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

Avaliação de parâmetros de estresse oxidativo em plantas de cana-de-açúcar tratadas com peróxico de hidrogênio

The genus Saccharum belongs to Poaceae family. Sugarcane has become important monocultures in Brazil due to their products: ethanol and sugar. The production may change between different regions from Brazil. This difference is related to soil, climatic conditions and temperature that promotes oxidative stress that may induce an early flowering. The aim of this work was to identify the effects of oxidative stress. In order to analyse this, sugarcane plants were submitted to oxidative stress using hydrogen peroxide. After this treatment, the oxidative stress were analyzed Then, the plant responses were analyzed under different approaches, using morphophysiological, biochemical and molecular tools. Thus, sugarcane plants were grown under controlled conditions and until two months they were subjected first to a hydroponics condition for 24 hours in order to acclimation. After this period, these plants were submitted to oxidative stresse using 0 mM, 10 mM, 20 mM and 30 mM hydrogen peroxide during 8 hours. The histomorphometric analysis allowed us to verify that both root and leaf tissues had a structural changes as it was observed by the increased in cell volume, lignin accumulation in cell walls. Besides, this observation suggested that there was a change in redox balance. Also, it was analyzed the activity of the SOD, CAT and APX enzymes. It was observed an increase in the SOD activity in roots and it was also observed a lipid peroxidation in leaves and roots. Then, in order to identify proteins that were differently expressed in this conditions it was used the proteomic tool either by bidimensional gel or by direct sequencing using the Q-TOF EZI. The results obtained with this approach identified more than 3.000 proteins with the score ranging from 100-5000 ions. Some of the proteins identified were: light Harvesting; oxygenevolving; Thioredoxin; Ftsh-like protein Pftf precusor; Luminal-binding protein; 2 cys peroxiredoxin e Lipoxygenase. All these proteins are involved in oxidative stress response, photsynthetic pathways, and some were classified hypothetical proteins and/or unknown (30% of total). Thus, our data allows us to propose that this treatment induced an oxidative stress and the plant in response changed its physiological process, it made changes in tissue, changed the redox response in order to survival to this new condition

Avaliação do potencial de mutagenicidade e toxidade da lectina hipoglicemiante de folha de Bauhinia monandra (pata-de-vaca)

Medicinal plants have been used since antiquity to treat various human diseases. The leaves of Bauhinia monandra are widely used in Brazil as herbal remedies in the treatment of Diabetes Mellitus. From the leaves of B. monandra was purified a galactose-specific lectin, called BmoLL, which also showed a significant hypoglycemic capacity. Following the proposed rules by decree No 116 of 1996/08/08 of the Ministry of Health of Brazil, the study aimed to evaluate the potential for toxicity and mutagenicity of BmoLL from the use of tests with Escherichia coli strain CC104 (Forward mutagenesis assay) with Salmonella typhimurium strain TA (Kado test), with plasmid pBCKS (Break occurrences in plasmid DNA) and enzyme exonuclease III (Search of abasic sites). The results demonstrated that the lectin was unable to increase the frequency of reverse mutation of strains of S. typhimurium, with and without metabolic activity. However, a significant decrease in the frequency of spontaneous mutation was observed in strains of E. coli, especially in poor repair (CC104mutMmutY), indicating an antioxidant potential of the lectin. BmoLL is unable to generate genotoxic and cytotoxic damage, based on the concentrations and the tests performed

Associação da frequência alélica dos polimorfismos dos genes do sistema renina angiotensina com a força muscular e pressão arterial de idosas

