Alimentos con menos colesterol

Alimentos con menos colesterol Investigadores del Conicet y de la Facultad de Farmacia y Bioquímica de la UBA desarrollaron un microorganismo unicelular (Tetrahymena) que al ser incorporado a alimentos como la leche y el huevo les reduce el colesterol en un 90 % transformándolo en provitamina D.Fil: Nudel, Berta Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D 3

Biocatalysis is a fundamental concept in biotechnology. The topic integrates knowledge of several disciplines; therefore, it was included in the course “design and optimization of biological systems” which is offered in the biochemistry curricula. We selected the ciliate tetrahymena as an example of a eukaryotic system with potential for the biotransformation of sterol metabolites of industrial interest; in particular, we focused on the conversion of cholesterol to provitamin D3. The students work with wild type and recombinant strains and learn how sterol pathways could be modified to obtain diverse sterol moieties. During the course the students identify and measure the concentration of sterols. They also search for related genes by bioinformatic analysis. Additionally, the students compare biotransformation rates, growing the ciliate in plate and in a bioreactor. Finally, they use fluorescence microscopy to localize an enzyme involved in biotransformation. The last day each team makes an oral presentation, explaining the results obtained and responds to a series of key questions posed by the teachers, which determine the final mark. In our experience, this course enables undergraduate students to become acquainted with the principles of biocatalysis as well as with standard and modern techniques, through a simple and robust laboratory exercise, using a biological system for the conversion of valuable pharmaceutical moieties.Fil: De Luca, Belén M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gonzalez, Rodrigo Horacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Blue-light-dependent inhibition of twitching motility in Acinetobacter baylyi ADP1: additive involvement of three BLUF-domain-containing proteins

Twitching motility in Acinetobacter baylyi ADP1 is inhibited by moderate intensities of blue light in a temperature-dependent manner (maximally at 20 °C). We analysed the involvement of four predicted blue-light sensing using flavin (BLUF)-domain-containing proteins encoded in the genome of this strain in the twitching motility phenotype. All four genes were expressed both in light and in darkness. A phylogenetic tree showed that one BLUF domain, ACIAD2110, grouped separately from the other three (ACIAD1499, ACIAD2125 and ACIAD2129). Individual knockout mutants of the latter three, but not of ACIAD2110, fully abolished the light dependency of the twitching motility response. Quantitative analysis of transcript level of the three genes showed a decreased expression in the light, with dark/light ratios of 1.65±0.28, 1.79±0.21 and 2.69±0.39, for ACIAD2125, ACIAD2129 and ACIAD1499, respectively. Double and triple knockouts of ACIAD1499, ACIAD2125 and ACIAD2129 confirmed the same phenotype as the corresponding single knockouts. Complementation of all the single knockouts and the triple knockout mutants with any of the three BLUF-domain-encoding genes fully restored the inhibition of twitching motility by blue light that is observed in the wild-type strain. A. baylyi ADP1 therefore shows a high degree of redundancy in the genes that encode BLUF-containing photoreceptors. Moreover, all plasmid-complemented strains, expressing any of the BLUF proteins irrespective of the specific set of deleted photoreceptors, displayed increased light-dependent inhibition of twitching motility, as compared to the wild-type (P<0.001). We conclude that the three genes ACIAD1499, ACIAD2125 and ACIAD2129 are jointly required to inhibit twitching motility under moderate blue-light illumination.Fil: Bitrian, Mariana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gonzalez, Rodrigo Horacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Paris, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Hellingwerf, Klaas. University Of Amsterdam; Países BajosFil: Nudel, Berta Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

La captación de colesterol por Tetrahymena thermophila se realiza principalmente por fagocitosis

The free-living ciliate Tetrahymena thermophila is a unicellular model organism in which landmark biological processes have been discovered, such as the first description of telomerase activity and the molecular structure of telomeres, the mechanism of self-splicing RNA and ribozymes, the function of histone acetylation in transcription regulation and a number of pioneer experiments on the interference (RNAi) mechanism for programmed genome rearrangements, among others...Fil: Elguero, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Sánchez Granel, María Luz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Montes, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Cid, Nicolás Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Favale, Nicolas Octavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

