks, the decrease in serum folate in the group of cleft patients and their mothers may reflect a disturbance in the metabolism of this metabolite, necessitating further studies such as studies methylation and expression to further elucidate the involvement of folate in the development of oral clefts

Estudo dos genes do complexo do antígeno leucocitário humano (hla) associados à susceptibilidade ao diabetes mellitus tipo 1

Of all of the genes associated with the development of Diabetes mellitus type 1 (T1D), the largest contribution comes from the genes in the Human Leukocyte Antigen (HLA) region, mostly the class II DR e DQ genes. Specific combinations of alleles DRB1, DQA1 and DQB1 constituting haplotypes, and further, a combination of more than one haplotype, providing multilocus genotypes are associated with susceptibility, protection and neutrality to DM1. Thus, the aim of present study was to verified the association of polymorphisms of HLA genes class II with susceptibility to type 1 diabetes mellitus (T1D). Ninety-two patients with T1D and 100 individuals normoglycemics (NG) aged between 6 and 20 years were studied. Genomic DNA was obtained from peripheral whole blood, collected in EDTA tube, using the extraction kit Illustra Triple Prep®, GE Healthcare. For HLA typing was used DNA LABType system by One Lambda kit applying Luminex® technology to the method of PCRSSO typing reverse. The alleles DRB1*03:01, *04:05, *04:01, *04:02, DQA1*03:01g, *05:01g, DQB1*02:01g, *03:02, the haplotypes DRB1*03:01-DQA1*05:01-DQB1*02:01, DRB1*04:05-DQA1*03:01g-DQB1*03:02, DRB1*04:02-DQA1*03:01g-DQB1*03:02, DRB1*04:01-DQA1*03:01g-DQB1*03:02 and DR3-DQ2/DR4-DQ8 genotype were significantly associated with the chance of developing T1D. The alleles DRB1*11:01, *15:03, *15:01, *13:01, DQA1*01:02, *04:01g, *01:03, DQB1*06:02, *03:01g, *06:03, *04:02, the haplotypes DRB1*11:01-DQA1*05:01-DQB1*03:01, DRB1*13:01-DQA1*01:03-DQB1*06:03 and DRX-DQX/DRX-DQX genotype, formed by other than the DR3-DQ2 or DR4-DQ8 haplotypes, were significantly associated with T1D protection Despite the major racial Brazilian, even at the regional level, these results are similar to the majority of alleles, genotypes and haplotypes of HLA class II-related susceptibility or resistance to T1D, extensively described in the literature for Caucasian population. Children with age at diagnosis less than 5 years of age had significantly higher frequency of the heterozygous genotype DR3-DQ2/DR4-DQ8 compared to children with age at diagnosis than 5 years old. These results also demonstrate strong association of the genetic profile of the class II HLA for this age group, possibly associated with the severity and rapid progression to the onset of T1D. The knowledge of HLA class II genes may be useful in genetic screens that allow the prediction of T1D

Caracterização molecular de isolados de Flavobacterium columnare de tilapia do nilo e piracanjuba por meio de RAPD-PCR

Flavobacterium columnare is a cosmopolite bacteria and it is one of the main problem in Brazilian aquaculture, causing high mortalities index and economic damage. The main factors that contribute to columnaris disease are inadequate water quality, excess handling, high density of fish and temperature variations. For a successful epidemiological study and disease control, it is essential to differentiate the F. columnare from other yellow pigmentation bacteria. The present study used molecular techniques to characterize, by RAPD-PCR, two strains of F. columnare isolated from Oreochromis niloticus and Brycon orbignyanus. Data were analyzed as binary (0 and 1) and a genetic similarity matrix was generated by Jaccard's coefficient. Cluster analysis was performed by the neighbor joining method. The RAPD-PCR technique confirmed to be a usefull tool to obtain genetic profiles from F. columnare isolates based on the oligonucleotides used and to verify genetic similarity.

Comparison of DNA-extraction methods and Selective Enrichment broths on the detection of Salmonella Typhimurium in swine feces by polymerase chain reaction (PCR)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the inuA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers. (c) 2006 Elsevier Ltd. All rights reserved.

Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sexagem de embriões bovinos fecundados in vitro pela técnica de PCR multiplex

Neste trabalho, a técnica de PCR (polymerase chain reaction) foi utilizada para a sexagem de 92 embriões bovinos fertilizados in vitro. Os embriões originaram-se de fertilização in vitro de oócitos aspirados de ovários de fêmeas bovinas, provenientes de abatedouros comerciais. Os oócitos foram maturados, fertilizados e cultivados até o estádio de blastocisto. Os embriões foram lavados em solução de PBS, transferidos para tubos de polipropileno contendo água ultrapura, e imediatamente congelados a -196ºC. Os embriões foram descongelados sobre isopor contendo gelo picado e tratados com proteinase K. Para a reação de PCR, utilizaram-se alíquotas de 34 µl de cada tudo, onde foram acrescidos dois pares de primers, seqüência BC1.2 e seqüência satélite 1.715, desoxinucleotídeos, MgCl2, tampão PCR 10X, TaqDNA polimerase e água, em um volume final de 50 µl. As amostras foram amplificadas e a eletroforese realizada em gel de poliacrilamida a 8%. Os géis foram corados com solução de brometo de etídio e analisados em transiluminador de luz ultravioleta. Um índice de 93,47% de amplificação foi atingido, com 41 embriões (47,67%) machos e 45 (52,32%) embriões fêmeas. O uso de gel de poliacrilamida a 8% foi eficaz na separação de fragmentos de DNA muito próximos.