Physiological changes induced by the aging process is dynamic and progressive, reducing the adaptability and independence of older people and may be influenced by genetic and environmental factors. Thus the aim of this thesis was to investigate the association between polymorphism of the ACE gene ID and the phenotypes of muscular strength and blood pressure of 62 elderly Brazilian (67.35 ± 5.66 years) during a 16-week program of supervised training. The elderly women were stratified by age, with the group 1 (G1, n = 34) <70 years and group 2 (G2 n = 28) ≥ 70 years, and in three groups by ACE, ACE-II (n = 8) ACE- DD (n = 35) and ACE-ID (n = 19). The level of muscle strength was evaluated by the method of maximum repetitions and measures of blood pressure (BP) were measured before and after training (PAPré1 and PAPós1) and before and after each training session (PAPre2 and PAPós2), in place of training. DNA samples were isolated from peripheral blood leukocytes polymorphism and insertion / deletion (ID) of the ACE gene (rs1800795) was genotyped by polymerase chain reaction (PCR) plus PCR-confirmatory. The genotype distribution of the polymorphism ID attended the prerogatives of Hardy-Weitíherg. There was variation in power levels before and after training and the age between groups (t-test) and the ACE polymorphism (ANOVA) (p <0.05). Depending on the results it was concluded that resistance training helps to reduce SBP and increased muscle strength of upper and lower limbs when considering the age and ACE polymorphism. In this study the Elderly carriers of the D allele were more reactive to changes in BP resistance training. This study was multidisciplinary project involving researchers in the areas Medical, Physical Education, Pharmacy, Nutrition, Gerontology and Statistics. This fulfilled the requirements of the multidisciplinary Graduate Program in Health Sciences

Avaliação de efeitos biológicos de antiinflamatórios derivados do ácido propiônico através de modelos experimentais em nível molecular e celular

Derivatives of propionic acid NSAIDs are irreversible inhibitors of cyclooxygenase enzyme widely used. The aim of this study was to evaluate, through different experimental models, biological effects of derivatives of propionic acid (fenoprofen, naproxen, ibuprofen and ketoprofen) in cellular and molecular level. The labeling of blood constituents with technetium-99m (99mTc) and morphological analysis of erythrocytes of blood of rats, as well as growth, survival of cultures of Escherichia coli (E. coli) and the assessment of bacterial plasmid electrophoretic profiles were models used for experimental evaluation of possible biological effects of antiinflammatory drugs. The results show that, in general, anti-inflammatory drugs evaluated were not able to alter the labeling of blood constituents with 99mTc, the morphology of red blood cells from blood of rats, as well as the growth of cultures of E. coli and the electrophoretic profile of plasmid DNA. However, naproxen appears to cytotoxic effect on bacterial cultures, plasmids and genotoxic effects in reducing the action of stannous chloride in cultures of E. coli. The use of experimental fast performance and low cost was important for assessment of biological effects, contributing to a better understanding of the properties of propionic acid derivatives studied. anti-inflammatory, blood constituents, technetium-99m, stannous chloride, Escherichia coli; DNA

Rastreamento populacional para Doença de Gaucher em Tabuleiro do Norte-CE

Background. Gaucher Disease (GD) is a hereditary lysosomal storage disorder characterized by the accumulation of glucosylceramide, mainly in the cells of the reticuloendothelial system, due to a deficiency of the enzyme acid β-glucosidase (GBA). Diagnosis is usually based on measurement of GBA activity in peripheral leukocytes. The purpose of this study was to evaluate the ability of screening for GBA and chitotriosidase activity using Dried Blood Spots on Filter Paper (DBS-FP) to identify individuals at high risk for GD in high-risk populations such as that of Tabuleiro do Norte, a small town in Northeastern Brazil. Methods. Between June 1, 2007 and May 31, 2008, 740 consented residents and descendants of traditional families from Tabuleiro do Norte were submitted to screening with DBS-FP. Subjects with GBA activity <2.19 nmol/h/mL were referred to analysis of GBA and chitotriosidase activity in peripheral leukocytes and in plasma, respectively. Subjects at highest risk for GD (GBA activity in peripheral leukocytes <5.6 nmol/h/mg protein) were submitted to molecular analysis to confirm diagnosis. Results. Screening with DBS-FP identified 135 subjects (18.2%) with GBA activity <2.19 nmol/h/mL, 131 of whom remained in the study. In 10 of these (7.6%), GBA activity in leukocytes was 2.6 5.5 nmol/h/mg protein. Subsequent molecular analysis confirmed 6 cases of heterozygosity and 4 normals for GD. Conclusion. DBS-FP assay was shown to be an effective initial GD screening strategy for high-prevalence populations in developing regions. Diagnosis could not be established from GBA activity in leukocytes alone, but required confirmation with molecular analysis

