Fundacion Institute Estudios Avanzados

Caracas, Venezuela

Fundacion Institute Estudios Avanzados

Caracas, Venezuela
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Chiurillo M.A.,University Centroccidental Lisandro Alvarado | Cortez D.R.,University of Sao Paulo | Lima F.M.,University of Sao Paulo | Cortez C.,University of Sao Paulo | And 5 more authors.
Infection, Genetics and Evolution | Year: 2016

Trans-sialidase (TS) is a polymorphic protein superfamily described in members of the protozoan genus Trypanosoma. Of the eight TS groups recently described, TS group I proteins (some of which have catalytic activity) are present in the distantly related Trypanosoma brucei and Trypanosoma cruzi phylogenetic clades, whereas other TS groups have only been described in some species belonging to the T. cruzi clade. In the present study we analyzed the repertoire, distribution and phylogenetic relationships of TS genes among species of the T. cruzi clade based on sequence similarity, multiple sequence alignment and tree-reconstruction approaches using TS sequences obtained with the aid of PCR-based strategies or retrieved from genome databases. We included the following representative isolates of the T. cruzi clade from South America: T. cruzi, T. cruzi Tcbat, Trypanosoma cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli and Trypanosoma conorhini. The cloned sequences encoded conserved TS protein motifs Asp-box and VTVxNVxLYNR but lacked the FRIP motif (conserved in TS group I). The T. conorhini sequences were the most divergent. The hybridization patterns of TS probes with chromosomal bands confirmed the abundance of these sequences in species in the T. cruzi clade. Divergence and relationship analysis placed most of the TS sequences in the groups defined in T. cruzi. Further examination of members of TS group II, which includes T. cruzi surface glycoproteins implicated in host cell attachment and invasion, showed that sequences of T. cruzi Tcbat grouped with those of T. cruzi genotype TcI. Our analysis indicates that different members of the T. cruzi clade, with different vertebrate hosts, vectors and pathogenicity, share the extensive expansion and sequence diversification of the TS gene family. Altogether, our results are congruent with the evolutionary history of the T. cruzi clade and represent a contribution to the understanding of the molecular evolution and role of TS proteins in trypanosomes. © 2015 Elsevier B.V..


Mukhopadhyay R.,Florida International University | Mandal G.,Florida International University | Atluri V.S.R.,Florida International University | Figarella K.,Wayne State University | And 7 more authors.
Molecular and Biochemical Parasitology | Year: 2011

Leishmania major aquaglyceroporin LmAQP1 allows adventitious passage of antimonite, an activated form of the drug Pentostam, which is used as the first line treatment for leishmaniasis. The extracellular C-loop of an aquaglyceroporin confers substrate specificity. Alteration of Glu125 to serine in the Plasmodium falciparum aquaglyceroporin PfAQP has been shown to selectively affect water but not glycerol permeability. The C-loop of LmAQP1 is twelve residues longer than PfAQP, and Ala163 is at an equivalent position as Glu125 of PfAQP. The role of Ala163 in LmAQP1 solute permeability was investigated. Alteration of Ala163 to serine or threonine did not significantly affect conduction of solutes. However, alteration to aspartate, glutamate, and glutamine blocked passage of water, glycerol, and other organic solutes. While LmAQP1 is a mercurial insensitive water channel, mutation of the adjacent threonine (Thr164) to cysteine led to inhibition of water passage by Hg2+. This inhibition could be reversed upon addition of β-mercaptoethanol. These data suggest that, unlike Glu125 (PfAQP), Ala163 is not involved in stabilization of the C-loop and selective solute permeability. Ala163 is located near the pore mouth of the channel, and replacement of Ala163 by bulkier residue sterically hinders the passage of solutes. Alteration of Ala163 to serine or threonine affected metalloid uptake in the order, wild-type>A163S>A163T. Metalloid conduction was near completely blocked when Ala163 was mutagenized to aspartate, glutamate, or glutamine. Mutations such as A163S and A163T that reduced the permeability to antimonite, without a significant loss in water or solute conductivity raises the possibility that, subtle changes in the side chain of the amino acid residue in position 163 of LmAQP1 may play a role in drug resistance. © 2010 Elsevier B.V.


