Cellular and Molecular Biology Unit

Medellín, Colombia

Cellular and Molecular Biology Unit

Medellín, Colombia
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Hernandez O.,University of Antioquia | Hernandez O.,Cellular and Molecular Biology Unit | Almeida A.J.,University of Minho | Gonzalez A.,Medical and Experimental Mycology Group | And 8 more authors.
Infection and Immunity | Year: 2010

One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Simboeck E.,Medical University of Vienna | Sawicka A.,Medical University of Vienna | Zupkovitz G.,Medical University of Vienna | Senese S.,Italian National Cancer Institute | And 6 more authors.
Journal of Biological Chemistry | Year: 2010

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anticancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Ruiz H.O.,University of Antioquia | Ruiz H.O.,Cellular and Molecular Biology Unit | Ruiz H.O.,Major College of Antioquia | Gonzalez A.,Medical and Experimental Mycology Group | And 7 more authors.
PLoS Neglected Tropical Diseases | Year: 2011

Background: Paracoccidioides brasiliensis is a human thermal dimorphic pathogenic fungus. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells. Aims and Methodology: In this study, we evaluated the role of alternative oxidase (AOX), an enzyme involved in the intracellular redox balancing, during host-P. brasiliensis interaction. We generated a mitotically stable P. brasiliensis AOX (PbAOX) antisense RNA (aRNA) strain with a 70% reduction in gene expression. We evaluated the relevance of PbAOX during interaction of conidia and yeast cells with IFN-γ activated alveolar macrophages and in a mouse model of infection. Additionally, we determined the fungal cell's viability and PbAOX in the presence of H2O2. Results: Interaction with IFN-γ activated alveolar macrophages induced higher levels of PbAOX gene expression in PbWt conidia than PbWt yeast cells. PbAOX-aRNA conidia and yeast cells had decreased viability after interaction with macrophages. Moreover, in a mouse model of infection, we showed that absence of wild-type levels of PbAOX in P. brasiliensis results in a reduced fungal burden in lungs at weeks 8 and 24 post-challenge and an increased survival rate. In the presence of H2O2, we observed that PbWt yeast cells increased PbAOX expression and presented a higher viability in comparison with PbAOX-aRNA yeast cells. Conclusions: These data further support the hypothesis that PbAOX is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis. © 2011 Hernandez Ruiz et al.

Mouton V.,Catholic University of Leuven | Toussaint L.,Catholic University of Leuven | Toussaint L.,Cellular and Molecular Biology Unit | Vertommen D.,Catholic University of Leuven | And 9 more authors.
Biochemical Journal | Year: 2010

On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo-2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [γ-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBβ-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBα/β siRNA or PKBα siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBα. © The Authors.

Peixoto P.,University of Liège | Castronovo V.,University of Liège | Matheus N.,University of Liège | Polese C.,University of Liège | And 7 more authors.
Cell Death and Differentiation | Year: 2012

Histone deacetylases (HDACs) form a family of enzymes, which have fundamental roles in the epigenetic regulation of gene expression and contribute to the growth, differentiation, and apoptosis of cancer cells. In this study, we further investigated the biological function of HDAC5 in cancer cells. We found HDAC5 is associated with actively replicating pericentric heterochromatin during late S phase. We demonstrated that specific depletion of HDAC5 by RNA interference resulted in profound changes in the heterochromatin structure and slowed down ongoing replication forks. This defect in heterochromatin maintenance and assembly are sensed by DNA damage checkpoint pathways, which triggered cancer cells to autophagy and apoptosis, and arrested their growth both in vitro and in vivo. Finally, we also demonstrated that HDAC5 depletion led to enhanced sensitivity of DNA to DNA-damaging agents, suggesting that heterochromatin de-condensation induced by histone HDAC5 silencing may enhance the efficacy of cytotoxic agents that act by targeting DNA in vitro. Together, these results highlighted for the first time an unrecognized link between HDAC5 and the maintenance/assembly of heterochromatin structure, and demonstrated that its specific inhibition might contribute to increase the efficacy of DNA alteration-based cancer therapies in clinic. © 2012 Macmillan Publishers Limited All rights reserved.

