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1.
Braz. j. med. biol. res ; 50(5): e5981, 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-839288

RESUMO

Different pathways act synergistically to participate in many biological processes. Thus, the purpose of our study was to extract dysregulated pathways to investigate the pathogenesis of colorectal cancer (CRC) based on the functional dependency among pathways. Protein-protein interaction (PPI) information and pathway data were retrieved from STRING and Reactome databases, respectively. After genes were aligned to the pathways, each pathway activity was calculated using the principal component analysis (PCA) method, and the seed pathway was discovered. Subsequently, we constructed the pathway interaction network (PIN), where each node represented a biological pathway based on gene expression profile, PPI data, as well as pathways. Dysregulated pathways were then selected from the PIN according to classification performance and seed pathway. A PIN including 11,960 interactions was constructed to identify dysregulated pathways. Interestingly, the interaction of mRNA splicing and mRNA splicing-major pathway had the highest score of 719.8167. Maximum change of the activity score between CRC and normal samples appeared in the pathway of DNA replication, which was selected as the seed pathway. Starting with this seed pathway, a pathway set containing 30 dysregulated pathways was obtained with an area under the curve score of 0.8598. The pathway of mRNA splicing, mRNA splicing-major pathway, and RNA polymerase I had the maximum genes of 107. Moreover, we found that these 30 pathways had crosstalks with each other. The results suggest that these dysregulated pathways might be used as biomarkers to diagnose CRC.


Assuntos
Humanos , Adenoma/genética , Adenoma/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Mapas de Interação de Proteínas/genética , Área Sob a Curva , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Análise de Componente Principal , Análise Serial de Proteínas , Valores de Referência , Processamento de RNA , Transdução de Sinais , Transcriptoma
2.
Biomédica (Bogotá) ; 36(supl.1): 128-136, dic. 2016. ilus, graf
Artigo em Espanhol | LILACS | ID: lil-783530

RESUMO

Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho. Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito. Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados. Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos. Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito.


Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.


Assuntos
Giardia lamblia , Spliceossomos , Parasitos , Processamento de RNA , Transfecção , Organismos Eucariotos Unicelulares
3.
Rev. bras. hematol. hemoter ; 38(4): 320-324, Oct.-Dec. 2016. tab, graf
Artigo em Inglês | LILACS | ID: biblio-829951

RESUMO

ABSTRACT Background: Myelodysplastic syndromes (MDS) comprise a group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis and propensity for progression to acute myeloid leukemia. Acquired mutations in the gene encoding RNA splicing factor 3B subunit 1 (SF3B1) are highly associated with the MDS subtypes presenting ring sideroblasts, and represent a specific nosological entity. The effects of these mutations on clinical outcomes are diverse and contrasting. Methods: A cohort of 91 Brazilian MDS patients, including patients with ring sideroblasts in the bone marrow, were screened for mutations in the SF3B1 hotspots (exons 12-15) by direct Sanger sequencing. Results: SF3B1 heterozygous mutations were identified in six patients (7%), all of them with ring sideroblasts, thus confirming the association between SF3B1 mutations and myelodysplastic syndrome subtypes bearing this morphologic feature (frequency of 6/13, p-value < 0.0001). Conclusion: This is the first screening of SF3B1 mutations in a cohort of Brazilian myelodysplastic syndrome patients. Our findings confirm that mutations in this splicing gene correlate with bone marrow ringed sideroblasts.


Assuntos
Humanos , Feminino , Anemia Sideroblástica , Mutação , Síndromes Mielodisplásicas , Processamento de RNA , Fatores de Processamento de RNA
4.
Medicina (B.Aires) ; 75(2): 91-94, abr. 2015. graf
Artigo em Espanhol | LILACS | ID: lil-750520

