Effects of bromopride on expression of metalloproteinases and interleukins in left colonic anastomoses: an experimental study

Anastomotic dehiscence is the most severe complication of colorectal surgery. Metalloproteinases (MMPs) and interleukins (ILs) can be used to analyze the healing process of anastomosis. To evaluate the effects of bromopride on MMP and cytokine gene expression in left colonic anastomoses in rats with or without induced abdominal sepsis, 80 rats were divided into two groups for euthanasia on the third or seventh postoperative day (POD). They were then divided into subgroups of 20 rats for sepsis induction or not, and then into subgroups of 10 rats for administration of bromopride or saline. Left colonic anastomosis was performed and abdominal sepsis was induced by cecal ligation and puncture. A colonic segment containing the anastomosis was removed for analysis of gene expression of MMP-1α, MMP-8, MMP-13, IL-β, IL-6, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). On the third POD, bromopride was associated with increased MMP-1α, MMP-13, IL-6, IFN-γ, and IL-10 gene expression. On the seventh POD, all MMP transcripts became negatively modulated and all IL transcripts became positively modulated. In the presence of sepsis, bromopride administration increased MMP-8 and IFN-γ gene expression and decreased MMP-1, TNF-α, IL-6, and IL-10 gene expression on the third POD. On the seventh POD, we observed increased expression of MMP-13 and all cytokines, except for TNF-α. In conclusion, bromopride interferes with MMP and IL gene expression during anastomotic healing. Further studies are needed to correlate these changes with the healing process.

Virulence insights from the Paracoccidioides brasiliensis transcriptome

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.

Molecular chaperones in the Paracoccidioides brasiliensis transcriptome

Paracoccidioides brasiliensis is a thermally dimorphic and a human pathogenic fungus. Our group has partially sequenced its transcriptome and generated a database of mycelial and yeast PbAESTs (P. brasiliensis assembled expressed sequence tags). In the present review we describe the identification of PbAESTs encoding molecular chaperones. These proteins, involved in protein folding and renaturation, are also implicated in several other biological processes, where the dimorphic transition is of particular interest. Another important issue concerning these proteins refers to their participation in the immunopathogenicity of infectious diseases. We have found 438 ESTs (184 in mycelium and 253 in yeast) encoding P. brasiliensis molecular chaperones and their co-chaperones, which were clustered in 48 genes. These genes were classified in families, corresponding to three small chaperones, nine HSP40s, 10 HSP60s, seven HSP70s, five HSP90s, four HSP100s, and 10 other chaperones. These results greatly increase the knowledge on P. brasiliensis molecular chaperones, since only eight of such proteins had been previously characterized.

Paracoccidioides brasiliensis RNA biogenesis apparatus revealed by functional genome analysis

The RNA biogenesis machinery of Paracoccidioides brasiliensis was assessed by comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags (ESTs)) with sequences from Saccharomyces cerevisiae MIPS database. PbAESTs related to almost all categories of S. cerevisiae RNA biogenesis were found. Two of the 12 S. cerevisiae RNA Pol II core subunits, Rpb3 and Rpb7, were found, probably reflecting the growth phase from which the cDNA libraries used in ESTs generation were constructed, as well as the low abundance of some of these transcripts. We have also found orthologs to TATA-box-binding protein (TBP), and at least one subunit of each TBP-associated factors (TFII) in P. brasiliensis transcriptome, except TFIIB. Genes associated to the chromatin remodeling complex, as well as transcription factors probably involved in the control of genes associated to a sexual cycle and virulence, were also identified. With respect to the pre-mRNA processing, 65 PbAEST orthologs to S. cerevisiae basal splicing machinery and 21 orthologs of 5'- and 3'-end formation processes were found. Components involved in RNA interference were detected, suggesting that this gene expression regulation mechanism is probably used by P. brasiliensis. Twelve PbAESTs related to Pol I and Pol III machineries were assigned as S. cerevisiae orthologs. Finally, 25 and 10 PbAESTs associated to rRNA and tRNA processing, respectively, were detected. Taken together, our results enable us to depict, for the first time, a global view of transcription and RNA processing in P. brasiliensis.

Paracoccidioides brasiliensis translation and protein fate machineries revealed by functional genome analysis

The translational and post-translational modification machineries of Paracoccidioides brasiliensis were assessed by means of comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags) with sequences deposited on different databases. Of the 79 sequences corresponding to cytosolic ribosomal proteins, we were able to find 78 in the P. brasiliensis transcriptome. Nineteen of the 27 Saccharomyces cerevisiae genes related to translation initiation were also found. All eukaryotic elongation factors were detected in P. brasiliensis transcriptome, with eEF1A as one of the most expressed genes. Translation termination is performed, in eukaryotes, by factors 1 and 3 (eRF1, eRF3). In P. brasiliensis transcriptome it was possible to identify eRF3, but not eRF1. Sixteen PbAESTs showing aminoacyl-tRNA synthetase-predicted activities were found in our analyses, but no cysteinyl-, leucyl-, asparagyl- and arginyl-tRNA synthetases were detected. Among the mitochondrial ribosomal proteins, we have found 20 and 18 orthologs to S. cerevisiae large and small ribosomal subunit proteins, respectively. We have also found three PbAESTs similar to Neurospora crassa mitochondrial ribosomal genes, with no similarity with S. cerevisiae genes. Although orthologs to S. cerevisiae mitochondrial EF-Tu, EF-G and RF1 have been found in P. brasiliensis transcriptome, no sequences corresponding to functional EF-Ts were detected. In addition, 64 and 28 PbAESTs associated to protein modification and degradation, respectively, were found. These results suggest that these machineries are well conserved in P. brasiliensis, when compared to other organisms.