Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

Presencia de anticuerpos IgM contra el virus del dengue en donantes de sangre, Medellín / Presence of IgM antibodies against dengue virus in blood donors, Medellin

Objetivo: determinar la frecuencia de anticuerpos IgM contra el virus del dengue en donantes del Banco de Sangre de la Cruz Roja Colombiana de Medellín. Métodos: se realizó un estudio descriptivo de corte del 27 marzo a 5 de mayo de 2009. Se colectaron 100 muestras de sangre proveniente de donantes. A cada persona le fue solicitado su consentimiento para permitir que 6 mL de la sangre que estaba donando, fuera estudiada para anticuerpos IgM contra el virus del dengue. La detección de anticuerpos IgM contra el virus del dengue se hizo mediante la técnica comercial Dengue IgM captura ELISA. Además se recolectó información socio-demográfica. Resultados: el 67% de los participantes eran hombres. La mediana de la edad fue 28,5 años (rango de 18 a 56 años). El 75% de los participantes procedía de Medellín. Fueron detectados anticuerpos IgM contra el virus de dengue en el 2% de las muestras estudiadas. Ambos donantes eran hombres, los cuales procedían de los municipios de Medellín y Caldas. Ninguno manifestó haber presentado dengue en los últimos tres meses, ni fiebre en los últimos siete días. Conclusiones: Los hallazgos de este estudio permitieron un primer acercamiento a este problema donde resulta posible que los donadores de sangre asintomáticos sean potencialmente infecciosos, con la posibilidad de transmitir la infección a sus receptores.Sin embargo se deben realizar otros estudios determinando, además de anticuerpos IgM, la presencia del virus.

Objective: to determine the frequency of IgM antibodies against the dengue virus in blood donors of the red cross blood bank of Medellin. Methods: A cross sectional descriptive study was performed trough 27th March to 25th May of 2009. Sampling was by convenience and it was constituted of 100 blood donors. Each person was asked for 6 mL of the donated blood sample. IgM antibody detection was performed with a commercially available technique; IgM Dengue captures ELISA. In addition, demographic information was collected. Results: 67% of the participants were men. The median was 28.5 years old, (range=18 to 56 years). 75% of the participants were from Medellin city. In the study, it was found that 2% of donors were reactive for anti-DENV IgM. Both donors were men. One donor lives in the city of Medellín and the other lives in the city of Caldas. None of them had a history of dengue fever three months before of donation or fever seven days before of donation. Conclusions: This findings sug that asymptomatic donors can transmit dengue infection through blood transfusion, but it is required to perform other survey with a bigger sample during different epidemiological periods and with laboratory techniques like RT-PCR for virus identification.

Anticorpos anti-intiminas α, β, e γ de Escherichia coli em soros e colostros de adultos saudáveis da Grande São Paulo / Antibodies reactive with α, β,and γ intimins of Escherichia coli in serum and colostrum samples of healthy adults living in São Paulo, Brazil

A diarréia é um importante problema de saúde pública no mundo inteiro e a Escherichía coli é um dos mais freqüentes microorganismos causadores desta doença. A Escherichia coli enteropatogênica (EPEC), um dos principais agentes etiológicos das diarréias infantis no nosso país, é genética e fenotipicamente relacionada com a E. colí enterohemorrágica (EHEC) que além de provocar diarréia é responsável por complicações como síndrome hemolítica urêmica (HUS) e colite hemorrágica (HC). Embora a EHEC seja considerada emergente pela OMS, no Brasil poucos casos de complicações como HUS e HC foram reportados. O mecanismo de patogenicidade comum entre EPEC e EHEC é conhecido como a lesão "attaching and effacing" nos microvilos do enterócito. Esta lesão é mediada por um conjunto de fatores de virulência, dentre eles a intimina. A intimina é uma proteína de membrana externa, responsável pelo íntimo contato da bactéria com o enterócito, possui uma região N-terminal que é altamente conservada e uma região C-terminal que é variável. De acordo com a região variável, existem vários subtipos de intimina, dentre eles as intiminas , α, β e γ...

Heterologous expression and biochemical characterization of an alpha 1, 2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Prokaryotic expression and preparation of polyantibody of human histydyl-tRNA synthetase related gene / 华中科技大学学报（医学）（英德文版）

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.

Estudio de un brote de encefalitis venezolana en el Distrito Páez, Estado Zulia, en octubre de 1968 / Study of an outbreak of venezuelan encephalitis in the Paez District, Zulia State, Veneuela, in october 1968

Se describe y analiza una epidemia de encefalitis venezolana en el Distrito Páez del Estado Zulia, ocurrida en el mes de octubre de 1968. Se registraron 1077 casos de la enfermedad, 150 de ellos con ataque evidente al sistema nervioso. Hubo dos muertes atribuibles a la encefalitis, ambas de niños menores de un año. Los casos se presentaron en mayor número en los menores de seis años; nacidos después de la última epidemia de encefalitis en la región, indicando la actividad cíclica del virus. Se concluye que no hay actividad del virus en los períodos interepidémicos. Aparentemente, la inmunidad conferida por el virus es de larga duración según lo indica el bajo número de enfermos en las edades más altas y en los niños menores de un año, estos últimos protegidos por anticuerpos maternos trasmitidos