RESUMEN
Sudden cardiac death (SCD) is a major public health concern worldwide, and genetic analysis may be useful in identifying the cause of death as well as in determining the possible genetic risk factors for SCD. This study analyzed eight SNPs (single nucleotide polymorphisms) highly correlated with cardiac sudden death in samples (blood and bone) from six Brazilian families with a history of cardiovascular diseases. Individuals with no family history of cardiovascular diseases were recruited as controls. Y chromosomes and mtDNA haplogroups belonging to these subjects were verified as well. We found that SNP rs16857031 showed significant differences between the group with a family history of cardiovascular diseases and the control group. Furthermore, the data obtained were compatible with known frequencies of SNPs for the haplogroups in which the samples were classified. A possible hereditary factor was identified for SNP rs4725982 in one family. These preliminary results suggest that identification of certain SNPs could be used to assess risk factors for SCD.
Asunto(s)
Cardiomiopatías/genética , Muerte Súbita Cardíaca , Marcadores Genéticos/genética , Adulto , Anciano , Brasil , Estudios de Casos y Controles , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Linaje , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
A fermentation system was continuously fed with sugar-cane syrup and operated with recycling of Saccharomyces cerevisiae cells at temperatures varying from 30 to 47 °C. The aim of the present work was to obtain and study the colonies of isolates showing elongated cells of yeasts which were sporadically observed at the end of this continuous process. Based on a sequence of assays involving methods of classical taxonomy and RAPD-PCR, two groups of isolates showing characteristics of non-Saccharomyces yeasts were identified in the yeast population where S. cerevisiae was the dominant yeast. The largest group of non-Saccharomyces yeasts, resulting from a slow proliferation over the 2 months, reached a final level of 29.6% at the end of the process. RAPD-PCR profiles obtained for the isolates of this dominant non-Saccharomyces yeast indicated that they were isolates of Issatchenkia orientalis. Pichia membranifaciens was the only species of non-Saccharomyces yeast detected together with I. orientalis but at a very low frequency. The optimum temperature for ethanol formation shown by the isolate 195B of I. orientalis was 42 °C. This strain also showed a faster ethanol formation and biomass accumulation than the thermotolerant strain of S. cerevisiae used as the starter of this fermentation process. Some isolates of I. orientalis were also able to grow better at 40 °C than at 30 °C on plates containing glycerol as carbon source. Yeasts able to grow and produce ethanol at high temperatures can extend the fermentation process beyond the temperature limits tolerated by S. cerevisiae.
Asunto(s)
Etanol/metabolismo , Fermentación , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/crecimiento & desarrollo , Biomasa , ADN de Hongos/análisis , Microbiología Industrial , Pichia/crecimiento & desarrollo , Técnica del ADN Polimorfo Amplificado Aleatorio , Saccharomycetales/clasificación , Saccharomycetales/aislamiento & purificación , TemperaturaRESUMEN
As data on X-chromosomal short tandem repeats (X-STRs) for the Brazilian population are scarse, the aim of this study was to determine the allele frequencies of five X-STRs (DXS6854, DXS7424, DXS101, DXS6808 and DXS7132) in the São Paulo State, Brazil. No deviations from the Hardy-Weinberg equilibrium were observed, with the exception of DXS101. The forensic efficiency parameters demonstrated that DXS101 was the most informative marker. Population comparisons revealed that the X-STR profile sampled in the state of São Paulo was more similar to European and African populations than Asiatic populations reported in this work.
Asunto(s)
Cromosomas Humanos X/genética , Variación Genética , Genética de Población , Brasil , Femenino , Frecuencia de los Genes/genética , Humanos , Masculino , Repeticiones de Microsatélite/genéticaRESUMEN
Allele frequency distributions and population data for 12 Y-chromosomal short tandem repeats (STRs) included in the PowerPlex Y Systems (Promega) were obtained for a sample of 200 healthy unrelated males living in São Paulo State (Southeast of Brazil). A total of 192 haplotypes were identified, of which 184 were unique and 8 were found in 2 individuals. The average gene diversity of the 12 Y-STR was 0.6746 and the haplotype diversity was 0.9996. Pairwise analysis confirmed that our population is more similar with the Italy, North Portugal and Spain, being more distant of the Japan.
Asunto(s)
Cromosomas Humanos Y , Frecuencia de los Genes , Genética de Población , Secuencias Repetidas en Tándem , Brasil , Dermatoglifia del ADN , Haplotipos , Humanos , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.