Identificação e fatores de virulência de Candida spp isoladas da cavidade bucal de transplantados renais do hospital universitário Onofre Lopes em Natal-RN

Despite Candida species are often human commensals isolated from various oral sites such as: tongue, cheek and palatal mucosa plus subgingival region, there are some properties linked to the organism commonly known as virulence factors which confer them the ability to produce disease. Oral candidiasis is one of the main oral manifestations reported in literature related to kidney transplant patients. The objectives of the present study were to identify and investigate virulence factors of yeasts isolated from the oral cavity of kidney transplant recipients admitted at the Hospital Universitário Onofre Lopes, in Natal RN. Seventy Candida species isolated from 111 kidney transplant recipients were investigated in this study. Identification of the isolates was performed by using the evidence of germ tube formation, hypertonic broth, tolerance to grow at 42°C, micromorphology and biochemical profiles. We observed a high rate of isolation of yeasts from the oral cavity of kidney transplant recipients (63.1%) being C. albicans was the most prevalent species. Oral candidiasis was diagnosed in 14.4% of transplant recipients. We evaluated virulence properties of the isolates regarding to: biofilm formation on polystyrene microplates as well as XTT reduction, adherence to acrylic resin and human buccal epithelial cells and proteinase activity. Most isolates were able to form biofilm by the method of adhesion to polystyrene. All isolates of Candida spp. remained viable during biofilm formation when analyzed by the method of XTT reduction. The number of CFU attached to the acrylic resin suggested high adherence for C. parapsilosis. C. albicans isolates showed higher median adherence to human buccal epithelial cells than non-C. albicans Candida isolates. Nevertheless, this difference was not statistically significant. C. dubliniensis showed low ability to adhere to plastic and epithelial cells and biofilm formation. Proteolytic activity was observed for all the isolates investigated, including the unique isolate of C. dubliniensis. There was a statistically significant association between proteinase production and the presence of oral candidiasis. Studies related to oral candidiasis in renal transplant recipients are limited to clinical and epidemiological data, but investigations concerning Candida spp. virulence factor for this group of individuals are still scarce. We emphasize the importance of studies related to virulence factors of yeasts isolated from this population to contribute to the knowledge of microbiological aspects of oral candidiasis

Avaliação do estado nutricional relativo ao selênio e da expressão gênica de selenoproteínas em pacientes com aterosclerose tratados com estatinas

The aim of this study was to determine the effects of the use of rosuvastatin in patients with atherosclerosis, in relation to blood parameters of selenium and selenoproteins, and also observe possible changes in gene expression of selenoproteins in these patients. The sample consisted of 27 adult and elderly patients with a clinical diagnosis of coronary artery disease undergoing angioplasty, treated at Natal Hospital Center hospital, Natal, RN. Patients were treated with rosuvastatin 10 mg/day during four months. Anthropometric variables such as body mass index (BMI) and Waist circumference (WC) were measured before and after treatment, as well as lipid profile, blood glucose and liver enzymes (AST and ALT). The diet of the patients was also analyzed using 24-hour diet recall. We analyzed the concentrations of selenium in plasma and erythrocytes, and also the activity of Glutathione Peroxidase and gene expression by Real Time PCR of selenoproteins GPx1, SelP1 and SelN1. Patients had mean age of 61.0 ± 9.4 years, 59.3% were men and 40.7% were women. After four months of treatment there was significant reduction of CA and, according to BMI, most were overweight. The intake of macronutrients, cholesterol, polyunsaturated fatty acids, monounsaturated and saturated was adequate, but the energy and fiber intake was below the recommendations. Regarding the selenium intake was observed a high prevalence of inadequacy. As expected, after treatment with rosuvastatin, a significant reduction in total cholesterol, LDL and glucose, which was not observed for HDL. Selenium concentrations in plasma and erythrocytes showed no changes, keeping within the established cutoffs. We observed a significant increase in GPx enzyme activity and mRNA expression of GPX1 and SEPN1, but not for gene SEPP1. Thus, it was found that treatment with rosuvastatin did not reduce the expression of selenoproteins. More studies are needed to clarify the effects of rosuvastatin on gene expression of selenoproteins in patients with atherosclerosis