Phylogenomic analysis of integral diiron membrane histidine motif-containing enzymes in ciliates provides insights into their function and evolutionary relationships

The Integral Membrane Histidine Motif-containing Enzymes (IMHME) are a class of binuclear non-heme iron proteins widely distributed among prokaryotes and eukaryotes. They are characterized by a conserved tripartite motif consisting of eight to ten histidine residues. Their known function is the activation of the dioxygen moiety to serve as efficient catalysts for reactions of hydroxylation, desaturation or reduction. To date most studies on IMHME were carried out in metazoan, phototrophic or parasitic organisms, whereas genome-wide analysis in heterotrophic free living protozoa, such as the Ciliophora phylum, has not been undertaken. In the seven fully sequenced genomes available we retrieved 118 putative sequences of the IMHME type, albeit with large differences in number among the ciliates: 11 sequences in Euplotes octocarinatus, 7 in Ichthyophthirius multifiliis, 13 in Oxytricha trifallax, 18 in Stylonychia lemnae, 25 in Tetrahymena thermophila, 31 in Paramecium tetraurelia and 13 in Pseudocohnilembus persalinus. The pool of putative sequences was classified in 16 orthologous groups from which 11 were related to fatty acid desaturase (FAD) and 5 to the fatty acid hydroxylase (FAH) superfamilies. Noteworthy, a large diversity on the number and type of FAD / FAH proteins were found among the ciliates, a feature that, in principle, may be attributed to peculiarities of the evolutionary process, such as gene expansion and reduction, but also to horizontal gene transfer, as we demonstrate in this work. We identified twelve putative enzymatic activities, from which four were newly assigned activities: sphingolipid Δ4-desaturase, ω3/Δ15 fatty acid desaturase, a large group of alkane 1-monooxygenases, and acylamide-delta-3(E)-desaturase, although unequivocal allocation would require additional experiments. We also combined the phylogenetics analysis with lipids analysis, thereby allowing the detection of two enzymatic activities not previously reported: a C-5 sterol desaturase in P. tetraurelia and a delta-9 fatty acid desaturase in Cohnilembus reniformis. The analysis revealed a significant lower number of FAD´s sequences in the spirotrichea ciliates than in the oligohymenophorea, emphasizing the importance of fatty acids trophic transfer among aquatic organisms as a source of variation in metabolic activity, individual and population growth rates, and reproduction.Fil: Cid, Nicolás Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Sánchez Granel, María Luz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Montes, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Elguero, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

The cytochrome b(5) dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae.

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes.Fil: Poklépovich Caride, Tomás Javier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rinaldi, Mauro A.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Tomazic, Mariela Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Favale, Nicolas Octavio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Turkewitz, Aaron P. University of Chicago. Department of Molecular Genetics and Cell Biology; Estados UnidosFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentin

Genome-wide Transcriptional Analysis of Tetrahymena thermophila Response to Exogenous Cholesterol

