Proteomic analysis of Saccharomyces cerevisiae to study the effects of red wine polyphenols on oxidative stress

Understanding the molecular mechanisms underlying the “French paradox” has contributed to a growing interest in the investigation of the biological activity of red wine polyphenols (RWP). The main goal of this research is to provide valuable information on how RWP could exert their biological action at the cellular level. So, we report a proteomic analysis of S. cerevisiae exposed to both pro-oxidant (H2O2) and antioxidant (wine) agents. Cellular proteome analysis shows that RWP modify the level of certain proteins. Under both normal conditions (Wine treatment) and oxidative stress situations (Wine + H2O2 treatment), the proteins involved in the metabolism and biosynthesis of biomolecules were down-regulated, while one ribosomal protein was up-regulated, probably performing its ribosome-independent functions, and so contributing to the stress defense system. Considering this action mechanism, we suggest that RWP may be acting as mild pro-oxidants and, therefore, exerting a hormetic effect that leads to the strengthening of cells’ antioxidant capacity.Fil: Lingua, Mariana Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Neme Tauil, Ricardo Martin. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Batthyány, Carlos. Universidad de la Republica, Facultad de Medicina, Departamento de Bioquímica, Unidad de Bioquímica y Proteómica Analítica, IPMON; UruguayFil: Wunderlin, Daniel Alberto. Universidad Nacional de Córdoba. Secretaría de Ciencia y Tecnología. Instituto Superior de Investigación, Desarrollo y Servicio de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Baroni, María Verónica. Universidad Nacional de Córdoba. Secretaría de Ciencia y Tecnología. Instituto Superior de Investigación, Desarrollo y Servicio de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentin

Insights into the genome and proteome of Sphingomonas paucimobilis strain 20006FA involved in the regulation of polycyclic aromatic hydrocarbon degradation

In order to study the mechanisms regulating the phenanthrene degradation pathway and the intermediate-metabolite accumulation in strain S. paucimobilis 20006FA, we sequenced the genome and compared the genome-based predictions to experimental proteomic analyses. Physiological studies indicated that the degradation involved the salicylate and protocatechuate pathways, reaching 56.3% after 15 days. Furthermore, the strain degraded other polycyclic aromatic hydrocarbons (PAH) such as anthracene (13.1%), dibenzothiophene (76.3%), and fluoranthene. The intermediate metabolite 1-hydroxy-2-naphthoic acid (HNA) accumulated during phenanthrene catabolism and inhibited both bacterial growth and phenanthrene degradation, but exogenous-HNA addition did not affect further degradation. Genomic analysis predicted 126 putative genes encoding enzymes for all the steps of phenanthrene degradation, which loci could also participate in the metabolism of other PAH. Proteomic analysis identified enzymes involved in 19 of the 23 steps needed for the transformation of phenanthrene to trichloroacetic-acid intermediates that were upregulated in phenanthrene cultures relative to the levels in glucose cultures. Moreover, the protein-induction pattern was temporal, varying between 24 and 96 h during phenanthrene degradation, with most catabolic proteins being overexpressed at 96 h—e. g., the biphenyl dioxygenase and a multispecies (2Fe–2S)-binding protein. These results provided the first clues about regulation of expression of phenanthrene degradative enzymes in strain 20006FA and enabled an elucidation of the metabolic pathway utilized by the bacterium. To our knowledge the present work represents the first investigation of genomic, proteomic, and physiological studies of a PAH-degrading Sphingomonas strain.Fil: Macchi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Martínez, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Neme Tauil, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Valacco, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Morelli, Irma Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Coppotelli, Bibiana Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin

Investigation of proteins in samples of a mid-18th century colonial mural painting by MALDI-TOF/MS and LC-ESI/MS (Orbitrap)

In this paper, we report the study of proteinaceous binders in samples from a South American Colonial 18th century painting using two complementary mass spectrometry techniques. Before restoration, seven micro-samples were extracted from representative colors of one of the mural paintings located inside the church of Our Lady of Copacabana de Andamarca built in 1723 in Bolivia. Previous analysis by gas chromatography (GC) of the amino acid derivatives of the extracted protein fraction of three of the samples, suggested the presence of animal glue and egg. In this work, we introduce a methodology that combines a protein extraction procedure with a typical treatment for analysis of proteins as tryptic peptides by mass spectrometry. For this study, we applied in the first stage, MALDI-TOF/MS and then, LC-ESI/MS (Orbitrap), an ultrahigh resolution mass spectrometry, to achieve more reliability in the identification of the protein binders. By LC-ESI/MS (Orbitrap), we detected several peptides from egg white proteins, particularly ovalbumin, ovotransferrin and lysozyme, and egg yolk proteins, vitellogenin-2 and apolipoprotein B, with high confidence peptides each. In all the samples, the presence of collagen from animal provenance was established by MALDI-TOF/MS and LC-ESI/MS (Orbitrap). To our knowledge, this is the first time that protein binders in Andean paintings are identified without ambiguity by MALDI-TOF/MS and LC-ESI/MS (Orbitrap) mass spectrometry using a proteomic approach.Fil: Levy, Ivana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Neme Tauil, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Valacco, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Moreno, Silvia Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Siracusano, Gabriela Silvana. Universidad Nacional de Tres de Febrero; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maier, Marta Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad Nacional de Tres de Febrero; Argentin

Assigning ecological roles to the populations belonging to a phenanthrene-degrading bacterial consortium using omic approaches.

