Enzymatic, immunological and phylogenetic characterization of Brucella suis urease

<p>Abstract</p> <p>Background</p> <p>The sequenced genomes of the <it>Brucella </it>spp. have two urease operons, <it>ure</it>-1 and <it>ure</it>-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from <it>Brucella suis </it>strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined.</p> <p>Results</p> <p>Urease encoded by the <it>ure</it>-1 operon of <it>Brucella suis </it>strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28–35°C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a <it>K</it>_{<it>m </it>}of 5.60 ± 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with K_iof 1.04 ± 0.31 mM and 26.12 ± 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130–135 kDa by gel filtration chromatography and 157 ± 7 kDa using 5–10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by <it>ureC1</it>. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, <it>ureC1 </it>was related to the <it>ureC </it>typically present in the <it>Rhizobiales</it>; in contrast, the <it>ureC2 </it>encoded in the <it>ure</it>-2 operon is more related to distant species.</p> <p>Conclusion</p> <p>We have for the first time purified and characterized an active urease from <it>B. suis</it>. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of <it>B. suis </it>is a product of <it>ure</it>-1 operon.</p

Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice

BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Δure2K (ureB and ureC in ure2 operon), strain 1330Δure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Δure1KΔure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Δure2K and 1330Δure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure1K and 1330Δure1KΔure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure1K and 1330Δure1KΔure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure1KΔure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure1K was completely killed, strain 1330Δure2C was partially killed, but strains 1330 and 1330Δure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro

Dietary diversity and nutritional adequacy among an older Spanish population with Metabolic Syndrome in the PREDIMED-Plus study: a cross-sectional analysis

Dietary guidelines emphasize the importance of a varied diet to provide an adequate nutrient intake. However, an older age is often associated with consumption of monotonous diets that can be nutritionally inadequate, increasing the risk for the development or progression of diet-related chronic diseases, such as metabolic syndrome (MetS). To assess the association between dietary diversity (DD) and nutrient intake adequacy and to identify demographic variables associated with DD, we cross-sectionally analyzed baseline data from the PREDIMED-Plus trial: 6587 Spanish adults aged 55–75 years, with overweight/obesity who also had MetS. An energy-adjusted dietary diversity score (DDS) was calculated using a 143-item validated semi-quantitative food frequency questionnaire (FFQ). Nutrient inadequacy was defined as an intake below 2/3 of the dietary reference intake (DRI) forat least four of 17 nutrients proposed by the Institute of Medicine (IOM). Logistic regression models were used to evaluate the association between DDS and the risk of nutritionally inadequate intakes. In the higher DDS quartile there were more women and less current smokers. Compared with subjects in the highest DDS quartile, those in the lowest DDS quartile had a higher risk of inadequate nutrient intake: odds ratio (OR) = 28.56 (95% confidence interval (CI) 20.80–39.21). When we estimated food varietyfor each of the food groups, participants in the lowest quartile had a higher risk of inadequate nutrient intake for the groups of vegetables, OR = 14.03 (95% CI 10.55–18.65), fruits OR = 11.62 (95% CI 6.81–19.81), dairy products OR = 6.54 (95% CI 4.64–9.22) and protein foods OR = 6.60 (95% CI 1.96–22.24). As DDS decreased, the risk of inadequate nutrients intake rose. Given the impact of nutrient intake adequacy on the prevention of non-communicable diseases, health policies should focus on the promotion of a healthy varied diet, specifically promoting the intake of vegetables and fruit among population groups with lower DDS such as men, smokers or widow(er)s. View Full-Tex

Fitorremediación de metales pesados

Capítulo del libro "Avances en Ciencias del Agua", págs. 493-509Los metales pesados son prioritarios en suelos y aguas debido a que representan un gran peligro para el ambienta y la salud humana, al ser altamente persistentes y presentar altos niveles de bioacumulación. Su procedencia es muy variada, y en México, se asocian principalmente con vertidos ilegales, falta de plantas de tratamiento y corrosión de tuberías y depósitos. La fitorremediación de aguas está tomando mucho auge recientemente dado que provee una solución económica, de fácil implementación y ambigable con el ambiente. En este capítulo se presenta la utilidad de E. crassipes tanto en tratamiento de aguas como en cuerpos de agua contaminados con metales pesadosCONACYT, a través del proyecto, 91184; FESE (proyecto 3379/2013E) y UAEM, proyectos 3120/2011 y 3449/2013CH