The ciliate Tetrahymena thermophila does not require sterols for growth and synthesizes pentacyclic triterpenoid alcohols, mainly tetrahymanol, as sterol surrogates. However, when sterols are present in the environment, T. thermophila efficiently incorporates and modifies them. These modifications consist of desaturation reactions at positions C5(6), C7(8), and C22(23), and de-ethylation at C24 of 29-carbon sterols (i.e. phytosterols). Three out of four of the enzymes involved in the sterol modification pathway have been previously identified. However, identification of the sterol C22 desaturase remained elusive, as did other basic aspects of this metabolism. To get more insights into this peculiar metabolism, we here perform a whole transcriptome analysis of T. thermophila in response to exogenous cholesterol. We found 356 T. thermophila genes to be differentially expressed after supplementation with cholesterol for 2 h. Among those that were upregulated, we found two genes belonging to the long spacing family of desaturases that we tentatively identified by RNAi analysis as sterol C22 desaturases. Additionally, we determined that the inhibition of tetrahymanol synthesis after supplementation with cholesterol occurs by a transcriptional downregulation of genes involved in squalene synthesis and cyclization. Finally, we identified several uncharacterized genes that are likely involved in sterols transport and signaling.Fil: Najle, Sebastián Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universitat Pompeu Fabra; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Hernandez, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ocaña Pallarès, Eduard. Universitat Pompeu Fabra; España. Consejo Superior de Investigaciones Científicas; EspañaFil: García Siburu, Nicolás Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Slamovits, Claudio H.. Dalhousie University Halifax; CanadáFil: Uttaro, Antonio Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Photoreceptors in Chemotrophic Prokaryotes: The Case of Acinetobacter spp. Revisited

A comprehensive description of blue light using flavin (BLUF) photosensory proteins, including preferred domain architectures and the molecular mechanism of their light activation and signal generation, among chemotrophic prokaryotes is presented. Light‐regulated physiological responses in Acinetobacter spp. from environmental and clinically relevant strains are discussed. The twitching motility response in A. baylyi sp. ADP1 and the joint involvement of three of the four putative BLUF‐domain‐containing proteins in this response, in this species, is presented as an example of remarkable photoreceptor redundancy.Fil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Hellingwerf, Klaas J.. University of Amsterdam; Países Bajo

Biotechnology in ciliates: an overview

Since their description and classification in the 19th century, ciliates have played an important role in science, leading to several fundamental discoveries in the areas of cellular and molecular biology. During the last decades, with the emergence of biotechnology, many new developments are also coming to light. In this review, we describe a range of applications in which ciliates have found a niche, ranging from the production of a vast array of proteins, lipids, metabolites, and antigens to their use in toxicity screening, biocontrol, bioremediation, and biotransformation of substrates into more valuable products. We highlight the benefits and drawbacks of their use in biotechnology, the latest developments in large-scale culture and state-of-the-art molecular-genetic techniques, as well as the estimations on the exploitation areas with better potential, i.e., the production of complex membrane proteins, and those less interesting or with less chances of success.Fil: Elguero, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

Optimization by factorial analysis of caprylic acid precipitation of non-immunoglobulins from hiperimmune equine plasma for antivenom preparation

Optimization of caprylic acid precipitation of equine plasma non-immunoglobulin proteins for antivenom preparation was achieved by regression analysis of the responses of three highly significant factors assayed by factorial design. The factors studied were caprylic acid concentration, plasma pH and temperature, and their response was assessed in terms of filtration speed, residual albumin, total protein content and turbidity. The results evidenced that the three variables are involved in the precipitation process. Moreover, the factors displayed significant interactions, indicating that their levels distinctly affect the optimization procedure. The best combination was 3% caprylic acid, 37 °C and plasma pH 4.9; under these conditions, all immunoglobulins and only 0.1% albumin remained in the supernatant, in a very fast and simple procedure. After formulation, the antivenom obtained by this procedure presented full lethality neutralizing activity and absence of protein aggregates.Fil: Nudel, Berta Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Microbiologia,inmunologia y Biotecnolog.. Catedra de Inmunologia; ArgentinaFil: Perdoménico, Carolina. Ministerio de Salud de la Nación. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Doctor Carlos G. Malbrán". Instituto Nacional de Producción de Biológicos; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentina. Ministerio de Salud de la Nación. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Doctor Carlos G. Malbrán". Instituto Nacional de Producción de Biológicos; ArgentinaFil: Cascone, Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Microbiologia,inmunologia y Biotecnolog.. Catedra de Inmunologia; Argentina. Ministerio de Salud de la Nación. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Doctor Carlos G. Malbrán". Instituto Nacional de Producción de Biológicos; Argentin