Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Investigação do papel modulador do Haloperidol sobre os níveis de proteínas relacionadas à plasticidade e preferência por objetos novos em camudongos

Dopamine (DA) is known to regulate both sleep and memory formations, while sleep plays a critical role in the consolidation of different types of memories. We believe that pharmacological manipulation of dopaminergic pathways might disrupt the sleep-wake cycle, leading to mnemonic deficits, which can be observed in both behavioral and molecular levels. Therefore, here we investigated how systemic injections of haloperidol (0.3 mg/kg), immediately after training in dark and light periods, affects learning assessed in the novel object preference test (NOPT) in mice. We also investigated the hippocampal levels of the plasticity-related proteins Zif-268, brain-derived neurotrophic factor (BDNF) and phosphorylated Ca2+/calmodulin-dependent protein kinases II (CaMKII-P) in non-exposed (naïve), vehicle-injected controls and haloperidol-treated mice at 3, 6 and 12 hours after training in the light period. Haloperidol administration during the light period led to a subsequent impairment in the NOPT. In contrast, preference was not observed during the dark period neither in mice injected with haloperidol, nor in vehicle-injected animals. A partial increase of CaMKII-P in the hippocampal field CA3 of vehicle-injected mice was detected at 3h. Haloperidol-treated mice showed a significant decrease in the dentate gyrus of CaMKII-P levels at 3, 6 and 12h; of Zif-268 levels at 6h, and of BDNF levels at 12h after training. Since the mnemonic effects of haloperidol were only observed in the light period when animals tend to sleep, we suggest that these effects are related to REM sleep disruption after haloperidol injection

Biodegradação de hidrocarbonetos aromáticos policíclicos: prospecção metagenômica e modelagem computacional 3-D de proteínas

Knowledge of the native prokaryotes in hazardous locations favors the application of biotechnology for bioremediation. Independent strategies for cultivation and metagenomics contribute to further microbiological knowledge, enabling studies with non-cultivable about the "native microbiological status and its potential role in bioremediation, for example, of polycyclic aromatic hydrocarbons (HPA's). Considering the biome mangrove interface fragile and critical bordering the ocean, this study characterizes the native microbiota mangrove potential biodegradability of HPA's using a biomarker for molecular detection and assessment of bacterial diversity by PCR in areas under the influence of oil companies in the Basin Petroleum Geology Potiguar (BPP). We chose PcaF, a metabolic enzyme, to be the molecular biomarker in a PCR-DGGE detection of prokaryotes that degrade HPA s. The PCR-DGGE fingerprints obtained from Paracuru-CE, Fortim-CE and Areia Branca-RN samples revealed the occurrence of fluctuations of microbial communities according to the sampling periods and in response to the impact of oil. In the analysis of microbial communities interference of the oil industry, in Areia Branca-RN and Paracuru-CE was observed that oil is a determinant of microbial diversity. Fortim-CE probably has no direct influence with the oil activity. In order to obtain data for better understanding the transport and biodegradation of HPA's, there were conducted in silico studies with modeling and simulation from obtaining 3-D models of proteins involved in the degradation of phenanthrene in the transport of HPA's and also getting the 3-D model of the enzyme PcaF used as molecular marker in this study. Were realized docking studies with substrates and products to a better understanding about the transport mechanism and catalysis of HPA s

Proteínas SAG1, SAG2, SAG3 de toxoplasma gondii como perspectivas para o desenvolvimento de protótipo vacinal contra a toxoplasmose

Toxoplasmosis is a zoonosis of worldwide distribution caused by the protozoan Toxoplasma gondii, triggering dangerous complications in immunocompromised patients and pregnant women, as well as having great economic impact for the livestock. So far the control of toxoplasmosis is made primarily by chemotherapy. However, most drugs used routinely have some limitations. In order to control this disease, several research groups, including ours, has been working to develop a medical-veterinary vaccine based on parasite antigens, vectors and protocols of immunization. In this study were implemented and standardized methodologies for amplification and cloning of recombinant immunogens in the system for the development of a prototype vaccine, based on the surface antigens of T. gondii and recombinant adenovirus encoding these antigens. Genes encoding BAG1, GRA2 and SAG1 proteins were amplified. We established a strategy for cloning SAG1, SAG2, SAG3 and TgAMA1- genes in recombinant system. The genes encoding SAG1 and SAG2 were cloned and their sequences showed high similarity with sequences from GenBank. The virtual translation of these proteins showed polymorphisms in the amino acid sequence, which can be correlated with levels of antigenicity. Simultaneously, the adenovirus encoding the SAGs (HAdSAGs) were expanded, purificated and characterizated. Immunization of C57bl/6 mice, using viral supernatant was not enought to elicit immune responses at high levels, being required HAdSAGs titration for future immunizations. Therefore, this study allowed the cloning of the two genes important for the development of a prototype vaccine. Besides, implementations methodologies that permit advancements in the development of a vaccine against toxoplasmosis using adenovirus to express proteins of the parasite

Extração, caracterização e atividades biológicas de proteínas da espécie cnidoscolus urens (L.) Arthur

The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocaseína and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The Aα chain and Bβ of fibrinogen were completely cleaved at a concentration of 0.18 μg/μL of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60ºC. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory

Perfil eletroforético de proteínas séricas do sangue do cordão umbilical de cães

A importância do estudo do sangue do cordão umbilical (SCU) tem sido verificada pela presença de células-tronco hematopoéticas no SCU humano. O modelo experimental em cão tem propiciado informações importantes nos transplantes em humanos. Entretanto, apesar da importância do SCU e do modelo experimental em cães, não existem estudos sobre a eletroforese de proteínas séricas do cordão umbilical canino. No presente protocolo experimental, foi feita a colheita de SCU de 20 neonatos caninos, o qual foi utilizado para o fracionamento eletroforético de suas proteínas. Os valores médios absolutos obtidos para proteínas séricas totais, albumina, alfa, beta e gamaglobulinas no sangue do cordão umbilical de cães ao nascimento foram 3,09±0,59; 1,51±0,38; 0,87±0,17; 0,68±0,12 e 0,03±0,01, respectivamente. Todos os resultados apresentaram-se abaixo dos níveis reportados para animais adultos, devido à passagem das proteínas e suas frações ocorrerem principalmente através do colostro e pela imaturidade hepática. em todos os traçados eletroforéticos do SCU de cães, foi observada uma pequena curva entre alfaglobulina 2 e betaglobulina 1, não relatada no soro de cães adultos. Portanto, neste experimento, foram observadas diferenças quantitativas no traçado eletroforético das proteínas séricas do SCU de cães, ao nascimento, diferindo-os, neste particular, de cães em diferentes fases da vida pós-colostral.

Hemograma e proteínas do soro sanguíneo de bezerros Canchim-Nelore e da raça Holandesa nos primeiros 30 dias de vida

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Leucograma e teores plasmáticos de proteínas de fase aguda de eqüinos portadores de abdômen agudo e submetidos à laparotomia

Foram examinados 20 eqüinos adultos portadores de abdômen agudo e submetidos à laparotomia. Dez recuperaram-se sem intercorrência pós-operatória (G1) e 10 foram a óbito sete a 10 dias após a cirurgia, com sinais de choque séptico (G2). Avaliaram-se temperatura retal, freqüências cardíaca e respiratória, tempo de preenchimento capilar e teores plasmáticos das proteínas de fase aguda - fibrinogênio, ceruloplasmina, proteína C-reativa, antitripsina, haptoglobina e glicoproteína ácida -, antes e até sete dias após a laparotomia. As leucometrias às 72h e no sétimo dia pós-operatório dos eqüinos que foram a óbito foram, respectivamente, 34,6% e 57,1%, mais altas que a dos animais curados. Os maiores valores de proteína de fase aguda ocorreram no sétimo dia após a cirurgia; os percentuais de elevação de fibrinogênio, antitripsina, glicoproteina ácida, proteína C-reativa, ceruloplasmina e haptoglobina de eqüinos do G2 em relação ao G1 foram 46,8%, 67,9%, 91,9%, 112,2%, 126,9% e 186,2%, respectivamente.

Proteínas séricas de potros da raça Puro Sangue Árabe recém-desmamados ou com mais de trinta dias de desmame

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteínas ligantes do insulin-like growth factor (IGFBPs) e dominância folicular em vacas Bos taurus indicus puras e cruzadas

O objetivo deste estudo foi identificar um marcador bioquímico de folículos bovinos com diâmetro maior do que 10 mm, o qual poderia ser utilizado para identificar folículos dominantes nesta espécie. Líquido folicular era aspirado de folículos com diâmetros menores do que 5 mm, entre 5 e 10 mm e maiores do que 10 mm, provenientes de ovários de 37 fêmeas bovinas abatidas. Após aspiração, o líquido folicular (LF) era acondicionado em ependorfs etiquetados e congelados. Os padrões eletroforéticos em SDS-PAGE das proteínas do LF foram determinados e verificou-se que os folículos com diâmetro maior do que 10 mm apresentaram um polipeptídeo com PM entre 39 e 43 KDa identificado como a proteína ligante de IGF-3 (IGFBP-3), o qual não foi observado em folículos com diâmetro menor do que 5 mm e entre 5 e 10 mm. Este estudo sugere que este polipeptídeo possa ser utilizado como marcador bioquímico de folículos maiores do que 10 mm.

Utilização de subprodutos de origem animal em dietas para frangos de corte com base no conceito de proteínas bruta e ideal, no período de 43 a 49 dias de idade

Foi conduzido um experimento para avaliar a utilização de subprodutos de origem animal em rações para frangos de corte, formuladas com base nas proteínas bruta e ideal no período de 43 a 49 dias de idade. Foram utilizados 600 frangos machos distribuídos em arranjo fatorial 2x2+1, com duas fontes de proteína de origem animal (farinha de vísceras de aves e farinha de sangue bovino), dois conceitos de formulação (proteínas bruta e ideal) e uma ração testemunha à base de milho e de farelo de soja, com quatro repetições cada. As características avaliadas foram ganho de peso, consumo de ração, conversão alimentar, rendimento de carcaça, peito e porcentagem de gordura abdominal. Os melhores ganho em peso e conversão alimentar foram obtidos quando a dieta à base de milho e farelo de soja foi utilizada. As farinhas de vísceras e de sangue e os conceitos de formulação não exerceram influência sobre as características avaliadas.

Composição química, fracionamento de carboidratos e proteínas e digestibilidade in vitro de forrageiras tropicais em diferentes idades de corte

Objetivou-se avaliar o valor nutritivo de três espécies forrageiras tropicais: capim-tanzânia (Panicum maximum Jacq.), capim-marandu (Brachiaria brizantha) e capim-tifton 85 (Cynodon spp), em duas épocas do ano (janeiro-março e abril-junho) e em três idades de rebrota (28, 35 e 42 dias), por meio da composição química, do fracionamento de proteínas e carboidratos e da digestibilidade in vitro da matéria seca (DIVMS) e da matéria orgânica (DIVMO). O capim-marandu destacou-se no período de janeiro-março, com menores conteúdos de parede celular e fração B2 dos carboidratos e maiores valores de proteína bruta, fração A + B1, DIVMS e DIVMO, em comparação aos capins tanzânia e tifton 85, independentemente da idade de corte. O aumento da concentração de parede celular em detrimento ao conteúdo celular com o avanço da maturidade das plantas foi evidente no capim-marandu no período de janeiro-março, quando foram observados maior valor da fração B2, maior conteúdo de fibra em detergente neutro (FDN) e menor concentração da fração carboidratos não-fibrosos. No período de abril-junho, a composição em parede celular não apresentou diferenças evidentes com aumento da idade, devido às condições ambientais observadas. O capim-tanzânia apresenta, de modo geral, baixos valores de parede celular e altos valores de carboidratos não-fibrosos, DIVMS e DIVMO nesse período, seguido pelos capins marandu e tifton 85, respectivamente.

Aplicação de técnicas de aprendizado de máquina no reconhecimento de classes estruturais de proteínas

Nowadays, classifying proteins in structural classes, which concerns the inference of patterns in their 3D conformation, is one of the most important open problems in Molecular Biology. The main reason for this is that the function of a protein is intrinsically related to its spatial conformation. However, such conformations are very difficult to be obtained experimentally in laboratory. Thus, this problem has drawn the attention of many researchers in Bioinformatics. Considering the great difference between the number of protein sequences already known and the number of three-dimensional structures determined experimentally, the demand of automated techniques for structural classification of proteins is very high. In this context, computational tools, especially Machine Learning (ML) techniques, have become essential to deal with this problem. In this work, ML techniques are used in the recognition of protein structural classes: Decision Trees, k-Nearest Neighbor, Naive Bayes, Support Vector Machine and Neural Networks. These methods have been chosen because they represent different paradigms of learning and have been widely used in the Bioinfornmatics literature. Aiming to obtain an improvment in the performance of these techniques (individual classifiers), homogeneous (Bagging and Boosting) and heterogeneous (Voting, Stacking and StackingC) multiclassification systems are used. Moreover, since the protein database used in this work presents the problem of imbalanced classes, artificial techniques for class balance (Undersampling Random, Tomek Links, CNN, NCL and OSS) are used to minimize such a problem. In order to evaluate the ML methods, a cross-validation procedure is applied, where the accuracy of the classifiers is measured using the mean of classification error rate, on independent test sets. These means are compared, two by two, by the hypothesis test aiming to evaluate if there is, statistically, a significant difference between them. With respect to the results obtained with the individual classifiers, Support Vector Machine presented the best accuracy. In terms of the multi-classification systems (homogeneous and heterogeneous), they showed, in general, a superior or similar performance when compared to the one achieved by the individual classifiers used - especially Boosting with Decision Tree and the StackingC with Linear Regression as meta classifier. The Voting method, despite of its simplicity, has shown to be adequate for solving the problem presented in this work. The techniques for class balance, on the other hand, have not produced a significant improvement in the global classification error. Nevertheless, the use of such techniques did improve the classification error for the minority class. In this context, the NCL technique has shown to be more appropriated

Ação de tinturas e óleos essenciais de plantas medicinais sobre o crestamento bacteriano comum do feijoeiro e na produção de proteínas de indução de resistência

A exploração da atividade biológica de compostos secundários presentes nas tinturas ou em óleos essenciais de plantas podem representar, ao lado da indução de resistência, mais uma forma potencial de controle de doenças em plantas cultivadas. O presente trabalho objetivou avaliar o potencial de tinturas de Lippia alba, Lippia sidoides, Mikania glomerata, Equisetum sp. e Hedera helix e óleos essenciais de Rosmarinus officinalis e Cinnamomum zeylanicum nas atividades in vitro, in vivo e na produção de proteínas na indução de resistência, em plantas de feijão vagem cultivar Bragança. Os resultados obtidos demonstraram que as tinturas de L. alba e L. sidoides e os óleos essenciais (R. officinalis e C. zeylanicum) apresentaram atividade in vitro aos isolados de Xanthomonas axonopodis pv. phaseoli. Todas as tinturas ensaiadas apresentaram menores valores do progresso da doença (AACPD), em relação à testemunha, merecendo destaque a tintura de L. alba, que estavam correlacionadas com os maiores teores de polifenoloxidase, peroxidase e proteínas solúveis totais, evidenciando uma possível indução de resistência. Os óleos essenciais não apresentaram diferença na AACPD e nem na indução de proteínas.

Efeito dos períodos de envelhecimento na lixiviação de íons e de proteínas solúveis em sementes de milho

Este trabalho foi conduzido no Laboratório de Análise de Sementes, do Departamento de Produção Vegetal, da Universidade Estadual Paulista (Botucatu/SP), com o objetivo de avaliar os efeitos causados por diferentes períodos de envelhecimento acelerado na lixiviação de íons e de proteínas solúveis em sementes de milho. Sementes de milho do híbrido BR 3123 foram colocadas sobre tela em gerbox, contendo 40mL de água destilada, e mantidas a temperatura de 42°C por períodos de 0, 24, 48, 72, 96, 120, 144 e 166 horas. Após o envelhecimento, parte das sementes foi colocada para germinar, empregando quatro repetições para cada período, obtendo-se a porcentagem de plântulas normais no quarto dia. em outra parte realizou-se, após manutenção em copos plásticos com 75mL de H2O destilada por 24 horas à 25°C, leituras de condutividade elétrica (µS.cm-1.g-1), determinação de íons (mg.L-1.g de semente-1) e de proteínas solúveis totais (µproteina.g-1.mL-1) lixiviadas na solução. Os resultados indicaram que a elevação dos valores de condutividade elétrica e de lixiviado de proteínas totais ocorreu a partir de 72 horas de envelhecimento acelerado. A lixiviação de potássio foi mais acentuada que a dos íons cálcio, zinco, manganês, cobre, ferro e magnésio.