RESUMO

La neurofibromatosis tipo 1 (NF1) es un desorden genético autosómico dominante, con una prevalencia de 1 en 2500-3000 nacidos vivos. La dificultad diagnóstica se debe al tamaño extenso del gen NF1 con pocos sitios hot-spot, la ausencia de una clara relación genotipo-fenotipo y rasgos clínicos con un espectro muy heterogéneo. Un caso sospechoso de NF1 procedente de la provincia de Jujuy fue analizado por MLPA (multiplex ligation-dependent probe amplification) en nuestro laboratorio. Mujer, adolescente mestiza (Amerindia/Europea), con un osteoma maxilar, lordosis lumbar, neurofibromas cutáneos y manchas café con leche. Por MLPA se detectó una alteración en el exón 13 del gen NF1. Por secuenciación del exón 13 se identificó una mutación "missense" en la posición 1466 del ARNm (NM_000267.3:c.1466A>G) que introduce un sitio de splicing aberrante. La patogenicidad de la mutación fue corroborada en la base de datos de variantes clínicas del National Center for Biotechnology Information. En nuestro conocimiento, este es el primer registro de una mutación NF1 en un paciente proveniente de poblaciones mestizas del Noroeste Argentino. La alteración ha sido reportada en individuos de otras poblaciones de origen muy disímil al del caso presentado, como la europea, sugiriendo que el sitio podría considerarse un sitio hot-spot del gen. Donde exista baja disponibilidad de diagnósticos moleculares, como en nuestro caso, se puede aplicar un algoritmo que comience por el estudio del gen NF1 por MLPA, metodología relativamente sencilla y de costo accesible. Con ella se evita enviar muestras al extranjero para análisis genéticos.


Neurofibromatosis type 1 (NF1) is a dominant autosomic genetic disorder, with a birth incidence of 1 in 2500-3000. Diagnosis is difficult because of the size of gene NF1 that has few hot-spots sites, the absence of a clear genotype-phenotype relation, and a heterogeneous clinical manifestation. A NF1 suspected case from Jujuy province was analyzed by multiplex ligation-dependent probe amplification (MLPA). Mestizo female teenage (Amerindian/European), with a maxilar osteoma, lumbar lordosis, cutaneous neurofibromas and café au lait spots. MLPA detected an alteration in exon 13 of the NF1 gene. By sequencing of exon 13, a missense mutation (NM_000267.3:c.1466A>G) was found which introduces an aberrant splicing site and is registered as pathogenic in the clinical variants database of NCBI. As far as we are aware, this is the first report of a NF1 mutation in mestizo population of Northwest Argentina. 1466A>G has been described before in patients of European origin, suggesting that the affected site could be a hot-spot site of the gene. For countries as Argentina, with limited availability of molecular diagnostic methods, we propose a diagnosis algorithm by starting the mutational analysis of NF1 with MLPA. This methodology is relatively simple and of low cost, avoiding to send samples abroad for genetic analyses.


Assuntos
Adolescente , Feminino , Humanos , Reação em Cadeia da Polimerase Multiplex , Mutação de Sentido Incorreto , Neurofibromatose 1/genética , Processamento de RNA , Análise de Sequência de DNA , Algoritmos , Argentina , Grupo com Ancestrais do Continente Europeu , Índios Sul-Americanos
6.
Rev. chil. pediatr ; 85(2): 213-221, abr. 2014. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-711583

RESUMO

Introducción: La Enfermedad Granulomatosa Crónica (EGC) se presenta como consecuencia de mutaciones en los genes que codifican 5 de las subunidades del sistema NADPH oxidasa humano. Su forma más común es causada por cambios en el gen CYBB que codifica gp91 phox. Objetivo: Identificar el defecto molecular que lleva a la presentación de la EGC. Caso clínico: Paciente de sexo masculino con antecedentes de enfermedad diarreica aguda y abscesos perianales recurrentes desde los 2 meses. A los 6 meses, presentó una inflamación crónica del colon y colitis bacteriana. A los 3 años tenía infecciones en las vías respiratorias inferiores y perianales. Estudio compatible con EGC. El análisis del ADNc identificó expresión anormal del ARNm, lo cual se confirmó al realizar la secuenciación. Específicamente se observó la ausencia del exón 2. Adicionalmente, los datos de la secuenciación del ADNg identificaron una alteración en el sitio aceptor de "splicing" del intrón 1, que incluye una deleción seguida de la inserción de 3 nucleótidos (c.46-14_-11delTTCT insGAA). Conclusiones: Se presenta el primer estudio molecular de un paciente con EGC por defecto de "splicing" reportado en Colombia. La definición de la mutación y su correlación con el fenotipo es importante para proveer una apropiada consejería genética al paciente y su familia.


