Propiedades de una proteinasa de Trypanosoma Cruzi

Fil: Bontempi, Esteban José. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Biosynthesis of 5-aminolevulic acid in trypanosoma cruzi

Trypanosoma cruzi requires heme-compounds for growing, due to its partially or totally deficient biosynthetic pathway of heme. There are reports that support the functionality of mitochondrial enzymes involved in this pathway, such as 5-aminolevúlico synthetase (ALA-S) and Heme synthetase (Heme-S). T. cruzi genome is known and two homologous genes, Tc00.1047053511899.40 y Tc00.1047053511071.140, were identified by bioinformatic studies. Both of them are candidates to code with high score (50%) for the ALA-S enzyme, responsible for synthesizing ALA from succinyl CoA and glycine. Our hypothesis is that the parasite is able to synthesize ALA (although it cannot be metabolized to heme) and the Tc00.1047053511899.40 y Tc00.1047053511071.140 sequences encodes for a protein with ALA-S activity. Using epimastigotes, we were able to detect and quantify, by spectrophotometric studies and HPLC chromatography, the presence of ALA in the parasite both intra and extracellularly. The mesasurements were made in 30ml of parasite culture which yielded about 608,31 ± 45,20 nmol of ALA. The extracellular content represents 96% of the total synthesized. Such excretion would be avoiding the citotoxicity of ALA since it cannot be metabolized to heme From bioinformatic studies using the Blast, ORF Finder, Mitoprop, Prosite and ClustalW platforms, it was determined that the above genes would code for a mitochondrial protein (98%) which is dependent on pyridoxal phosphate and shown a KBL domain, which is characteristic of enzymes as ALA-S. Both, ALA detection and the computer analysis would support our hypothesis and encourages us to continue trying to confirm it.Fil: Puente, Vanesa Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Martinez, Maria del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Abou Assali, Lubna. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Benlolo, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Bontempi, Esteban. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Lombardo, Maria Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaLXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; IX Reunión Anual de la Asociación Argentina de Nanomedicinas y VI Reunión Científica Regional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioMar del PlataArgentinaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentalSociedad Argentina de BiologíaSociedad Argentina de ProtozoologíaAsociación Argentina de NanomedicinasAsociación Argentina de Ciencia y Tecnología de Animales de Laboratori

ALCHIMIA D2.1 Requirements and human-centric recommendation

This report constitutes Deliverable 2.1: Requirements and human-centric recommendations of the ALCHIMIA project. ALCHIMIA is a tool that will help companies in the steel and metal sector to continuously optimise production process by exploiting a broad range of production-related data. The ALCHIMIA project adopts a human-centred approach to design that guides the design and development process in specific ways to ensure the views, interests and needs of users and stakeholders are taken during the design phase of the technology. The report provides an overview and summary of activities conducted and findings obtained during the initial stages of the development of the AI-based ALCHIMIA system. It starts by providing an overview of current production processes in two participating companies, one an EAF-based steel producer and the other a manufacturer of disk brakes for the automotive industry (Part 2: Setting the Scene). The description of current approaches leads to a description of efficiency problems in both industrial settings that ALCHIMIA is aiming to solve (Part 3: ALCHIMIA and its Functions). To effectively take user and stakeholder views, interests and needs into account, they need to be systematically elicited and ultimately translated into system requirements that constrain and guide the actual technical design of the ALCHIMIA system. Part 4 of the report (User and Stakeholder Requirements and Expectations) provides an overview of the requirement elicitation activities conducted as part of the ALCHIMIA project. Findings and outcomes of these activities are also presented. In Part 5 (Human-Centred Design Recommendations), we reflexively analyse and evaluate the alignment of the design and development activities in the ALCHIMIA project with six human-centred design principles. While this evaluation indicates that the ALCHIMIA project is well aligned with the human-centred design principles, there are aspects that can be improved. We thus make three recommendations that, if implemented, should ensure improved alignment of the ALCHIMIA project with human-centred design prescriptions

