Seroprevalencia de Brucella canis en perros de un refugio para animales de compañía en Bogotá, Colombia / Seroprevalence of Brucella canis in canines from a dog shelter in Bogotá, Colombia

Resumen | Introducción. El riesgo de infección con Brucella canis en humanos y perros aumenta con la exposición constante a perros portadores asintomáticos. En Colombia hay evidencia de infección con B. canis en personas que conviven con perros. Una preocupación adicional en Bogotá es la falta de información actualizada sobre la prevalencia de la infección en perros destinados a programas de adopción. Objetivo. Establecer la seroprevalencia de la infección por B. canis en perros de un refugio para animales de compañía destinados a la adopción en Bogotá. Materiales y métodos. Se hizo un estudio descriptivo de corte transversal en un refugio para animales de Bogotá. Se detectaron anticuerpos contra B. canis en el suero de 51 perros (28 hembras y 23 machos) mediante una prueba inmunocromatográfica de flujo lateral. Asimismo, los individuos positivos se analizaron con PCR para la detección del ADN de Brucella spp. Resultados. La seroprevalencia de B. canis fue del 1,96 % (1/51). El perro seropositivo correspondió a una hembra asintomática de tres años de edad en la cual no se detectó ADN bacteriano en sangre mediante la PCR. Conclusiones. La seroprevalencia representada por un solo perro con IgG anti-B. canis puede considerarse un riesgo potencial para las poblaciones de perros y humanos, ya que podría tratarse de un animal con infección persistente capaz de diseminar la bacteria.

Abstract | Introduction: The risk of Brucella canis infection in humans and dogs has increased due to the permanent exposure to asymptomatic carrier dogs. In Colombia, there is evidence of B. canis infection in humans living with dogs. In the case of Bogotá, an additional concern is the lack of updated information related to the prevalence of the infection in dogs. Objective: To determine the seroprevalence of infection by B. canis in dogs intended for adoption programs in Bogotá. Materials and methods: By means of a descriptive cross-sectional study carried out in a dog shelter in Bogotá, anti-B. canis IgG antibodies were detected in the serum from 51 dogs (28 females and 23 males) using a lateral-flow immunochromatographic test. Additionally, seropositive animals were analyzed with PCR to detect Brucella spp DNA. Results: Brucella canis seroprevalence was 1.96% (1/51). The seropositive dog was an asymptomatic three-year-old she-dog in which no bacteria DNA was detected in the blood through PCR. Conclusions: The seroprevalence determined in this study represented by a single dog with anti-B. canis IgG can be considered a potential risk both for canine and human populations since this single dog could have a persistent infection capable of spreading the bacteria.

Immunochromatography and laboratory serologies: an evaluation of immunodiagnoses in prenatal care / Inmunocromatográficos y serologías laboratoriales: evaluación de inmunodiagnósticos en el cuidado prenatal / Imunocromatográficos e sorologias laboratoriais: avaliação de imunodiagnósticos no cuidado pré-natal

ABSTRACT Objectives: to describe the use of affinity chromatographies and laboratory serologies in the evaluation of immunodiagnoses during prenatal. Methods: quantitative study, characterized as a descriptive observational research. Data was collected from the records of 46 pregnant women who were in prenatal follow up in the Primary Health Care in a capital in the South of Brazil. The data found was codified and analyzed using descriptive statistics. Results: a mean of 43.1 days was found to take place between the request of laboratory serology and the evaluation by a professional. It was also found that 21.7% of pregnant women did not collect the serologies requested during the first prenatal consultation, and that the affinity chromatographies were only applied in 10.8% of the participants. Conclusions: in spite of the studies for the improvement of prenatal consultations, for the provision of new technologies and for the permanent education offered to the professionals, there are still questions that make the actual implementation of affinity chromatographies more difficult.