Chronic granulomatous disease (CGD) is caused by mutations in the genes that encode five of the subunits of the human NADPH oxidase. The most common form is caused by mutations in CYBB, the human gene encoding gp 91 phox. Objective: To identify the molecular defects causing CGD. Case report: A male patient with a history of acute diarrhea and recurrent perianal abscess since two months old. At 6 months, the patient presented a chronic inflammatory disease of the colon and bacterial colitis. After three years, he developed infections in the lower and perianal respiratory tract. The cDNA analysis identified abnormal mRNA expression, which was confirmed by sequencing. Specifically the exclusion of exon 2 was observed. Additionally, gDNA sequencing identified an alteration in the acceptor splice site of intron 1, including a deletion followed by insertion of three nucleotides (c.46-14_-11delTTCT insGAA). Conclusions: The first molecular study of a patient with CGD due to splicing pattern change, reported in Colombia, is presented. The definition of the mutation and its correlation with the phenotype is essential to provide appropriate genetic counseling to patients and their families.


Assuntos
Humanos , Masculino , Lactente , Cromossomos Humanos X , Doença Granulomatosa Crônica/genética , Mutação , NADPH Oxidases/genética , DNA Complementar/genética , RNA Mensageiro/genética , Sequência de Bases , Separação Celular , Éxons , Doença Granulomatosa Crônica/diagnóstico , Citometria de Fluxo , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Processamento de RNA
7.
ABCS health sci ; 38(3)set.-dez. 2013.
Artigo em Português | LILACS | ID: lil-698570

RESUMO

A principal e mais conhecida função plaquetária ainda está relacionada à parada de sangramento após um dano vascular. No entanto, plaquetas estão envolvidas em diversos processos, tais como iniciar e amplificar a inflamação, interagir com células da resposta imune, além de participar na progressão tumoral, angiogênese e metástase. Neste sentido, está claro que plaquetas apresentam funções no processo inflamatório e podem influenciar respostas imune, além de desordens plaquetárias autoimune erelacionadas a presença de auto-anticorpos após transfusões, comopor exemplo, na lesão pulmonar aguda associada à transfusão. Após muita especulação, recentes observações têm estabelecido novos paradigmas relacionando plaquetas à biologia molecular. Plaquetas humanas contêm fatores de spliceossomo, incluindo pequenos RNAs nucleares, proteínas de splicing e pre-mRNA endógenos. Outro ponto importante é o controle do número de plaquetas circulantes, resultado do equilíbrio entre a produção e destruição dessas células. Assim, é proposto um processo de morte programada da célula anucleada que determina seu tempo de vida. Esse processo é alvo de especulações desde a década de 60 e ainda permanece em discussão. A noção geral de que plaquetas funcionais são importantes para o sucesso de processos hematogênicos corroboram com inovações experimentais e também ligam a processos de interação plaquetas-células tumorais e seu microambiente que regula a progressão maligna. Plaquetas contribuem na sobrevivência e disseminação de células tumorais. Desta forma, discutimos aqui os mecanismos pelos quais as plaquetas atuam na imunidade, na inflamação e no câncer, uma vez que estas pequenas células são mais versáteis do que se pensava.


The principal and the most known function of platelets still remains stopping hemorrhage following vascular injury. However, platelets are involved in diverse processes such as triggering inflammation, participating in the immune response, besides tumor progression, angiogenesis, and metastasis. In this sense, it is becoming increasingly clear that platelets display inflammatory functions and can influence both innate and adaptive immune responses, such as autoimmune and alloimmune platelet disorders, and transfusion-related acute lung injury (TRALI). Despite much speculation recent observations have established new paradigms relevant to influence of platelets on molecular biology. Primary human platelets contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. Other point is, like all lineages of blood cells, the steady state number of mature platelets is the result of a balance between their production and destruction. Thus, it isproposed a programmed anuclear cell death delimits platelet lifespan is subject of speculation since the 1960s and has remainedelusive. The general notion that functional platelets are importantfor successful hematogenous tumor metastasis dates more than 4 decades and has been corroborated in numerous experimentalsettings. The dynamic crosstalk between tumors and their microenvironment is increasingly recognized as a key regulator ofmalignant progression. These contributions of platelets to tumorcell survival and spread suggest platelets as a new avenue forresearch. Here, we discuss the mechanisms by which plateletscontribute to immunity, inflammation, and cancer, since thesesmall cells are more versatile than we once thought.