Estatus funcional de adultos mayores del Tláhuac, Ciudad de México

La identificación de la capacidad de funcionalidad en el adulto mayor es una valoración conveniente. Se concibe que la funcionalidad está relacionada con lograr satisfacer sus necesidades básicas, bienestar emocional y social, significa independencia y, por lo tanto, con calidad de vida. El objetivo de este estudio fue identificar el grado de funcionalidad que presentan los adultos mayores de 60 años y mayores que asisten a consulta en 4 centros de salud ubicados en la delegación Tláhuac, Ciudad de México durante el periodo de junio a diciembre de 2014. Fue una investigación exploratoria, en una muestra por conveniencia de 754 adultos mayores; se utilizaron 5 ítems del apartado A del instrumento «Evaluación Funcional del Adulto Mayor» el cual fue aplicado en los centros de salud. Para el procesamiento de la información se utilizó estadística descriptiva. Resultados: El 82% de los adultos mayores fueron independientes y el 18% están en riesgo de dependencia. Las actividades que presentaron menor porcentaje de dependencia fueron preparar sus alimentos y realizar tareas domésticas. Conclusión: Los resultados arrojados en el presente estudio implican sensibilizar a los profesionales de los centros de salud para que incorporen en sus programas propuestas para mantener la funcionalidad en este grupo etario como son ejercicio mental y físico, hábitos alimentarios, ocupación del tiempo y el manejo de situaciones estresantes

<it>Brucella suis </it>urease encoded by <it>ure</it>1 but not <it>ure</it>2 is necessary for intestinal infection of BALB/c mice

Abstract Background In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. Results The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Δure2K (ureB and ureC in ure2 operon), strain 1330Δure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Δure1KΔure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Δure2K and 1330Δure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure1K and 1330Δure1KΔure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure1K and 1330Δure1KΔure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure1KΔure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure1K was completely killed, strain 1330Δure2C was partially killed, but strains 1330 and 1330Δure2K were not killed. Conclusion These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro.</p

Estatus funcional de adultos mayores de Tláhuac, Ciudad de México

La identificación de la capacidad de funcionalidad en el adulto mayor es una valoración conveniente. Se concibe que la funcionalidad está relacionada con lograr satisfacer sus necesidades básicas, bienestar emocional y social, significa independencia y, por lo tanto, con calidad de vida. El objetivo de este estudio fue identificar el grado de funcionalidad que presentan los adultos mayores de 60 anos y mayores que asisten a consulta en 4 centros de salud ubicados en la delegación Tláhuac, Ciudad de México durante el periodo de junio a diciembre de 2014. Fue una investigación exploratoria, en una muestra por conveniencia de 754 adultos mayores; se utilizaron 5 ítems del apartado A del instrumento «Evaluación Funcional del Adulto Mayor» el cual fue aplicado en los centros de salud. Para el procesamiento de la información se utilizó estadística descriptiva. Resultados: El 82% de los adultos mayores fueron independientes y el 18% están en riesgo de dependencia. Las actividades que presentaron menor porcentaje de dependencia fueron preparar sus alimentos y realizar tareas domésticas. Conclusión: Los resultados arrojados en el presente estudio implican sensibilizar a los profesionales de los centros de salud para que incorporen en sus programas propuestas para mantener la funcionalidad en este grupo etario como son ejercicio mental y físico, hábitos alimentarios, ocupación del tiempo y el manejo de situaciones estresantes

The schematic representations of the operons with corresponding Ure subunits, and deletion sites of mutant strains 1330Δ1K, 1330Δ2K and 1330Δ2C

<p><b>Copyright information:</b></p><p>Taken from "urease encoded by 1 but not 2 is necessary for intestinal infection of BALB/c mice"</p><p>http://www.biomedcentral.com/1471-2180/7/57</p><p>BMC Microbiology 2007;7():57-57.</p><p>Published online 19 Jun 2007</p><p>PMCID:PMC1983905.</p><p></p> A: 1 operon. B: 2 operon. The numbers represent the location of the genes in the chromosome I