RESUMEN Objetivos: describir la utilización de los inmunocromatográficos y de las serologías laboratoriales en la evaluación de inmunodiagnósticos durante el prenatal. Métodos: estudio de abordaje cuantitativo, caracterizado como una investigación observacional del tipo descriptiva. Recogidos datos de los prontuarios de 46 gestantes que realizan acompañamiento prenatal en la Atención Primaria de Salud del Sur de Brasil. Datos obtenidos tuvieron su contenido codificado y analizados mediante estadística descriptiva. Resultados: identificada una mediana de 43,1 días desde la solicitud de las serologías laboratoriales hasta la evaluación profesional. Así, también se verificó que 21,7% de las gestantes no recogieron las serologías solicitadas durante la primera consulta prenatal y que aplicaron inmunocromatográficos en solo 10,8% de los participantes. Conclusiones: sin embargo los estudios para perfeccionamiento de consulta prenatal, provisión de nuevas tecnologías y educación permanente ofertada a los profesionales, aún persisten cuestiones que dificultan la concreta implementación de inmunocromatográficos.

RESUMO Objetivos: descrever a utilização dos testes imunocromatográficos e das sorologias laboratoriais na avaliação de imunodiagnósticos durante o pré-natal. Métodos: estudo de abordagem quantitativa, caracterizado como uma pesquisa observacional do tipo descritiva. Foram coletados dados dos prontuários de 46 gestantes que realizam acompanhamento pré-natal na Atenção Primária à Saúde de uma capital do Sul do Brasil. Os dados obtidos tiveram seu conteúdo codificado e analisados mediante estatística descritiva. Resultados: foi identificada uma média de 43,1 dias desde a solicitação das sorologias laboratoriais até a avalição profissional. Nesse sentido, também foi verificado que 21,7% das gestantes não coletaram as sorologias solicitadas durante a primeira consulta pré-natal e que foram aplicados testes imunocromatográficos em apenas 10,8% dos participantes. Conclusões: apesar dos estudos para o aprimoramento da consulta pré-natal, do fornecimento de novas tecnologias e da educação permanente ofertada aos profissionais, ainda persistem questões que dificultam a concreta implementação dos testes imunocromatográficos.

Actividad inhibitoria de dihidroxifenil propenona sobre betalactamasas de Enterobacter cloacae: estudio preliminar en el desarrollo de fármacos para enfrentar la resistencia bacteriana / Inhibitory activity of dihydroxy-phenyl-propenone on betalactamase of Enterobacter cloacae : Preliminary study for drug development to overcome bacterial resistance

Introducción . Los microorganismos patógenos como Enterobacter cloacae producen betalactamasas que les confieren resistencia frente a los antibióticos betalactámicos; se ha identificado, además, la actividad limitada de los inhibidores enzimáticos, de modo que la única posibilidad de enfrentar la resistencia es el diseño de nuevos fármacos y su uso racional. Objetivo. Evaluar el efecto de la chalcona dihidroxifenil propenona sobre un aislamiento clínico de E. cloacae y sobre la betalactamasa aislada a partir de este microorganismo resistente como un aporte en la búsqueda de compuestos inhibidores de las betalactamasas. Materiales y métodos. Se sintetizó la chalcona dihidroxifenil propenona y se evaluó su efecto sobre el aislamiento clínico de E. cloacae para determinar la concentración inhibitoria mínima mediante el método de microdilución en caldo y con la betalactamasa purificada mediante cromatografía de afinidad se realizaron estudios espectrofotométricos de cinética enzimática. Resultados. La concentración inhibitoria mínima de la dihidroxifenil propenona sobre E. cloacae fue de 35 µg/ml; el porcentaje de recuperación de la betalactamasa a partir del microorganismo fue de 31,75 %; en el estudio cinético se evidenció actividad inhibitoria de acuerdo con los parámetros cinéticos de V max =1,7 x 10 -3 µM/minuto y K M´ =2330 µM. Conclusión. La chalcona dihidroxifenil propenona ejerce su actividad inhibitoria por medio de la interacción con la betalactamasa y, de esta manera, protege la integridad estructural de los antibióticos betalactámicos; dicho efecto sinérgico la convierte en un compuesto promisorio en la búsqueda de alternativas para enfrentar la resistencia bacteriana.