Assuntos
Humanos , Masculino , Feminino , Plaquetas , Transfusão de Sangue , Hemostasia , Inflamação , Neoplasias , Processamento de RNA , Biologia Molecular
8.
Electron. j. biotechnol ; 14(3): 12-12, May 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-602989

RESUMO

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the research of life science, but it is difficult to obtain by traditional reverse transcription. In this study, we employed a novel strategy to clone the human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA directly isolated from the mucous membrane of mouth. The hLIF sequence can be acquired within a few hours by means of amplification of each exon and splicing using overlap-PCR. Thus, the new approach developed in this study is simple, time- and cost-effective, and it is not limited to particular gene expression levels of each tissue.


Assuntos
Humanos , DNA Complementar/genética , Fator Inibidor de Leucemia/genética , Mucosa Bucal , Sequência de Bases , Clonagem Molecular , Processamento de RNA/genética , Éxons/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Mem. Inst. Oswaldo Cruz ; 106(2): 130-138, Mar. 2011. ilus
Artigo em Inglês | LILACS | ID: lil-583935

RESUMO

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.


Assuntos
DNA de Protozoário , Precursores de RNA , Processamento de RNA , Trypanosoma , Sequência de Aminoácidos , Dados de Sequência Molecular
10.
Arq. bras. endocrinol. metab ; 53(6): 709-715, ago. 2009. ilus
Artigo em Inglês | LILACS | ID: lil-529947

RESUMO

RNA splicing is an essential, precisely regulated process that occurs after gene transcription and before mRNA translation, in which introns may be removed and exons, retained. Variability in splicing patterns is a major source of protein diversity from the genome and function to generate a tremendously diverse proteome from a relatively small number of genes. Changes in splice site choice can determine different effects on the encoded protein. Small changes in peptide sequence can alter ligand binding, enzymatic activity, allosteric regulation, or protein localization. Errors in splicing regulation have been implicated in a number of different disease states. This study reviewed the mechanisms of splicing and their repercussion in endocrinology, emphasizing its importance in some thyroid physiological and pathological conditions.


Após a transcrição genética e antes da tradução do mRNA, ocorre o splicing do RNA, que consiste em um processo essencial, precisamente regulado, por meio do qual podem ocorrer excisões de íntrons e retenções de éxons. A variabilidade dos padrões de splicing é a principal fonte de diversidade de proteínas geradas por um pequeno número de genes. Alterações na escolha do sítio de splicing podem determinar efeitos diferentes nas proteínas codificadas. Pequenas alterações na sequência peptídica podem alterar o seu sítio de ligação de substratos, sua atividade enzimática, a regulação alostérica ou a localização proteica. Erros na regulação do splicing têm sido implicados em grande número de doenças. Nessa revisão, foram descritos os mecanismos de splicing enfatizando sua importância em algumas condições fisiológicas e patológicas envolvendo a tireoide.


Assuntos
Humanos , Processamento de RNA/genética , Glândula Tireoide , Hormônios Tireóideos/genética , Neoplasias da Glândula Tireoide/genética , Processamento Alternativo/genética , Receptores dos Hormônios Tireóideos/fisiologia , Glândula Tireoide/fisiologia , Hormônios Tireóideos/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Tireotropina/fisiologia
11.
Biol. Res ; 42(2): 189-198, 2009. ilus, tab
Artigo em Inglês | LILACS | ID: lil-524889

RESUMO

We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivaríate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.


Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diabetes Mellitus Tipo 1/complicações , /complicações , Nefropatias Diabéticas/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único/genética , Biópsia , Estudos de Casos e Controles , Genótipo , Íntrons/genética , Análise Multivariada , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Processamento de RNA/genética , Transcrição Genética/genética
13.
Mem. Inst. Oswaldo Cruz ; 102(1): 97-106, Feb. 2007. tab, ilus
Artigo em Inglês | LILACS | ID: lil-440625

RESUMO

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.


Assuntos
Animais , Genoma de Protozoário/genética , RNA Nuclear Pequeno/genética , Trypanosoma cruzi/genética , Southern Blotting , Clonagem Molecular , Reação em Cadeia da Polimerase/métodos , Processamento de RNA
14.
Braz. j. med. biol. res ; 40(1): 33-39, Jan. 2007. ilus
Artigo em Inglês | LILACS | ID: lil-439671

RESUMO

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.