The present study describes the behavior of a natural phenanthrene-degrading consortium (CON), a synthetic consortium (constructed with isolated strains from CON) and an isolated strain form CON (Sphingobium sp. AM) in phenanthrene cultures to understand the interactions among the microorganisms present in the natural consortium during phenanthrene degradation as a sole carbon and energy source in liquid cultures. In the contaminant degradation assay, the defined consortium not only achieved a major phenanthrene degradation percentage (> 95%) but also showed a more efficient elimination of the intermediate metabolite. The opposite behavior occurred in the CON culture where the lowest phenanthrene degradation and the highest HNA accumulation were observed, which suggests the presence of positive and also negative interaction in CON. To consider the uncultured bacteria present in CON, a metagenomic library was constructed with total CON DNA. One of the resulting scaffolds (S1P3) was affiliated with the Betaproteobacteria class and resulted in a significant similarity with a genome fragment from Burkholderia sp. HB1 chromosome 1. A complete gene cluster, which is related to one of the lower pathways (meta-cleavage of catechol) involved in PAH degradation (ORF 31-43), mobile genetic elements and associated proteins, was found. These results suggest the presence of at least one other microorganism in CON besides Sphingobium sp. AM, which is capable of degrading PAH through the meta-cleavage pathway. Burkholderiales order was further found, along with Sphingomonadales order, by a metaproteomic approach, which indicated that both orders were metabolically active in CON. Our results show the presence of negative interactions between bacterial populations found in a natural consortium selected by enrichment techniques; moreover, the synthetic syntrophic processing chain with only one microorganism with the capability of degrading phenanthrene was more efficient in contaminant and intermediate metabolite degradation than a generalist strain (Sphingobium sp. AM)

Finding of muscle proteins in art samples from mid-18th century murals by LC-MSMS

In this work, we studied protein binders in micro-samples from mural paintings from two 18th century Colonial Andean churches located in the northernmost region of Chile using a proteomic approach. The protein fraction of the samples was extracted with ammonia following a previously optimized methodology and analyzed by LC–MSMS. We identified collagen as well as egg white and yolk proteins, in accordance with a tempera painting technique. In addition, we detected for the first time the presence of muscle proteins in two of the micro-samples. The collagen and muscle proteins could be ascribed to bovine (Bos taurus) and llama (Vicugna pacos), a domesticated camelid used as food source and for transportation of goods in the Andes since pre-Hispanic times. These findings provide new aspects on the knowledge of old animal glues composition and painting practices in the Andean region with reliable information for restoration and conservation work.Fil: Levy, Ivana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Neme Tauil, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Rosso, Ana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Valacco, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Moreno, Silvia Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Guzmán, Fernando. Universidad Adolfo Ibañez; ChileFil: Siracusano, Gabriela Silvana. Universidad Nacional de Tres de Febrero. Instituto de Investigaciones en Arte y Cultura "Dr. Norberto Griffa"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maier, Marta Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad Nacional de Tres de Febrero. Instituto de Investigaciones en Arte y Cultura "Dr. Norberto Griffa"; Argentin

Bacterial counts in CON and SC cultures in phenanthrene-supplemented cultures.

<p>Heterotrophic viable bacteria (A) and PAH-degrading bacteria (B) counts in CON and SC cultures growing in LMM with phenanthrene as a sole carbon and energy source during 15 days of incubation. Yellow colony (YC) counts in CON and SC during phenanthrene degradation (C). The results are the means of triplicate independent experiments. The bars represent standard deviations.</p

Mascot results of identified and non-identified proteins found in 2-DE electrophoresis gels of CON cultures.

<p>Summary of the Mascot search results of identified (shaded grey) (high score and/or high sequence coverage) and non-identified proteins (low score) found in 2-DE electrophoresis gels of CON cultures after 4 and 15 days of incubation and analyzed by MALDI TOF/TOF MS/MS. The “x” indicates proteins present in each condition. The lower the expectation value (e-value), the more significant is the score.</p

2-DE electrophoresis gels of the soluble fractions of proteins extracted from CON after 4 and 15 days of incubation.

<p>The marked spots were analyzed by MALDI TOF/TOF MS/MS. The identified spots are shown in red, and the ones with a low score shown in green were unidentified. The numbers match with the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184505#pone.0184505.t002" target="_blank">Table 2</a>.</p

Metabolic reactions that could involve proteins codified in the ORFs found in S1P3.

<p>(1) The upper degradation pathway of aromatic compounds: A1 to A6, which were initial dioxygenase, dehydrogenase, extradiol dioxygenase, isomerase, hydratase/aldolase and aldehyde dehydrogenase, respectively. (2) The lower degradation pathway (meta-cleavage of catechol): B1 to B8, which were catechol 2, 3-dioxygenase, hydroxymuconic- semialdehyde dehydrogenase, hydroxymuconic-semialdehyde hydrolase, 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-keto-4-pentenoate hydratase, 2-oxo-4-hydroxypentenoate aldolase and acetaldehyde dehydrogenase, respectively.</p

Dioxygenase enzymes codified in the genome of <i>Sphingobium</i> sp. AM strain and its putative substrates.

<p>Dioxygenase enzymes codified in the genome of <i>Sphingobium</i> sp. AM strain and its putative substrates.</p