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

Avaliação do status antioxidante, expressão gênica e polimorfismos dos genes SOD1, SOD2 e GPx1 em crianças, adolescentes e adultos jovens com diabetes tipo 1

Studies report that the pathophysiological mechanism of diabetes complications is associated with increased production of Reactive Oxygen Species (ROS)-induced by hyperglycemia and changes in the capacity the antioxidant defense system. In this sense, the aim of this study was to evaluate changes in the capacity of antioxidant defense system, by evaluating antioxidant status, gene expression and polymorphisms in the genes of GPx1, SOD1 and SOD2 in children, adolescents and young adults with type 1 diabetes. We studied 101 individuals with type 1 diabetes (T1D) and 106 normoglycemic individuals (NG) aged between 6 and 20 years. Individuals with type 1 diabetes were evaluated as a whole group and subdivided according to glycemic control in DM1G good glycemic control and DM1P poor glycemic control. Glycemic and metabolic control was evaluate by serum glucose, glycated hemoglobin, triglycerides, total cholesterol and fractions (HDL and LDL). Renal function was assessed by measurement of serum urea and creatinine and albumin-to-creatinine ratio (ACR) in spot urine. Antioxidant status was evaluate by content of reduced glutathione (GSH) in whole blood and the activity of erythrocyte enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). We also analyzed gene expression and gene polymorphisms of GPx1 (rs1050450), SOD1 (rs17881135) and SOD2 (rs4880) by the technique of real-time PCR (Taqman®). Most individuals with DM1 (70.3%) had poor glycemic control (glycated hemoglobin> 8%). Regarding the lipid profile, individuals with type 1 diabetes had significantly elevated total cholesterol (p <0.001) and LDL (p <0.000) compared to NG; for triglycerides only DM1NC group showed significant increase compared to NG. There was an increase in serum urea and RAC of individuals with DM1 compared to NG. Nine individuals with type 1 diabetes showed microalbuminuria (ACR> 30 mg / mg). There was a decrease in GSH content (p = 0.006) and increased erythrocyte GPx activity (p <0.001) and SOD (p <0.001) in DM1 group compared to NG. There was no significant difference in the expression of GPx1 (p = 0.305), SOD1 (.365) and SOD2 (0.385) between NG and DM1. The allele and genotype frequencies of the polymorphisms studied showed no statistically significant difference between the groups DM1 and NG. However, the GPx1 polymorphism showed the influence of erythrocyte enzyme activity. There was a decrease in GPx activity in individuals with type 1 diabetes who had a polymorphic variant T (p = 0.012). DM1 patients with the polymorphic variant G (AG + GG) for polymorphism of SOD2 (rs4880) showed an increase in the RAC (p <0.05). The combined data suggest that glucose control seems to be the predominant factor for the emergence of changes in lipid profile, renal function and antioxidant system, but the presence of the polymorphisms studied may partly contribute to the onset of complications

Avaliação citológica em base líquida de fármacos moduladores estrogênicos

Hormone therapy is an important tool in the treatment of breast cancer and tamoxifen represents one of the most important drugs used in this type of treatment. Recently other drugs based on the inhibition of aromatase had been developed, this enzyme is responsible for the synthesis of estrogenic esteroids from the androgenic ones. The objective of this study would be the development of a quantitative cytological model of murine estral analysis that allowed the characterization of different hormone drugs effect over vaginal epithelium. The technique of monochromatic staining with Evans blue (C.I. 23860) showed to be efficient in the qualitative and quantitative classification of the cycle. It had been observed differences in the cytological standard of animals submitted to the studied drugs; tamoxifen presented a widening of phases of lesser maturation (diestrais), while anastrozole and exemestane increased the duration of the phases of larger maturation (estrais). The data were analysed through a cubical non linear regression (spline) which allowed a better characterization of the drugs, suggesting a proper cytological profile to the antagonism of the estrogen receptor (tamoxifen), aromatase competition (anastrozole) and inhibition of the enzyme (exemestane)