Assuntos
Animais , Nitrofurazona/análogos & derivados , Nitrofurazona/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA de Protozoário/efeitos dos fármacos , Tripanossomicidas/farmacologia , Trypanosoma cruzi/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Processamento de RNA/efeitos dos fármacos , Fatores de Tempo , Transcrição Genética/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento
15.
Genet. mol. res. (Online) ; 6(1): 105-115, 2007. tab, ilus
Artigo em Inglês | LILACS | ID: lil-456755

RESUMO

This paper presents a novel approach to the problem of splice site prediction, by applying stochastic grammar inference. We used four grammar inference algorithms to infer 1465 grammars, and used 10-fold cross-validation to select the best grammar for each algorithm. The corresponding grammars were embedded into a classifier and used to run splice site prediction and compare the results with those of NNSPLICE, the predictor used by the Genie gene finder. We indicate possible paths to improve this performance by using Sakakibara’s windowing technique to find probability thresholds that will lower false-positive predictions.


Assuntos
Humanos , Algoritmos , Inteligência Artificial , Modelos Moleculares , Processamento de RNA/genética , Processos Estocásticos
16.
Mem. Inst. Oswaldo Cruz ; 99(6): 617-620, Oct. 2004. ilus
Artigo em Inglês | LILACS | ID: lil-387911

RESUMO

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.


Assuntos
Animais , Processamento de RNA , RNA Mensageiro , RNA de Protozoário , Tripanossomicidas , Trypanosoma cruzi , Permeabilidade da Membrana Celular , Éxons , Íntrons , Fatores de Tempo , Transcrição Genética
17.
Rev. ciênc. farm ; 22(2): 295-306, 2001. ilus, graf
Artigo em Português | LILACS | ID: lil-314690

RESUMO

A maturaçäo dos pré-mRNAs em tripanosomatídeos ocorre por meio de um mecanismo denominado trans-splicing, que envolve a excisäo dos íntrons e a uniäo dos éxonsde dois transcritos independentes. Um transcrito curto , splice leader (SL) RNA, é trans-spliceado a pré-mRNAs aceptores, originando o RNA maduro. O objetivo deste trabalho foi padronizar as condiçöes necessárias que favoreçam essa reaçäo in vitro, empregando, para este fim, extratos nucleares de formas epimastigotas de T. cruzi e pré-mRNA de alfa-tubulina de tripanosoma africano. Nas condiçöes experimentais para a reaçäo de trans-splicing, em que se utilizou extrato nuclear de cepa Bolíviae o pré-mRNA a-tubulina (aceptor) marcado radioativamente, na ausência do pré-mRNA SL exógeno, foi obtida uma banda acima do pré-mRNA de alfa-tubulina (possível produto), sugerindo que esta reagiu com o SL presente no extrato nuclear. A banda foi processada, mas näo se detectou a sequência do produto esperado(SL + éxon alfa-tubulina). Entretanto, o tamanho da banda na reaçäo de trans-splicing (acima do pré-mRNA) pode trtar-se do lariat, cuja estrutura molecular näo possibilitaria seu sequenciamento pela metodologia empregada. Interessante salientar que a banda somente aparece na reaçäo de trans-splicing contendo ATP, o que sugere tratar-se de uma reaçäo de transesterificaçäo.


Assuntos
Animais , Técnicas In Vitro , Processamento de RNA , Trans-Splicing , Trypanosoma cruzi , Estrutura Molecular
19.
Adelantos microbiol. enfermedades infecc ; 6: IX-XVII, sept. 1987. ilus
Artigo em Espanhol | LILACS | ID: lil-71667

RESUMO

Este brevísimo resumen sobre la organización y la expresión génica tiene dos finalidades: introducir los conceptos más básicos sobre este tema de modo de ir familiarizando al lector sobre la terminología específica y segundo, la de hacer evidente hasta qué nivel de profundidad se conoce el mecanismo molecular de los procesos involucrados en la expresión génica. Todo el esclarecimiento de esta maravilha se hizo tan sólo en 20 años. No es de estrañar entonces la velocidad con que se van dilucidando actualmente las interrupciones en la normalidad de esta expresión génica como son las enfermedades genéticas, el cáncer, las infecciones virales, lo que hace tener esperanzas en el pronto hallazgo de soluciones para dichas enfermedades


Assuntos
Animais , Humanos , Replicação do DNA , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Genética , Processamento de RNA
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