Expression of TPK1 and TPK2 genes encoding PKA catalytic subunits during growth and morphogenesis in Candida albicans.

The transcript levels of Candida albicans TPK1 and TPK2 genes, encoding PKA catalytic subunits, as well as phosphotransferase activity, were measured in the parental strain CAI4 and in homozygous tpk1Delta and tpk2Delta mutants during vegetative growth and during yeast-to-mycelial transition in N-acetylglucosamine liquid inducing medium at 37 degrees C. We observed two TPK2 transcripts, a major one of 1.8 kb and a minor one of 1.4 kb, and established by 3'-RACE that they originate from the recognition of the three polyadenylation signals present in the 3' untranslated region of the gene. During vegetative growth of CAI4 strain, the expression profiles of TPK1 and TPK2 varied similarly, reaching maximal expression at the late logarithmic phase. TPK1 mRNA levels were lower than those of TPK2 at all stages measured. In the corresponding homozygous tpk mutants, mRNA levels and the expression patterns of TPK1 and TPK2 were similar to those of CAI4, suggesting that the loss of one catalytic isoform is not compensated by overexpression of the other. Changes in PKA specific activity roughly correlated with fluctuations of mRNA expression levels. During yeast-to-mycelial transition, a sharp increase in TPK1 mRNA levels and in PKA-specific activity correlated with the onset of germ-tube formation in strain tpk2Delta. We also showed that tpk1Delta strain exhibited a delayed morphogenetic shift in comparison with CAI4 and tpk2Delta strains in several liquid inducing media, reinforcing the idea that Tpk1p is important for faster germ-tube appearance.

Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior.

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.

La neumonía del niño hospitalizado de cinco a catorce años de edad / Pneumonia of the hospitalized child from five to fourteen years old

Introducción: a nivel mundial las neumonías en niños menores de cinco años causan morbilidad severa y mortalidad, lo cual amerita se realicen esfuerzos para su control. Sin embargo, niños con neumonía mayores de esas edades también representan una carga importante para los servicios de salud y en ellos la neumonía bacteriana, por frecuencia neumocócica tiene gran impacto. Un estudio de base poblacional se llevó a cabo durante tres años para determinar la carga de enfermedad que representan las neumonías en los niños hospitalizados de 0 a 14 años de edad. Objetivo: analizar los datos sobre las características clínico-radiográficas y la tasa de incidencia de la neumonía a fin de recomendar estrategias preventivas para los niños de cinco años y mayores de esa edad. Métodos: un estudio prospectivo, cubriendo una población expuesta de 43.103 de 5 a 14 años, se realizó en los departamentos de Paysandú y Salto. Todos los pacientes hospitalizados por una infección respiratoria aguda baja, que requirieron una radiografía de tórax, fueron incluidos en el estudio. La interpretación estandarizada de las radiografías de tórax clasificó a las neumonías. Resultados: de 2.658 pacientes captados, 474 (17,8 por ciento) eran niños de 5 años y más. Las neumonías predominaron en el segundo semestre del año así como en los niños de cinco a siete años (59,7 por ciento). Se diagnosticaron 228 neumonías consolidantes y 134 no consolidantes: las hospitalizaciones previas y el uso de antibióticos difirieron significativamente en ambos grupos. En el segundo año del estudio se registró la mayor tasa de incidencia que fue de 222/100.000. Diez niños requirieron cuidados intensivos y cinco fallecieron. Conclusiones: la tasa de incidencia de la neumonía consolidante, la severidad de los casos y los fallecidos señalan la necesidad de implementar medidas preventivas para los niños de 5 años y mayores de esa edad. La inmunización de estos niños con la vacuna antineumococo 23-valente resultaría...