PubMed | Virginia Commonwealth University, Fundacion Institute Estudios Avanzados, University Centroccidental Lisandro Alvarado and University of Sao Paulo
Type: | Journal: Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases | Year: 2015

Trans-sialidase (TS) is a polymorphic protein superfamily described in members of the protozoan genus Trypanosoma. Of the eight TS groups recently described, TS group I proteins (some of which have catalytic activity) are present in the distantly related Trypanosoma brucei and Trypanosoma cruzi phylogenetic clades, whereas other TS groups have only been described in some species belonging to the T. cruzi clade. In the present study we analyzed the repertoire, distribution and phylogenetic relationships of TS genes among species of the T. cruzi clade based on sequence similarity, multiple sequence alignment and tree-reconstruction approaches using TS sequences obtained with the aid of PCR-based strategies or retrieved from genome databases. We included the following representative isolates of the T. cruzi clade from South America: T. cruzi, T. cruzi Tcbat, Trypanosoma cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli and Trypanosoma conorhini. The cloned sequences encoded conserved TS protein motifs Asp-box and VTVxNVxLYNR but lacked the FRIP motif (conserved in TS group I). The T. conorhini sequences were the most divergent. The hybridization patterns of TS probes with chromosomal bands confirmed the abundance of these sequences in species in the T. cruzi clade. Divergence and relationship analysis placed most of the TS sequences in the groups defined in T. cruzi. Further examination of members of TS group II, which includes T. cruzi surface glycoproteins implicated in host cell attachment and invasion, showed that sequences of T. cruzi Tcbat grouped with those of T. cruzi genotype TcI. Our analysis indicates that different members of the T. cruzi clade, with different vertebrate hosts, vectors and pathogenicity, share the extensive expansion and sequence diversification of the TS gene family. Altogether, our results are congruent with the evolutionary history of the T. cruzi clade and represent a contribution to the understanding of the molecular evolution and role of TS proteins in trypanosomes.


Gonzalez-Bacerio J.,University of Habana | Osuna J.,National Autonomous University of Mexico | Ponce A.,University of Habana | Fando R.,CSIC - National Center for Metallurgical Research | And 4 more authors.
Protein Expression and Purification | Year: 2014

Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo proteases, is a promising target for malaria, a devastating human parasitic disease. We report the high-level expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector. Optimal expression was achieved by induction with 1 mM IPTG at 37°C for 18 h. This allowed obtaining 100 mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass was from rPFAM1. rPfAM1, fused to an amino-terminal 6×His tag, was purified in a single step by immobilized metal ion affinity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition profile typical of metallo-aminopeptidases, and inhibition from Zn2+ excess, were similar to those of the native PfAM1. We have thus optimized an expression system that should be useful for identifying new PfAM1 inhibitors. © 2014 Elsevier Ltd. All rights reserved.


PubMed | CSIC - National Center for Metallurgical Research, National Autonomous University of Mexico, Fundacion Institute Estudios Avanzados and University of Habana
Type: | Journal: Protein expression and purification | Year: 2016

Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo proteases, is a promising target for malaria, a devastating human parasitic disease. We report the high-level expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector. Optimal expression was achieved by induction with 1mM IPTG at 37C for 18h. This allowed obtaining 100mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass was from rPFAM1. rPfAM1, fused to an amino-terminal 6His tag, was purified in a single step by immobilized metal ion affinity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition profile typical of metallo-aminopeptidases, and inhibition from Zn(2+) excess, were similar to those of the native PfAM1. We have thus optimized an expression system that should be useful for identifying new PfAM1 inhibitors.


Lobo G.,Central University of Venezuela | Zuleta E.,Central University of Venezuela | Charris K.,Central University of Venezuela | Capparelli M.V.,Fundacion Institute Estudios Avanzados | And 3 more authors.
Journal of Chemical Research | Year: 2011

A highly regiospecific synthesis and crystal structure of (4bRS,9bRS)-5-(2,4-dimethoxyphenyl)-7,7-dimethyl-4b,9bdihydroxy-4b,5,6,7,8, 9b-hexahydroindeno[1,2-b]indole-9,10-dione is reported. It was tested in vitro against six human tumour cell lines and two nontumourogenic cell lines. Their in vitro activity against Mycobacterium tuberculosis is also reported. In general, it was found to possess a marginal activity.


Loro M.,Simon Bolivar University of Venezuela | Valero-Jimenez C.A.,University of Zulia | Nozawa S.,Central University of Venezuela | Marquez L.M.,Simon Bolivar University of Venezuela | Marquez L.M.,Fundacion Institute Estudios Avanzados
Journal of Arid Environments | Year: 2012

Fungal endophytes grow asymptomatically within the tissues of all vascular plants and some are known to provide their host plants with tolerance to different types of environmental stress. The diversity and incidence of fungal endophytes has been negatively correlated with latitude. However, fungal endophyte communities from arid and semiarid environments do not follow this pattern, arguably due to their extreme conditions. In this study, fungal endophytes were cultured from dominant grasses and sedges growing at three different environments in the neotropical semiarid region of Northwest Venezuela. Operational Taxonomic Units were clearly differentiated using phylogenetic analysis of the ITSrDNA region. The results indicated that the incidence and diversity of fungal endophytes in these samples were comparable with those in other tropical plant communities and those in semiarid temperate grasslands. The most common OTUs within the grasses and sedges were related to Cochliobolus sp. and Phoma sp. (Order Pleosporales). This supports the notion that dark septate fungal endophytes dominate semiarid grasslands worldwide. © 2012 Elsevier Ltd.


Nino C.H.,National University of Colombia | Forero-Baena N.,National University of Colombia | Contreras L.E.,National University of Colombia | Sanchez-Lancheros D.,National University of Colombia | And 2 more authors.
Memorias do Instituto Oswaldo Cruz | Year: 2015

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite’s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites. © 2015, Fundacao Oswaldo Cruz. All rights reserved.


Velasco-Ramirez A.P.,University of Guadalajara | Torres-Moran M.I.,University of Guadalajara | Molina-Moret S.,Fundacion Institute Estudios Avanzados | de Jesus Sanchez-Gonzalez J.,University of Guadalajara | Santacruz-Ruvalcaba F.,University of Guadalajara
Electronic Journal of Biotechnology | Year: 2014

Background: At present, species known as camote de cerro (Dioscorea spp.) are found only in the wilderness in Mexico, but their populations are extremely depleted because they are indiscriminately collected, it is urgent to evaluate the conservation status of these plants in order to design conservation genetics programs. In this study, genetic diversity parameters along with cluster analysis based on Jaccard's coefficient were estimated 16 with the objective to assess the efficiency of RandomAmplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP) and Inverse Sequence Tagged Repeat (ISTR) molecular DNA markers in the Dioscorea genus. Results: The polymorphic information contents were quite similar for all markers (≈0.48). Genetic variation of Dioscorea spp., in terms of average heterozygosity was lower with ISTR (0.36), and higher when other markers were used (RAPD = 0.43; ISSR = 0.45 and AFLP = 0.47). Conclusion: This indicates an important level of genetic differences despite the fact that the plant is asexually propagated. Based on the diversity statistics, any marker tested in present work can be recommended for use in large-scale genetic studies of populations. However, the low correlations among different molecular marker systems show the importance of the complementarity of the information that is generated by different markers for genetic studies involving estimation of polymorphism and relationships. © 2014 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.


PubMed | National University of Colombia and Fundacion Institute Estudios Avanzados
Type: Journal Article | Journal: Memorias do Instituto Oswaldo Cruz | Year: 2015

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasites drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

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