Hernandez O.,Major College of Antioquia | Araque P.,University of Antioquia | Tamayo D.,University of Medellín | Restrepo A.,Cellular and Molecular Biology Unit | And 4 more authors.
Medical mycology | Year: 2015

Paracoccidioides brasiliensis is the etiologic agent of one of the most common systemic mycoses in Latin America. As a dimorphic fungus, it must adapt to different environments during its life cycle, either in nature or within the host, enduring external stresses such as temperature or host-induced oxidative stress. In this study we addressed the role of alternative oxidase (PbAOX) in cellular homeostasis during batch culture growth and the morphological transition of P. brasiliensis. Using a PbAOX-antisense-RNA (PbAOX-aRNA) strain with a 70% reduction in gene expression, we show that PbAOX is crucial for maintaining cell viability and vitality during batch culture growth of yeast cells, in what appears to be a pH-dependent manner. We also show that silencing of PbAOX drastically reduced expression levels of other detoxifying enzymes (PbY20 and PbMSOD). In addition, our data indicate that PbAOX plays a role during the morphological transition, namely, during the yeast-to-mycelia germination and mycelia/conidia-to-yeast transition, essential events during the establishment of infection by dimorphic fungal pathogens. Altogether, our findings support the hypothesis that PbAOX is important for the maintenance of cellular homeostasis, possibly by assisting redox balancing during cell growth and the morphological switch of P. brasiliensis. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PubMed | Cellular and Molecular Biology Unit, The Broad Institute of MIT and Harvard, University of Medellín and University of Antioquia
Type: | Journal: Fungal genetics and biology : FG & B | Year: 2017

Dimorphic human pathogenic fungi interact with host effector cells resisting their microbicidal mechanisms. Yeast cells are able of surviving within the tough environment of the phagolysosome by expressing an antioxidant defense system that provides protection against host-derived reactive oxygen species (ROS). This includes the production of catalases (CATs). Here we identified and analyzed the role of CAT isoforms in Paracoccidioides, the etiological agent of paracoccidioidomycosis. Firstly, we found that one of these isoforms was absent in the closely related dimorphic pathogen Coccidioides and dermatophytes, but all of them were conserved in Paracoccidioides, Histoplasma and Blastomyces species. We probed the contribution of CATs in Paracoccidioides by determining the gene expression levels of each isoform through quantitative RT-qPCR, in both the yeast and mycelia phases, and during the morphological switch (transition and germination), as well as in response to oxidative agents and during interaction with neutrophils. PbCATP was preferentially expressed in the pathogenic yeast phase, and was associated to the response against exogenous H

Clay O.K.,El Rosario University | Clay O.K.,Cellular and Molecular Biology Unit | Clay O.K.,Stazione Zoologica Anton Dohrn | Bernardi G.,Stazione Zoologica Anton Dohrn | Bernardi G.,Rome University 3
Molecular Biology and Evolution | Year: 2011

In an article published in these pages, Elhaik et al. (Elhaik E, Landan G, Graur D. 2009. Can GC content at third-codon positions be used as a proxy for isochore composition? Mol Biol Evol. 26:1829-1833) asked if GC3, the GC level of the third-codon positions in protein-coding genes, can be used as a "proxy" to estimate the GC level of the surrounding isochore. We use available data to directly answer this simple question in the affirmative and show how the use of indirect methods can lead to apparently conflicting conclusions. The answer reasserts that in human and other vertebrates, genes have a strong tendency to reside in compositionally corresponding isochores, which has far-reaching implications for genome structure and evolution. © 2010 The Author.

Munoz J.F.,Cellular and Molecular Biology Unit | Munoz J.F.,University of Antioquia | Gallo J.E.,Cellular and Molecular Biology Unit | Gallo J.E.,El Rosario University | And 6 more authors.
FEBS Letters | Year: 2013

In recent years, readily affordable short read sequences provided by next-generation sequencing (NGS) have become longer and more accurate. This has led to a jump in interest in the utility of NGS-only approaches for exploring eukaryotic genomes. The concept of a static, 'finished' genome assembly, which still appears to be a faraway goal for many eukaryotes, is yielding to new paradigms. We here motivate an object-view concept where the raw reads are the main, fixed object, and assemblies with their annotations take a role of dynamically changing and modifiable views of that object. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Hernandez O.,University of Antioquia | Hernandez O.,Cellular and Molecular Biology Unit | Hernandez O.,Major College of Antioquia | Almeida A.J.,Cellular and Molecular Biology Unit | And 8 more authors.
Medical Mycology | Year: 2012

Adherence of the dimorphic pathogenic fungus Paracoccidioides brasiliensis to lung epithelial cells is considered an essential event for the establishment of infection. We have previously shown that the PbHAD32 hydrolase is important in this early stage of the host-P. brasiliensis yeast cells interaction. The aim of this study was to further elucidate the role of PbHAD32 in conidial thermodimorphism and their interaction with lung epithelial cells. Analysis of the PbHAD32 gene expression revealed higher mRNA levels during the conidia to mycelia (CM) germination when compared to the conidia to yeast (CY) transition. Moreover, PbHAD32 was consistently expressed at higher levels upon infection of lung epithelial cells, but to a greater extent when conidia germinated to produce mycelia. Interestingly, at this particular transitional stage, more conidia adhered to epithelial cells than when they were transiting to the yeast form. Altogether our data further corroborates the importance of PbHAD32 during initial adherence to host cells and suggest that the 32-KDa hydrolase may also participate at different stages of the CM and CY conversions. © 2012 ISHAM.

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