A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi

Fil: Ferella, Marcela. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Montalvetti, Andrea. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Rohloff, Peter. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Miranda, Kildare. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Fang, Jianmin. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Reina, Silvia. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Kawamukai, Makoto. University Matsue. Faculty of Life and Environmental Science. Department of Applied Bioscience and Biotechnology; Japón.Fil: Bua, Jacqueline. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Nilsson, Daniel. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Pravia, Carlos. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Katzin, Alejandro. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Casera, María B. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Áslund, Lena. Uppsala University. Department of Genetics and Pathology; Suecia.Fil: Andersson, Björn. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Docampo, Roberto. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Bontempi, Esteban. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén"; Argentina.We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ∼ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target

Propiedades de una proteinasa de Trypanosoma Cruzi

Fil: Bontempi, Esteban José. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A page-classification approach to web usage semantic analysis

info:eu-repo/semantics/publishedSpecial Issue on Soft computing in Artificial Intelligence, Data and Web Mining, Machine Learnin

Category-based audience metrics for Web site content improvement using ontologies and page classification

SCOPUS: cp.kinfo:eu-repo/semantics/publishe

Characterisation of a cyclophilin isoform in Trypanosoma cruzi

Fil: Bua, Jacqueline. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Aslund, Lena. Rudbeck Laboratory. Department of Genetics and Pathology; Suecia.Fil: Pereyra, Natalia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Ruiz, Andrés Mariano. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Bontempi, Esteban J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.The immunosuppressive drug cyclosporin A (CsA) has shown antiparasitic activity against several protozoans and helminths, when complexed to proteins called cyclophilins (CyPs). In this paper, the molecular characterisation of one member of the CyP family in Trypanosoma cruzi is reported. TcCyP19 gene proved to be highly conserved compared to CyPs from other organisms and was highly homologous to a Trypanosoma brucei brucei CyPA. This gene was expressed in Escherichia coli and the purified recombinant protein exhibited a peptidyl prolyl cis-trans isomerase activity that was inhibited by CsA (IC(50) = 18.4 + /-0.8 nM). The TcCyP19 gene was located on two chromosomal bands in T. cruzi CL Brener clone

Proteomics in Trypanosoma cruzi--localization of novel proteins to various organelles

The completion of the genome sequence of Trypanosoma cruzi has been followed by several studies of protein expression, with the long-term aim to obtain a complete picture of the parasite proteome. We report a proteomic analysis of an organellar cell fraction from T. cruzi CL Brener epimastigotes. A total of 396 proteins were identified by LC-MS/MS. Of these, 138 were annotated as hypothetical in the genome databases and the rest could be assigned to several metabolic and biosynthetic pathways, transport, and structural functions. Comparative analysis with a whole cell proteome study resulted in the validation of the expression of 173 additional proteins. Of these, 38 proteins previously reported in other stages were not found in the only large-scale study of the total epimastigote stage proteome. A selected set of identified proteins was analyzed further to investigate gene copy number, sequence variation, transmembrane domains, and targeting signals. The genes were cloned and the proteins expressed with a c-myc epitope tag in T. cruzi epimastigotes. Immunofluorescence microscopy revealed the localization of these proteins in different cellular compartments such as ER, acidocalcisome, mitochondrion, and putative cytoplasmic transport or delivery vesicles. The results demonstrate that the use of enriched subcellular fractions allows the detection of T. cruzi proteins that are undetected by whole cell proteomic methods

The tyrosine aminotransferase from Trypanosoma rangeli: sequence and genomic characterization

Fil: Bontempi, Esteban. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Garcia, G. A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Buschiazzo, A. Instituto de Investigaciones Biotecnológicas, Universidad Nacional de Gral. San Martín, INTI; Argentina.Fil: Henriksson, J. Department of Medical Genetics and Pathology, Uppsala University, Suecia.Fil: Pravia, Carlos. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Ruiz, Andrés Mariano. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Pettersson, Ulf. Department of Medical Genetics and Pathology, Uppsala University; Suecia.Fil: Pszenny, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates