RESUMO
Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.
Assuntos
Vírus Junin/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas da Matriz Viral/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/metabolismo , Infecções por Arenaviridae/virologia , Western Blotting , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Spodoptera/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Assuntos
Fracionamento Celular/métodos , Separação Celular/métodos , Fezes/parasitologia , Giardia/isolamento & purificação , Oocistos , Oocistos/isolamento & purificação , Animais , DNA de Protozoário/isolamento & purificação , Cães , Eletroforese em Gel de Ágar , Endopeptidase K/farmacologia , Giardia/citologia , Giardia/genética , Temperatura Alta , Humanos , Oocistos/química , Oocistos/efeitos dos fármacos , Pressão Osmótica , Cloreto de Sódio/farmacologia , Soluções , Estresse Mecânico , Sacarose/farmacologiaRESUMO
El objetivo de este trabajo fue optimizar y evaluar las técnicas de purificación, aislamiento y ruptura de quistes de Giardia spp a partir de heces formoladas para la obtención de ADN. La materia fecal filtrada fue sometida a 3 técnicas de purificación, utilizando soluciones de formol-éter, sacarosa y formol-éter más sacarosa. La solución de sacarosa permitió aislar los quistes con menos detritos. Los quistes purificados fueron tratados con 3 técnicas para la ruptura de los mismos: shock osmótico y calor, degradación química y shock térmico, acción enzimática y efecto mecánico. Solamente con la técnica de shock térmico, acción enzimática y efecto mecánico se observaron bandas fluorescentes en geles de agarosa. Los resultados de este trabajo permiten contar con una metodología de rutina, simple, que podría ser usada en los pasos previos a la técnica de PCR para la genotipificación de este parásito.
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Assuntos
Animais , Cães , Humanos , Fracionamento Celular/métodos , Separação Celular/métodos , Fezes/parasitologia , Giardia/isolamento & purificação , Oocistos , Oocistos/isolamento & purificação , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Ágar , Endopeptidase K/farmacologia , Giardia/citologia , Giardia/genética , Temperatura Alta , Pressão Osmótica , Oocistos/química , Oocistos/efeitos dos fármacos , Soluções , Estresse Mecânico , Cloreto de Sódio/farmacologia , Sacarose/farmacologiaRESUMO
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
RESUMO
OBJECTIVE: To compare patients with Coagulase Negative Staphylococcus bacteremia (CoNS-B) and pseudobacteremia (CoNS-PB) in a pediatric hospital. METHODS: Descriptive and comparative study between children diagnosed with CoNS-B and CoNS-PB. RESULTS: A total of 159 children with CoNS positive blood cultures were evaluated. 66 children were classified as CoNS-B (41.5%) and 93 as CoNS-PE3 (58.5%). On the average CoNS was isolated on day 21 among children with bacteremia (B) and on day 2 in those with PB (p < 0.01). Excluding newborns, patients with B and PB had on average 2.6 and 1.1 positive cultures respectively. Most children with bacteremia were at the intensive care unit (67.2%), while patients with PB were mostly detected at the emergency room. Using logistic regression analysis, we found four factors independently associated with CoNS bacteremia: total parenteral nutrition (OR 5.4; 95% CI 2.2-12.9), low birth weight (OR 2.6; 95% CI 1.1-5.9), catheters placed by cut-down technique (OR 1.9; 95% CI 1.1-3.8), and inmuno-compromised patients (OR 2.7; 95% CI 1.1-6.7). Resistance to oxacilin was reported in 71.7% of the CoNS isolated. The overall mortality associated to CoNS-B was 6%. Among children with CoNS-PB, 10% received antibiotics, half of them vancomycin. CONCLUSIONS: These results show that CoNS-B occurs mainly as a nosocomial episode. CoNS-PB more likely resulted from specimen contamination at collection, being responsible for almost 60% of all positive blood cultures. The false-positive results caused unnecessary administration of antibiotics in a significant proportion of CoNS-PB events and have a potential impact upon the emergence of resistant pathogens.
Assuntos
Bacteriemia/microbiologia , Infecções Estafilocócicas/microbiologia , Adolescente , Bacteriemia/epidemiologia , Criança , Pré-Escolar , Coagulase/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Infecções Estafilocócicas/epidemiologia , Staphylococcus/enzimologiaRESUMO
RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junín virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.
Assuntos
Arenavirus/fisiologia , Células Eucarióticas/virologia , Proteínas do Nucleocapsídeo/fisiologia , Animais , Arenavirus/genética , Arenavirus/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular , Cricetinae , Vírus Junin/química , Vírus Junin/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/análise , RNA Viral/genética , Transcrição Gênica , Ativação Transcricional , Transfecção , Replicação Viral/genéticaRESUMO
DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.
Assuntos
Impressões Digitais de DNA/métodos , Lactobacillus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Streptococcus/isolamento & purificação , Animais , Sequência de Bases , DNA Bacteriano , Lactobacillus/classificação , Lactobacillus/genética , Leite/microbiologia , Dados de Sequência Molecular , Fenótipo , Sequências Repetitivas de Ácido Nucleico , Streptococcus/classificação , Streptococcus/genética , Iogurte/microbiologiaRESUMO
Bread from wheat flour is one of the most widely consumed products by the Mexican population. In this study, proximate composition, and mineral content were determined, and cost per/gram of protein and energy of traditional and industrial bread were compared. Seven types of bread were analyzed: bolillo, virginia, white bread and pastries conchita, croissant, breadroll and donuts. Products were analyzed for proximate composition by official methods, and content of Na, K, Ca, Mg, Fe, y Zn by atomic absorption spectrometry. Based on 24 hour recall interviews, the consumption of each type of bread as well as the percent of protein and energy allowances were calculated. Significant differences (p < 0.05) were found with respect to nutrient content between traditional and industrial breads. White bread and pastries from industrial origin showed higher costs (p < 0.05) per gram of protein and energy than traditional products in most cases. Both industrial and traditional breads showed higher fat content than that established by Official Mexican regulations. A high content of Ca was found in bread from the industrial origin and K, Mg, Fe y Zn were higher (p < 0.05) than those from the traditional bakery.
Assuntos
Pão/análise , Ingestão de Energia , Indústria Alimentícia , Proteínas/química , México , Valor NutritivoRESUMO
Arenaviridae is a worldwide distributed family, of enveloped, single stranded, RNA viruses. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as the geographic distribution. In this study the phylogenetic relationship among the members of the Arenaviridae was examined, using the reported genomic sequences. The comparison of the aligned nucleotide sequences of the S RNA and the predicted amino acid sequences of the GPC and N proteins, together with the phylogenetic analysis, strongly suggest a possible kinship of Pichindé and Oliveros viruses, with the Old World arenavirus group. This analysis points at the evolutive relationships between the arenaviruses of the Americas and can be used to evaluate the different hypotheses about their origin.
Assuntos
Arenavirus/genética , Filogenia , Arenavirus/classificação , Sequência de Bases , Evolução Molecular , Genes Virais , Vírus Pichinde/genética , Análise de Sequência de RNA , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
The Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper we report the nucleotide sequences of S RNA of Candid #1 and its more virulent ancestors XJ#44 and XJ (prototype). Their relationship to Junin virus wild-type MC2 strain and other closely and distantly related arenaviruses was also examined. Comparisons of the nucleotide and amino acid sequences of N and GPC genes from Candid #1 and its progenitor strains revealed some changes that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype.