Navegando por Autor "Baracat-Pereira, Maria Cristina"
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Item Acyl homoserine lactone changes the abundance of proteins and the levels of organic acids associated with stationary phase in Salmonella Enteritidis(Microbial Pathogenesis, 2016-12-01) Almeida, Felipe Alvesde; Pimentel-Filho, Natan de Jesus; Carrijo, Lanna Clícia; Bento, Claudia Braga Pereira; Baracat-Pereira, Maria Cristina; Pinto, Uelinton Manoel; Oliveira, Leandro Licursi de; Vanetti, Maria Cristina DantasQuorum sensing (QS) is cell-cell communication mechanism mediated by signaling molecules known as autoinducers (AIs) that lead to differential gene expression. Salmonella is unable to synthesize the AI-1 acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. Our study aimed to evaluate the influence of AI-1 on the abundance of proteins and the levels of organic acids of Salmonella Enteritidis. The presence of N-dodecyl-homoserine lactone (C12-HSL) did not interfere on the growth or the total amount of extracted proteins of Salmonella. However, the abundance of the proteins PheT, HtpG, PtsI, Adi, TalB, PmgI (or GpmI), Eno, and PykF enhanced while the abundance of the proteins RplB, RplE, RpsB, Tsf, OmpA, OmpC, OmpD, and GapA decreased when Salmonella Enteritidis was anaerobically cultivated in the presence of C12-HSL. Additionally, the bacterium produced less succinic, lactic, and acetic acids in the presence of C12-HSL. However, the concentration of extracellular formic acid reached 20.46 mM after 24 h and was not detected when the growth was in the absence of AI-1. Considering the cultivation period for protein extraction, their abundance, process and function, as well as the levels of organic acids, we observed in cells cultivated in presence of C12-HSL a correlation with what is described in the literature as entry into the stationary phase of growth, mainly related to nitrogen and amino acid starvation and acid stress. Further studies are needed in order to determine the specific role of the differentially abundant proteins and extracellular organic acids secreted by Salmonella in the presence of quorum sensing signaling molecules.Item Anti-hypertensive peptides derived from caseins: mechanism of physiological action, production bioprocesses, and challenges for food applications(Applied Biochemistry and Biotechnology, 2018-01-03) Oliveira, Mara Rose de; Silva, Thaís Jordânia; Barros, Edvaldo; Guimarães, Valéria Monteze; Baracat-Pereira, Maria Cristina; Eller, Monique Renon; Coimbra, Jane Sélia dos Reis; Oliveira, Eduardo Basílio deThis review is focused on the state-of-art of peptides with inhibitory activity towards angiotensin I-converting enzyme (ACE) — thus, with anti-hypertensive potential — derived from enzymatic hydrolysis of caseins. Firstly, molecular characteristics of caseins relevant to a better understanding of this subject were concisely commented. Next, a brief description of the pathophysiology of hypertension was explained, focusing on the ACE role in regulation of blood pressure in human body. Then, casein-derived peptides with ACE inhibitory capacity were specifically addressed. The main in vitro and in vivo bioassays often reported in literature to assess the anti-hypertensive potential of peptides were presented, illustrated with recently published studies, and discussed in terms of advantages and limitations of both approaches. Characteristics related to amino acid composition and sequence of peptides with high ACE-inhibitory potential were also commented. Process parameters of enzymatic hydrolysis (types and origins of casein substrates, types of enzymes, pH, temperature, and times of reactions) were discussed. Patents dealing with casein-derived anti-hypertensive peptides were examined not only in terms of amino acid sequences, but also regarding their novelty claims in hydrolysis process parameters. Finally, some trends, challenges, and opportunities inferred from this literature analysis were commented, emphasizing the importance of this research topic in food products development.Item Atividade de enzimas associadas ao estado de indução em mudas de cacaueiro expostas a dois actinomicetos residentes de filoplano(Summa Phytopathologica, 2008-01) Romeiro, Reginaldo da Silva; Baracat-Pereira, Maria Cristina; Lanna-Filho, Roberto; Batista, Gisele Sales; Pomella, Alan Willian Vilela; Macagnan, DirceuDois antagonistas selecionados para o biocontrole da vassoura-de-bruxa do cacaueiro foram avaliados quanto à capacidade em ativar mecanismos de defesa de plantas contra patógenos. Para tanto, mudas seminais de cacaueiro "comum" foram cultivadas em casa-de-vegetação por 30 dias e expostas aos antagonistas aplicados a mudas de cacaueiro por atomização, individualmente e em associação. O primeiro par de folhas das mudas dos diferentes tratamentos foi coletado aos dois, quatro, 12 e 24 dias após a exposição aos antagonistas. Foi quantificada a atividade de peroxidases, polifenoloxidases, quitinases e b-1,3-glucanases no material coletado. Observou-se um aumento na atividade de peroxidases e polifenoloxidases nos primeiros dias após a exposição das mudas, especialmente ao isolado Ac26. Não foi observado efeito aditivo ou sinergístico nas mudas expostas aos dois isolados simultaneamente.Item Biochemical properties of soybean leaf lipoxygenases: Presence of soluble and membrane-bound forms(Plant Physiology and Biochemistry, 2001-02) Baracat-Pereira, Maria Cristina; Oliveira, Maria Goreti de Almeida; Barros, Everaldo Gonçalves de; Moreira, Maurílio Alves; Santoro, Marcelo MatosLipoxygenases (EC 1.13.11.12, LOX) extracted from soybean leaves (Glycine max [L.] Merrill cv. IAC-100) at pH 6.5 showed low stability. Given the importance of correlating the biochemical roles with the physiological characteristics of each LOX isoenzyme, this work evaluates biochemical characteristics and stability conditions of these enzymes in order to plan a purification procedure. LOX activity (A234 at pH 6.0) increased four to five times when 0.25 % (v/v) Triton X-100, 1 % (w/v) polyvinylpyrrolidone, and 1 mM phenylmethylsulfonyl fluoride were added to leaf macerates. Fe2+ (1 mM) stabilised LOX (70.3 % of activity recovered after 48 h storage). Ammonium sulphate fractionation (35–65 % saturation) increased specific LOX activity five times and stabilised the enzymes. Two optimum LOX activities were observed at pH 6.0–6.5 and 4.0–5.0, and the greater storage stability was at pH 6.5 (after 24–28 h storage at different pH values). The results suggest the presence of at least two different forms of the enzyme. The forms of LOX that are active at acidic pH are more stable than the ones that are active at neutral pH. These stable forms were extracted in absence of detergents (soluble forms), while the forms of LOX that are active at pH 6.0–6.5 are unstable forms specially extracted in presence of Triton X-100, and possibly correspond to membrane-bound proteins.Item Computer aided identification of a Hevein-like antimicrobial peptide of bell pepper leaves for biotechnological use(BioMed Central Genomics, 2016-12-15) Games, Patrícia Dias; Silva, Elói Quintas Gonçalves da; Barbosa, Meire de Oliveira; Almeida-Souza, Hebréia Oliveira; Fontes, Patrícia Pereira; Magalhães-Jr, Marcos Jorge de; Pereira, Paulo Roberto Gomes; Prates, Maura Vianna; Franco, Gloria Regina; Faria-Campos, Alessandra; Campos, Sérgio Vale Aguiar; Baracat-Pereira, Maria CristinaAntimicrobial peptides from plants present mechanisms of action that are different from those of conventional defense agents. They are under-explored but have a potential as commercial antimicrobials. Bell pepper leaves (‘Magali R’) are discarded after