In silico characterization of hypothetical proteins from Paracoccidioides lutzii.

Nearly 60% of Paracoccidioides lutzii genes encode products annotated as hypothetical or predicted proteins (HPs). In this study, we describe the global detection and functional inference of HPs, using computational methods based on sequence similarity, identification of targeting signals, presence of known protein domains, and use of the Gene Ontology functional classification scheme. Our analysis enabled a high-throughput characterization of predicted cellular localization and presence of protein domains, clustering HPs into different functional categories including metabolism, localization, cell cycle, response to stimulus, and signaling. To investigate P. lutzii HP expression profiles, we used data obtained from the expressed sequence tag database (dbEST). These analyses revealed 2364 HPs expressed in different situations, namely in mycelial and yeast forms, during the transition from mycelium to yeast, and under conditions mimicking infection. Based on this transcriptomic data, we performed a functional enrichment analysis according to the domains present in the HPs expressed in each condition. The most overrepresented functional domains were those involved in the regulation of gene expression, suggesting important and as yet undescribed roles for these HPs in the adaptation of P. lutzii to environmental conditions. In addition, the expression profiles of six randomly selected HPs were analyzed by quantitative real-time polymerase chain reaction in order to verify their expression in the complementary DNA libraries analyzed in this investigation. The approach used in this study should improve functional characterization of P. lutzii HPs.

SpotWhatR: a user-friendly microarray data analysis system

SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella fastidiosa Microarray Projects. SpotWhatR is freely available at http://blasto.iq.usp.br/~tkoide/SpotWhatR, in English and Portuguese versions. In addition, the user can choose between [quot ]single experiment[quot ] and [quot ]batch processing[quot ] versions.

Expression of glycoprotein gp43 in stage-specific forms and during dimorphic differentiation of Paracoccidioides brasiliensis.

Expression of the 43 kDa glycoprotein (gp43) was analysed in several Paracoccidioides brasiliensis isolates. Using one- and two-dimensional analysis of crude cellular extracts, it was shown that protein expression in yeast and mycelium was dependent on the isolate analysed. In two strains, in both yeast and mycelium cells. gp43 was present, whereas expression was restricted to the yeast phase of two other strains. The clinical implications of this phase-specific gp43 expression are uncertain.

Protein synthesis patterns of Paracoccidiodes brasiliensis isolates in stage-specific forms and during cellular differentiation.

In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates. The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms. The examined isolates exhibited highly variable cellular morphology at 36 degrees C, when typical yeast cells were expected. On the other hand, at 26 degrees C all the isolates showed mycelial morphology. The analysis of protein synthesis profiles made it possible to cluster the P. brasiliensis isolates into groups that correlated with the morphological data. Interestingly, growth at 36 degrees C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates. It was possible to cluster the isolates grown at 36 degrees C in three groups based on their two-dimensional protein synthesis analysis. The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogenous gene expression pattern in the host-adapted form than in the saprobic phase.

In vitro amphotericin B effects on growth, viability and dimorphism of Paracoccidioides brasiliensis: reversal of the treatment.

The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 micrograms/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of 35S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.