Asunto(s)
Genoma de Protozoos/genética , ARN Nuclear Pequeño/genética , Trypanosoma cruzi/genética , Animales , Southern Blotting , Clonación Molecular , Reacción en Cadena de la Polimerasa/métodos , Empalme del ARN , Análisis de Secuencia de ADNRESUMEN
Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.
Asunto(s)
Animales , Genoma de Protozoos/genética , ARN Nuclear Pequeño/genética , Trypanosoma cruzi/genética , Southern Blotting , Clonación Molecular , Reacción en Cadena de la Polimerasa/métodos , Empalme del ARNRESUMEN
No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 microM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.
Asunto(s)
Nitrofurazona/análogos & derivados , Nitrofurazona/farmacología , Profármacos/farmacología , ARN Mensajero/efectos de los fármacos , ARN Protozoario/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos , Electroforesis en Gel de Poliacrilamida , Empalme del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , ARN Nuclear Pequeño/efectos de los fármacos , ARN Nuclear Pequeño/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Trypanosoma cruzi/genéticaRESUMEN
No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 æM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.
Asunto(s)
Animales , Nitrofurazona/análogos & derivados , Nitrofurazona/farmacología , ARN Mensajero/efectos de los fármacos , ARN Protozoario/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma cruzi/genética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Empalme del ARN/efectos de los fármacos , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.
Asunto(s)
Nitrofurazona/análogos & derivados , Nitrofurazona/farmacología , Empalme del ARN/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Protozoario/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma cruzi/genética , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Exones/genética , Intrones/genética , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.
Asunto(s)
Animales , Empalme del ARN , ARN Mensajero , ARN Protozoario , Tripanocidas , Trypanosoma cruzi , Permeabilidad de la Membrana Celular , Exones , Intrones , Factores de Tiempo , Transcripción GenéticaRESUMEN
We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95 percent of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31 percent reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.
Asunto(s)
Animales , Humanos , Autoanticuerpos , Enfermedad de Chagas , Reacciones Cruzadas , Ribonucleoproteínas Nucleares Pequeñas , Trypanosoma cruzi , Autoanticuerpos , Western Blotting , Enfermedad Crónica , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Pruebas de PrecipitinaRESUMEN
We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.
Asunto(s)
Autoanticuerpos/sangre , Enfermedad de Chagas/inmunología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Trypanosoma cruzi/inmunología , Animales , Autoanticuerpos/inmunología , Western Blotting , Enfermedad Crónica , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Células HeLa/inmunología , Humanos , Pruebas de PrecipitinaRESUMEN
Small nuclear ribonucleoproteins (snRNPs) are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.
Asunto(s)
Autoanticuerpos/sangre , Autoinmunidad/inmunología , Enfermedad de Chagas/inmunología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Proteínas Ribosómicas/inmunología , Trypanosoma cruzi/genética , Secuencia de Aminoácidos , Animales , Autoantígenos/inmunología , Autoinmunidad/genética , Southern Blotting , Western Blotting , Clonación Molecular , Reacciones Cruzadas , ADN Complementario/química , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Alineación de Secuencia , Trypanosoma cruzi/inmunología , Proteínas Nucleares snRNPRESUMEN
The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.
Asunto(s)
Genoma Bacteriano , Plantas/microbiología , Xanthomonas/genética , Xanthomonas/fisiología , Orden Génico/genética , Interacciones Huésped-Parásitos , Datos de Secuencia Molecular , Filogenia , Regulón/genética , Origen de Réplica/genética , Especificidad de la Especie , Virulencia/genética , Xanthomonas/clasificación , Xanthomonas/patogenicidad , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidad , Xanthomonas campestris/fisiologíaRESUMEN
A protein from Trypanosoma cruzi bloodstream trypomastigotes that binds IgG from man and several other animal species was isolated and characterized. The 52-kDa protein obtained from different T. cruzi cell extracts showed saturable binding with a K of 3.72 nM. Immunoblot analysis revealed that Fab, but not Fc, fragments of the Ig were recognized. When the protein was added to an unrelated C-fixation reaction, lysis was abolished in a dose-dependent fashion. When freshly prepared T. cruzi extracts were run in a 10% acrylamide SDS gel into which normal rabbit IgG was incorporated before polymerization, proteolytic activity, as seen by a transparent band after Coomassie blue staining, migrated in the same m.w. range of the 52-kDa protein. These data provide further clues to the mechanisms through which this pathogen escapes the host's immune response, thus maintaining a long-standing infection.