Introduction: Enterobacter cloacae is a pathogenic microorganism with the ability to produce betalactamase enzymes, which makes them resistant to betalactamic antibiotics. Additionally, the limited activity of enzymatic inhibitors has been identified, and, therefore, the design of new drugs and the promotion of their rational use are the only possibilities to overcome this problem. Objective: The aim of this research was to evaluate the effect of dihydroxy-phenyl-propenone on a clinical isolate of E. cloacae , as well as its activity on a betalactamase isolated from this resistant microorganism in order to contribute to the search for new betalactamase inhibitors. Materials and methods: Dihydroxy-phenyl-propenone chalcone was synthesized and evaluated on a clinical isolate of E. cloacae to determine the minimum inhibitory concentration by broth microdilution; once the betalactamase enzyme was purified by affinity chromatography, a spectrophotometric analysis was done to evaluate its kinetic activity. Results: The minimum inhibitory concentration value of dihydroxy-phenyl-propenone on E. cloacae was 35 µg/ml; the recovery percentage of the betalactamase from the microorganism was 31.75% and the kinetic parameters were V max =1.7 x 10 -3 µM/min and K M = 2330 µM, which show an important inhibitory activity. Conclusion: Dihydroxy-phenyl-propenone has shown inhibitory activity on betalactamase enzymes and the ability to protect the chemical integrity of betalactamic antibiotics; this synergistic effect turns it into a promising compound in the search for new alternatives to overcome bacterial resistance.

Mass Spectrometry Analysis for Nitration of Proteins / 한양의대학술지

Various proteomics and immunological methods including mass spectrometry combined with both liquid and 2-D PAGE, and immunodetection have been employed to identify and characterize nitrated proteins from pathological samples. Nitrosative modifications regulate cellular signal transduction and pathogenesis of inflammatory responses and neurodegenerative diseases. Nitric oxide generates reactive nitrosative species, such as peroxynitrite (ONOO-) that may be involved in a number of diseases. ONOO- can mediate protein tyrosine nitration which causes structural changes of affected proteins and leads to their inactivation. Protein tyrosine nitration is a biomarker of oxidative stress and also influences protein structure and function. Recent advances in mass spectrometry have made it possible to identify modified proteins and specific modified amino acid residues. This review focuses on the significance of protein tyrosine nitration and the progress achieved in analytical methods. Although mass spectrometry of nitrated peptides has become a powerful tool for the analysis of nitrated peptides, the low stoichiometry of protein tyrosine nitration clearly demands the use of affinity chromatography to enrich modified proteins (or peptides).

Prokaryotic expression, purification and polyclonal antibody preparation of ferritin light chain / 第二军医大学学报

Objective: To construct prokaryotic expression vector pET30a-FTL, obtain purified FTL protein and to prepare anti-FTL polyclonal antibody, so as to provide evidence for studying the biological function of the antibody. Methods: Gene recombination technology was used to link FTL gene (PCR product) and prokaryotic expression vector pET30a(+); the recombinant plasmid was then transformed into E. coli BL21 and the positive clones were identified by restriction enzyme digestion and PCR. Expression FTL protein was induced with IPTG and the expression product was purified. The purified FTL protein was used to immunize rabbits to obtain the antiserum. The specificity of the antibodies was examined by Western blotting. Results: The prokaryotic expression vector pET30a-FTL was successfully constructed and a fusion protein, with a molecular weight of 25 800, was obtained after induction with IPTG and affinity chromatography. The anti-FTL antibody was obtained from the immunized rabbits. The results of Western blotting indicated that the polyclonal antibody had high specificity to FTL protein. Conclusion: We have successfully obtained the purified FTL protein and anti-FTL polyclonal antibody, which lays a foundation for further research on the biological function of FTL and expression of FTL in lung cancer tissues.

