Cross-talk between Rac1 and Cdc42 GTPases regulates formation of filopodia required for dengue virus type-2 entry into HMEC-1 cells.

Infection with dengue virus type-2 (DENV-2) begins with virus adherence to cell surface receptors. In endothelial cells (HMEC-1), a cell model for DENV-2 infection, alpha 5 beta 3 integrin has been identified as a putative receptor for the virus. Previous work had suggested that the actin cytoskeleton of HMEC-1 cells plays an important role in virus entry and infection. In the present work, fixed and living HMEC-1 cells expressing enhanced green fluorescent protein-actin were monitored for actin reorganization after virus inoculation, utilizing fluorescence and time lapse microscopy. Cell infection and production of infective viruses were quantified using an anti-E protein antibody and by measuring the p.f.u. ml(-1). Specific drugs that antagonize actin organization and regulate actin-signalling pathways were tested in viral adhesion and infection assays, as were the expression of dominant-negative Rac1 and Cdc42 proteins. Disorganization of actin precluded infection, while microtubule depolymerization had no effect. Activation of Rac1 and Cdc42 signalling, which occurs upon virus binding, induced reorganization of actin to form filopodia in the cellular periphery. Formation of filopodia was a requirement for virus entry and further cell infection. Expression of the dominant-negative proteins Rac1 and Cdc42 confirmed the role of these GTPases in the actin reorganization that is required to form filopodia. In addition, inhibition of the ATPase activity of myosin II greatly decreased infection, suggesting its participation in filopodial stability. We show here, for the first time, that internalization of DENV-2 into endothelial cells requires viral induction of dynamic filopodia regulated by Rac1 and Cdc42 cross-talk and myosin II motor activities.

HPV+ cervical carcinomas and cell lines display altered expression of caspases.

OBJECTIVE: Loss of expression of apoptotic regulatory proteins in many neoplasias might result in defective or delayed apoptosis, thus facilitating tumor growth or survival. We analyzed here, the basal expression of precursors of apoptotic Caspases in normal cervical epithelium, HPV+ cervical tumor samples and HPV+ tumor-derived cell lines. METHODS: Expression of initiator and effector Caspases was analyzed by immunochemistry in normal cervical epithelium and three types of cervical tumors (squamous cell carcinoma, adenocarcinoma and adenosquamous cell carcinoma) whereas expression of Caspases in HeLa, SiHa and CaSki cells was by immunofluorescence, Western blot and RT-PCR. Besides, the effect of the HPV-16 E6/E7 oncogenes on Caspases expression in cervical cells was evaluated by transfecting C33-A (HPV-) cells. RESULTS: Expression of Caspases 3 and 9 was undetectable in adenocarcinoma and adenosquamous cell carcinoma, respectively. Whereas in squamous cell carcinoma, the expression of Caspases was similar those observed in normal samples. Expression of Caspases 3 and 6 was low in HeLa and CaSki cells, while Caspase 8 was low in SiHa and it was not detected in C33-A cells. All Caspases were detected in the cytoplasm and nucleus of the cells. We did not observe an effect of the E6/E7 oncogenes on the expression of Caspases in C33-A cell. CONCLUSION: Our results showed a differential expression of several Caspases in carcinoma samples and cell lines, suggesting multiple alterations of the Caspase pathways in cervical cancer.

Cell proliferation and inhibition of apoptosis are related to c-Kit activation in leukaemic lymphoblasts.