harvesting the fruit and are sources of bioactive peptides. This work reports the isolation by peptidomics tools, and the identification and partially characterization by computational tools of an antimicrobial peptide from bell pepper leaves, and evidences the usefulness of records and the in silico analysis for the study of plant peptides aiming biotechnological uses. Aqueous extracts from leaves were enriched in peptide by salt fractionation and ultrafiltration. An antimicrobial peptide was isolated by tandem chromatographic procedures. Mass spectrometry, automated peptide sequencing and bioinformatics tools were used alternately for identification and partial characterization of the Hevein-like peptide, named HEV-CANN. The computational tools that assisted to the identification of the peptide included BlastP, PSI-Blast, ClustalOmega, PeptideCutter, and ProtParam; conventional protein databases (DB) as Mascot, Protein-DB, GenBank-DB, RefSeq, Swiss-Prot, and UniProtKB; specific for peptides DB as Amper, APD2, CAMP, LAMPs, and PhytAMP; other tools included in ExPASy for Proteomics; The Bioactive Peptide Databases, and The Pepper Genome Database. The HEV-CANN sequence presented 40 amino acid residues, 4258.8 Da, theoretical pI-value of 8.78, and four disulfide bonds. It was stable, and it has inhibited the growth of phytopathogenic bacteria and a fungus. HEV-CANN presented a chitin-binding domain in their sequence. There was a high identity and a positive alignment of HEV-CANN sequence in various databases, but there was not a complete identity, suggesting that HEV-CANN may be produced by ribosomal synthesis, which is in accordance with its constitutive nature. Computational tools for proteomics and databases are not adjusted for short sequences, which hampered HEV-CANN identification. The adjustment of statistical tests in large databases for proteins is an alternative to promote the significant identification of peptides. The development of specific DB for plant antimicrobial peptides, with information about peptide sequences, functional genomic data, structural motifs and domains of molecules, functional domains, and peptide-biomolecule interactions are valuable and necessaryItem Concentração de proteína solúvel por Bradford releva diferenças no metabolismo de plantas de Ora-pro-nobis em diferentes doses de nitrogênio(Revista Brasileira de Agropecuária Sustentável, 2013-07-18) Guimarães, Dimitrius Santiago P. Simões Fróes; Souza, Maria Regina de Miranda; Hirano, Rafael T.; Pereira, Paulo Roberto Gomes; Baracat-Pereira, Maria CristinaO ora-pro-nobis (Pereskia aculeata Mill.) é um alimento rico em proteínas, muito utilizado no meio rural e com importância crescente na indústria de alimentos e farmacológica. O objetivo deste trabalho foi determinar a concentração de proteínas pelos métodos de Bradford e Kjeldahl, em folhas de plantas de ora-pro-nobis submetidas a diferentes doses de adubação nitrogenada, comparando estes métodos. As folhas das plantas de ora-pro-nobis adubadas com diferentes doses de N (0, 50, 100, 200 e 400 kg de N/ha) foram coletadas aos 423 dias após o plantio (DAP). Para o método de Bradford, as folhas foram trituradas com nitrogênio líquido e maceradas em Tris-HCl 50 mM, pH 7,0, o homogenato centrifugado e a proteína solúvel determinada no sobrenadante. Para avaliar o perfil proteico, as amostras dos diferentes tratamentos foram separadas por SDS-Tricina-PAGE 14%. O método de Kjeldahl tradicional foi realizado usando-se o fator de correção 6,25. Os resultados por ambos os métodos indicaram que houve alterações nas concentrações e composição de proteínas presentes em função da disponibilidade de N no solo. A proteína total por Kjeldahl aumentou até a dose de 100 kg de N/ha, e a proteína solúvel por Bradford aumentou nas doses de N entre 50 e 200 kg/ha. Pelo SDS-Tricina-PAGE, verificou-se aumento da intensidade das bandas consonante com o método de Bradford. Estes resultados sugerem que a avaliação de proteínas solúveis pelo método de Bradford permite detectar diferenças no metabolismo das plantas de ora-pro-nobis, expressando informações biológicas relevantes para estudos fisiológicos e nutricionais.Item Concentração de proteína solúvel por bradford revela diferenças no metabolismo de plantas de ora-pro-nobis em diferentes doses de nitrogênio(Revista Brasileira de Agropecuária Sustentável, 2013-07) Guimarães, Dimitrius Santiago P. Simões Fróes; Souza, Maria Regina de Miranda; Hirano, Rafael T.; Pereira, Paulo Roberto Gomes; Baracat-Pereira, Maria CristinaO ora-pro-nobis (Pereskia aculeata Mill.) é um alimento rico em proteínas, muito utilizado no meio rural e com importância crescente na indústria de alimentos e farmacológica. O objetivo deste trabalho foi determinar a concentração de proteínas pelos métodos de Bradford e Kjeldahl, em folhas de plantas de ora-pro-nobis submetidas a diferentes doses de adubação nitrogenada, comparando estes métodos. As folhas das plantas de ora-pro-nobis adubadas com diferentes doses de N (0, 50, 100, 200 e 400 kg de N/ha) foram coletadas aos 423 dias após o plantio (DAP). Para o método de Bradford, as folhas foram trituradas com nitrogênio líquido e maceradas em Tris-HCl 50 mM, pH 7,0, o homogenato centrifugado e a proteína solúvel determinada no sobrenadante. Para avaliar o perfil proteico, as amostras dos diferentes tratamentos foram separadas por SDS-Tricina-PAGE 14%. O método de Kjeldahl tradicional foi realizado usando-se o fator de correção 6,25. Os resultados por ambos os métodos indicaram que houve alterações nas concentrações e composição de proteínas presentes em função da disponibilidade de N no solo. A proteína total por Kjeldahl aumentou até a dose de 100 kg de N/ha, e a proteína solúvel por Bradford aumentou nas doses de N entre 50 e 200 kg/ha. Pelo SDS-Tricina-PAGE, verificou-se aumento da intensidade das bandas consonante com o método de Bradford. Estes resultados sugerem que a avaliação de proteínas solúveis pelo método de Bradford permite detectar diferenças no metabolismo das plantas de ora-pro-nobis, expressando informações biológicas relevantes para estudos fisiológicos e nutricionais.Item Differences in beef quality between Angus (Bos taurus taurus) and Nellore (Bos taurus indicus) cattle through a proteomic and phosphoproteomic approach(Public Library of Science One, 2017-01-19) Rodrigues, Rafael Torres de Souza; Chizzotti, Mario Luiz; Vital, Camilo Elber; Baracat-Pereira, Maria Cristina; Barros, Edvaldo; Busato, Karina Costa; Gomes, Rafael Aparecido; Ladeira, Márcio Machado; Martins, Taiane da SilvaProteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days. Proteins were extracted from Longissimus lumborum samples collected immediately after slaughter and separated by two-dimensional electrophoresis. Pro-Q Diamond stain was used in phosphoproteomics. Proteins identification was performed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Tropomyosin alpha-1 chain, troponin-T, myosin light chain-1 fragment, cytoplasmic malate dehydrogenase, alpha-enolase and 78 kDa glucose-regulated protein were more abundant in Nellore, while myosin light chain 3, prohibitin, mitochondrial stress-70 protein and heat shock 70 kDa protein 6 were more abundant in Angus (P<0.05). Nellore had higher phosphorylation of myosin regulatory light chain-2, alpha actin-1, triosephosphate isomerase and 14-3-3 protein epsilon. However, Angus had greater phosphorylation of phosphoglucomutase-1 and troponin-T (P<0.05). Therefore, proteins involved in contraction and muscle organization, myofilaments expressed in fast or slow-twitch fibers and heat shock proteins localized in mitochondria or sarcoplasmic reticulum and involved in cell flux of calcium and apoptosis might be associated with differences in beef quality between Angus and Nellore. Furthermore, prohibitin appears to be a potential biomarker of intramuscular fat in cattle. Additionally, differences in phosphorylation of myofilaments and glycolytic enzymes could be involved with differences in muscle contraction force, susceptibility to calpain, apoptosis and postmortem glycolysis, which might also be related to differences in beef quality among Angus and Nellore.Item Differential abundances of four forms of binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability(Theriogenology, 2016-02-27) Magalhães Jr, Marcos Jorge; Martins, Leonardo Franco; Senra, Renato Lima; Santos, Thaís Ferreira do; Okano, Denise Silva; Pereira, Paulo Roberto Gomes; Faria-Campos, Alessandra; Campos, Sérgio Vale Aguiar; Guimarães, José Domingos; Baracat-Pereira, Maria CristinaThe Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P < 0.05). The protein spot weighing 15.52 ± 0.53 kDa with a pI value of 5.78 ± 0.12 is highlighted by its high abundance in bulls with low semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process.Item Enzimas marcadoras de indução de resistência diferencialmente reguladas em soja resistente e suscetível à ferrugem-asiática-da-soja(Pesquisa Agropecuária Brasileira, 2012-01-09) Almeida, Hebréia Oliveira; Barbosa, Meire de Oliveira; Marques, Ana Ermelinda; Pereira, Tânus Henrique Abdalla; Magalhães Júnior, Marcos Jorge; Tessarollo, Nayara Gusmão; Games, Patrícia Dias; Barros, Everaldo Gonçalves de; Stolf-Moreira, Renata; Marcelino-Guimarães, Francismar Corrêa; Abdelnoor, Ricardo Vilela; Pereira, Paulo Roberto Gomes; Baracat-Pereira, Maria CristinaO objetivo deste trabalho foi avaliar, por meio de enzimas marcadoras, a indução de resistência à ferrugem-asiática-da-soja em genótipos de soja contrastantes quanto à suscetibilidade a Phakopsora pachyrhizi. Aproteína total e as atividades de cinco enzimas marcadoras da indução de resistência (lipoxigenases, peroxidases, fenilalanina amônia-liase, quitinases e β-1, 3-glucanases) foram avaliadas em extratos de folhas de plantas de soja dos genótipos Embrapa 48 (suscetível) e PI 561356 (resistente), submetidas à inoculação ou não com o patógeno. Foram observadas respostas de defesa discrepantes entre os dois genótipos e entre os tempos de coleta (12, 72 e 168 horas após inoculação). A resposta de indução dessas enzimas assemelha-se à defesa bifásica, para Embrapa 48, e é consistente com o observado para outros patossistemas. No entanto, o genótipo PI 561356 respondeu com diminuição da concentração de proteína total e das atividades enzimáticas, o que indica redução do metabolismo geral das plantas infectadas. Há um importante mecanismo de resistência do genótipo PI 561356, ainda não relatado, embasado em vias que envolvem essas enzimas marcadoras e em mecanismos que utilizam menor concentração de proteínas, como os de via metabólica de resposta em cascata.Item Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein(Virology, 2004-09-26) Mariano, Andrea C.; Andrade, Maxuel O.; Santos, Anésia A.; Carolino, Sonia M.B.; Oliveira, Marli L.; Baracat-Pereira, Maria Cristina; Brommonshenkel, Sergio H.; Fontes, Elizabeth P.B.Despite extensive studies in plant virus – host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N- terminal portion. The potential significance of the NSP – NIK interaction is discussed.Item Induction of systemic resistance in tomato by the autochthonous phylloplane resident Bacillus cereus(Pesquisa Agropecuária Brasileira, 2006-08) Halfeld-Vieira, Bernardo de Almeida; Vieira Júnior, José Roberto; Romeiro, Reginaldo da Silva; Silva, Harllen Sandro Alves; Baracat-Pereira, Maria CristinaThe objective of this work was to verify if the induced resistance mechanism is responsible for the capacity of a phylloplane resident bacteria (Bacillus cereus), isolated from healthy tomato plants, to control several diseases of this crop. A strain of Pseudomonas syringae pv. tomato was used as the challenging pathogen. The absence of direct antibiosis of the antagonist against the pathogen, the significant increase in peroxidases activity in tomato plants exposed to the antagonist and then inoculated with the challenging pathogen, as well as the character of the protection, are evidences wich suggest that biocontrol efficiency presented by the antagonist in previous works might be due to induced systemic resistance (ISR).Item Mineral, protein and nitrate contents in leaves of Pereskia aculeata subjected to nitrogen fertilization(Pesquisa Agropecuária Tropical, 2016-02) Pereira, Paulo Roberto Gomes; Souza, Maria Regina de Miranda; Magalhães, Ivan de Paiva Barbosa; Sediyama, Maria Aparecida Nogueira; Vidigal, Sanzio Mollica; Milagres, Cléverson Silva Ferreira; Baracat-Pereira, Maria CristinaConsidering that nitrogen is directly related to leaf protein content, the nitrogen fertilization in Pereskia aculeata plants may affect the protein content and increase its nutritional potential. This study aimed at assessing the effect of nitrogen fertilization on mineral, protein and nitrate contents, as well as the yield of P. aculeata leaves. A randomized blocks design was used, with three replications and five treatments, consisting of increasing topdressing nitrogen doses (0-400 kg ha^-1), in soil with organic matter content of 4.0 dag kg^-1. Three harvests were performed for leaf analysis. No significant effect was observed for mineral and protein content or leaf fresh mass yield. The mean values for mineral composition were: 3.52 dag kg^-1 of N, 0.47 dag kg^-1 of P, 4.65 dag kg^-1 of Ca, 0.71 dag kg^-1 of Mg, 0.25 dag kg^-1 of S, 36.64 mg kg^-1 of Zn and 174.13 mg kg^-1 of Fe. The mean content for protein was 21.86 % and the leaf fresh mass yield was 0.971 kg plant^-1. K levels decreased from 50 kg ha^-1 of N. Nitrate increased linearly with the nitrogen fertilization, reaching a maximum value of 78.2 mg kg^-1 of fresh mass, well below the health risk threshold. It was concluded that a soil with high organic matter content does not require nitrogen fertilization. However, doses up to 400 kg ha^-1 of nitrogen ensure adequate leaf yield and protein and mineral contents within the desired range for the species, being a food rich in proteins, iron and calcium.Item Plantas de pimentão submetidas à injúria mecânica modificam a expressão de proteínas em plantas vizinhas não injuriadas(Revista Brasileira de Agropecuária Sustentável, 2013-07-25) Games, Patrícia Dias; Fontes, Patrícia Pereira; Carrijo, Lanna Clicia; Magalhães Júnior, Marcos Jorge; Pereira, Paulo Roberto Gomes; Baracat-Pereira, Maria CristinaA proteômica avalia eventos biológicos por análise das proteínas expressas em células ou tecidos em situações fisiológicas diferentes. Na proteômica de plantas, visando manter padrões de cultivo, plantas tratadas e não tratadas são muitas vezes cultivadas no mesmo ambiente, sem considerar alterações por plantas tratadas sobre a expressão de proteínas de plantas não tratadas. O objetivo desse trabalho foi, por proteômica, avaliar a expressão diferencial de proteínas em folhas de plantas de pimentão (Capsicum annuum L. ́Magali R‘) não-feridas (não-tratadas) e feridas (tratadas) por injúria mecânica, quando cultivadas ou não próximas fisicamente. Aos 40 dias após o plantio, parte das plantas foi submetida à injúria por ferimentos nas folhas totalmente expandidas (feridas, F), e parte foi reservada como plantas não-feridas, ou cultivadas perto das plantas F (NFP, a 0,15 m) ou cultivadas longe das plantas F (NFL, a 10 m). As folhas das plantas NFP e NFL foram coletadas às 12, 48 e 168 h após ferimentos das plantas F, e os extratos NFL e NFP foram analisados por eletroforese bidimensional (2-DE). Os perfis proteicos foram comparados por Image Master, e as proteínas identificadas usando espectrometria de massas. Houve menor concentração de proteína em extratos de NFP 12 h; em 48 h e 168 h, a proteína foi semelhante entre NFL e NFP. Por 2-DE, não houve diferenças significativas entre NFP e NFL 12 h, porém para NFP 48 h foi observada maior expressão de proteínas envolvidas na defesa de plantas. Para NFP 168 h, houve aumento da expressão de proteínas envolvidas no metabolismo normal. Esses resultados indicam ter havido respostas de defesa das plantas NFP, decorrentes da proximidade do cultivo dessas plantas com as plantas F, possivelmente por ação de voláteis. Coloca-se assim a importância da escolha das condições de cultivo dos tratamentos-controle, não apenas para análises proteômicas.Item Proteomic analysis of Aedes aegypti midgut during post-embryonic development and of the female mosquitoes fed different diets(Parasitology International, 2016-09-03) Fernandes, Kenner Morais; Magalhães-Júnior, Marcos Jorge de; Baracat-Pereira, Maria Cristina; Martins, Gustavo FerreiraIn this work we analyzed protein expression in the Aedes aegypti midgut during the larval (fourth instar, L4), pupal, and adult stages [including newly emerged (NE), sugar-fed (SF) and blood-fed (BF) females]. Two-dimensional electrophoresis showed 13 spots in the midgut of larvae, 95 in the midgut of pupae, 90 in the midgut of NE, and 76 in the midgut of SF or BF females. In the larval midguts, high serpin expression was noted, while in the pupae, protein abundance was lower than in the NE, SF, and BF females. The spots related to proteins linked to energy production, protein metabolism, signaling, and transport were highly expressed in the NE stage, while spots related proteins involved in translation were abundant in SF and BF females. The differential abundance of proteins in the midgut of A. aegypti at different developmental stages supports the necessity for midgut development during immature stage followed by the necessity of proteins related to digestion in adults.Item Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae)(Archives of Insect Biochemistry and Physiology, 2017-10-10) Santos, Pollyanna Pereira; Games, Patricia Dias; Azevedo, Dihego Oliveira; Barros, Edvaldo; Oliveira, Leandro Licursi de; Ramos, Humberto Josué de Oliveira; Baracat-Pereira, Maria Cristina; Serrão, José EduardoThe ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two-dimensional gel electrophoresis and separation by ion-exchange and reverse-phase high-performance liquid chromatography followed by mass spectrometry using tanden matrix-assisted laser desorption/ionization with time-of-flight (MALDI-TOF/TOF) mass spectrometry and electrospray ionization-quadrupole with time-of-flight (ESI-Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10-ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.Item Separomics applied to the proteomics and peptidomics of low-abundance proteins: choice of methods and challenges - a review(Genetics and Molecular Biology, 2011-01-24) Baracat-Pereira, Maria Cristina; Barbosa, Meire de Oliveira; Magalhães Júnior, Marcos Jorge; Carrijo, Lanna Clicia; Games, Patrícia Dias; Almeida, Hebréia Oliveira; Sena Netto, José Fabiano; Pereira, Matheus Rodrigues; Barros, Everaldo Gonçalves deThe enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes.Item Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity(Microbiology, 2012-09-03) Paiva, Aline Dias; Oliveira, Michelle Dias de; Paula, Sérgio Oliveira de; Baracat-Pereira, Maria Cristina; Breukink, Eefjan; Mantovani, Hilário CuquettoBacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC50 of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC50 obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.