Purificación de IgG1, IgG2 e IgG3 mediante cromatografía de afinidad a partir de intacglobín / Purification of IgG1, IgG2 and IgG3 by affinity chromatography starting from Intacglobin

Se describe un método de purificación de las subclases IgG1, IgG2 e IgG3 a partir de Intacglobin mediante cromatografía de afinidad con el empleo de proteína A Sepharose y la aplicación de un gradiente lineal de pH. Se detectó la presencia de IgG1, IgG2 e IgG3 en las fracciones eluidas mediante doble inmunodifusión e inmunoelectroforesis, utilizando sueros comerciales anti IgG, anti IgG1, anti IgG2 y anti IgG3. El procedimiento desarrollado se caracterizó por su sencillez, elevada resolución y la relativa pureza de las subclases aisladas. Se logró un rendimiento total del 53,7 %, lo que indica la eficiencia del método.

A method for the purification of IgG1, IgG2, and IgG3 subclasses starting from Intacglobin by affinity chromatography with the use of protein A Sepharose and the application of a lineal gradient of pH is described. IgG1, IgG2 and IgG3 were detected in the eluted fractions by doble immunodifussion and immunoelectrophoresis by using anti IgG, anti IgG1, anti IgG2 and anti IgG3 commercial sera. The procedure developed was characterized by its simplicity, high resolution and the relative purity of the isolated subclasses. A total yield of 53.7 % was attained, which demonstrates the efficiency of this method.

Preparación de un conjugado peroxidasa-anti IgG humana (cadena ) en conejo / Preparation of a peroxidase-anti human IgG conjugate (chain g) in rabbit

Se preparó un conjugado con peroxidasa a partir de los anticuerpos específicos aislados de un suero de conejo anti cadenas g de la IgG humana. Los anticuerpos específicos se aislaron por cromatografía de afinidad, y el conjugado se preparó por el método de oxidación con peryodato. El conjugado obtenido presentó una relación molar IgG/peroxidasa de 1,07, un valor de RZ de 0,33 y resultó evaluado satisfactoriamente en cuanto a su especificidad y reactividad en los ensayos inmunoenzimáticos realizados.

A peroxidase conjugate was prepared starting from the specific antibodies isolated from an anti-chain rabbit serum and from human IgG. The specific antibodies were isolated by affinity chromatography and the conjugate was prepared by the method of oxidation with periodate. The conjugate obtained presented a molar IgG/peroxidase relation of 1.07, a RZ value of 0.33 and it was satisfactorily evaluated as regards its specificity and reactivity in the immunoenzimatic assays carried out.

Application of molecular recognition theory in thyrotropin and its receptor system / 中国病理生理杂志

AIM: The selective recognition of the sense peptides which are located in special regions of thyrotropin receptor (TSHR) by their corresponding antisense peptides has been investigated. Three pairs of sense and antisense peptides were named TR1 (aa37-45) and RT1 (aa45-37), TR2 (aa353-366) and RT2 (aa366-353), TR3 (aa648-655) and RT3 (aa655-648). METHODS: To prepare three affinity chromatography columns, antisense peptides were immobilized, called RT1-sepharose 4B, RT2-sepharose 4B and RT3-sepharose 4B, respectively and investigate the retardative behavior for each of native peptide TR1, TR2 or TR3 on above columns with stepwise elution. RESULTS: Each of the three immobilized antisense peptides recognized and retarded its corresponding sense peptide-TR1, TR2 or TR3 instead of those non-complementary peptides. Immobilized RT1 recognized free TSHR protein molecule as well. In additional, bovine thyrotropin was recognized by immobilized TR1. CONCLUSION: The results indicate that molecular recognition theory exsits in thyrotropin receptor system. It may be useful to isolate biological molecules and to locate epitopes of TSH on TSHR molecule. Otherwise, antisense peptide may be used for treatment of experimental autoimmunolized thyroid disease (AITD) in the rat. [