Receptor tyrosine kinase (RTK) activity may contribute to carcinogenesis. The c-Kit receptor, a member of the RTK family, is expressed in immature haematopoietic system cells. Acute lymphoblastic leukaemia (ALL) presents incompletely differentiated lymphoblasts, and consequently, c-Kit expression can be detected in these cells. The BCR-ABL kinase, which is usually present in both ALL and chronic myeloid leukaemia, can trigger signalling pathways with neoplastic effects. However, a certain number of ALL patients and chronic myeloid leukaemia patients do not express this kinase, raising the question of which other proteins that intervene in signalling pathways may be involved in the development of these diseases. OBJECTIVES: To test whether c-Kit has proliferative effects and affects the inhibition of apoptosis of leukaemic lymphoblasts that do not express BCR-ABL. METHODS: We cultured RS4:11 lymphoblasts and analysed the expression and activation of c-Kit by immunofluorescence, and flow cytometry, evaluation of cell proliferation, apoptosis, cyclin D1 and Bak expression were carried out by flow cytometry; activation of AKT and survivin expression were tested by immunoblot. RESULTS: The c-Kit receptor was found to induce proliferation and to increase the expression of cyclin D1 via the PI3K/AKT/NF-kB signalling pathway. Additionally, the c-Kit/PI3K/AKT pathway increased the inhibition of apoptosis and survivin expression. Similarly, c-Kit was observed to reduce the expression of the pro-apoptotic Bak protein. CONCLUSION: These results suggest that, in leukaemic lymphoblasts, c-Kit triggers a signalling pathway with proliferative and anti-apoptotic effects; information to this effect has not yet been reported in the literature.

Marcadores fenotípicos en cepas de Aeromonas aisladas en Cuba de niños con enfermedad diarreica aguda

Se estudiaron 27 casos de Aeromonas aisladas de 300 niños menores de 5 años con enfermedad diarreica aguda (EDA), con el objetivo de demostrar la presencia de algunos marcadores fenotípicos asociados con la enteropatogenicidad, entre ellos: descarboxilación de la lisina, producción de acetil metil carbinol, enteroxigenicidad, citoxicidad y hemólisis. El ciento por ciento de las cepas estudiadas poseía 2 o más de los marcadores investigados y 13 (48 %) lisaron los eritrocitos de conejo con títulos mayor de 1:16. Se demostró la presencia de los marcadores en las especies Aeromonas hydrophila, Aeromonas sobria y Aeromonas caviae.

Obtención de un clono de la línea celular AP-61. Su utilidad para la multiplicación de los virus dengue 1 y 2 / Obtetion o a cell line clone AP-61. Su utilidad para la multiplication of dengue 1 and 2 viruses

Se describe la obtención de un clono de la línea celular continua AP-61 (Aedes pseudoscutellaris). Se discuten detalles del clonaje y de los medios de cultivo empleados. Se prueba la utilidad del clono para la multiplicación de los virus dengue 1 y 2 comparativamente con la línea original, aplicando la técnica de inmunofluorescencia indirecta

Obtención de una línea celular diploide de pulmón humano (PHuE-1) / Obtention of a diploid cell line (PHuE-1) from the human lung

Se describe la obtención de una línea celular diploide de pulmón humano de intéres para el diagnóstico virológico y las investigaciones. Se discuten algunos aspectos de su aplicación y caracterización

Efecto de varios factores durante la criopreservación sobre la morfología y la viabilidad de algunas líneas celulares poikilotérmicas / Effect of several factors during cryopreservation on the morphology and viability of some poikilothermic cell lines

Se estudió el efecto de varios factores durante la criopreservación sobre 3 líneas celulares poikilotérmicas utilizando como indicadores la morfología y la viabilidad. Se comprobó que evitar la toxicidad en el medio de suspensión es de vital importancia en el éxito de la congelación. Además, uno de los sistemas estudiados y de gran aplicación en Virología no soporta largas hibernaciones. Finalmente se discute el método más adecuado de congelación para este tipo de células

Utilización del suero caprino como sustituto del suero de ternera donante en el crecimiento de algunos cultivos primarios de vertebrados / Use of goats serum as a substitute for doner calf serum in the growth of some primary cultures of vertebrates

Se utilizó el suero caprino para la preparación de cultivos primarios de fibroblastos de embrión de pollo y riñón de hámster, los cuales fueron posteriormente inoculados con el virus de la encefalomielitis equina del Este en comparación con los cultivos obtenidos mediante el suero de ternera donante. La utilización del suero caprino para nuestros propósitos en este trabajo, constituye una opción de gran interés para el desarrollo en el campo de las investigaciones científicas y la economía del país