Clinical evaluation of enalapril maleate and furosemide usage in dogs with degenerative myxomatous mitral valve, CHF functional class Ib

A doença degenerativa mixomatosa da válvula mitral (DDMVM) é uma cardiopatia de alta incidência na clínica médica de pequenos animais, acometendo mormente cães idosos e raças de pequeno porte. Desta forma, foi realizada uma investigação científica objetivando avaliar clinicamente a utilização dos fármacos maleato de enalapril e furosemida em cães com a referida enfermidade na classe funcional Ib da ICC, antes e após a terapêutica implantada. Para isso, utilizaram-se 16 cães portadores da valvulopatia supracitada, distribuídos em dois grupos; com o primeiro recebendo furosemida (n=8) e o segundo maleato de enalapril (n=8), durante 56 dias. Os cães foram avaliados em quatro momentos (T0, T14, T28 e T56 dias) quanto aos sinais clínicos e parâmetros hematológicos e bioquímico-séricos, que incluíram concentrações séricas da enzima conversora da angiotensina (ECA) e aldosterona, como também avaliações radiográficas, eletrocardiográficas, ecodopplercardiográficas e da pressão arterial. Os resultados quanto aos parâmetros clínicos, avaliações hematológicas e bioquímicas séricas não revelaram alterações significativas em ambos os grupos, mas reduções significativas nos valores de ECA e aldosterona no grupo que recebeu o maleato de enalapril foram identificadas. Ao exame radiográfico observou-se reduções nos valores de VHS e na variável onda Pms do eletrocardiograma em ambos os grupos, mas sem alterações nos valores da pressão arterial. Por sua vez, o ecodopplercardiograma evidenciou diminuição significativa das variáveis DIVEd/s nos grupos estudados e na FEC% nos cães que receberam somente enalapril. Portanto, a análise dos resultados encontrados indicou que a monoterapia fundamentada no maleato de enalapril apresentou melhor eficiência no controle do quadro clínico em pacientes da classe funcional Ib da ICC.

Survey of chicken abattoir for the presence of Campylobacter jejuni and Campylobacter coli

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

Molecular differentiation between Salmonella enterica subsp enterica serovar Pullorum and Salmonella enterica subsp enterica serovar Gallinarum

A S. Pullorum (SP) é muito semelhante à S. Gallinarum (SG), agentes da Pulorose e Tifo aviário, respectivamente, sendo que as duas enfermidades são responsáveis por perdas econômicas no setor avícola. SP e SG são de difícil diferenciação em procedimento laboratorial rotineiro, mas uma prova bioquímica muito utilizada na distinção das duas refere-se à capacidade de assimilar o aminoácido ornitina: SP descarboxila este aminoácido enquanto SG não. No entanto, o isolamento de cepas com comportamento bioquímico atípico, tem dificultado tal diferenciação. Um dos genes relacionados à assimilação do aminoácido ornitina, denomina-se gene speC, o qual está presente nos dois sorovares. Analisando 21 amostras de SP e 15 de SG com a utilização da PCR não foi possível realizar a diferenciação dos dois sorovares pois os fragmentos gerados eram idênticos. Posteriormente, com o uso da técnica de tratamento enzimático com a enzima de restrição Eco RI, foi possível observar que o padrão de bandas gerado em cada sorovar era diferente, mesmo quando amostras que apresentavam comportamento bioquímico atípico eram analisadas. Tal fato permitiu a padronização da técnica para ser utilizada na diferenciação entre os sorovares Pullorum e Gallinarum de maneira rápida e segura.

Soroprevalência da infecção por Trypanosoma cruzi em cães de uma área rural do Estado de Mato Grosso do Sul

Doença de Chagas é uma antropozoonose causada por Trypanosoma cruzi que tem os cães como importante reservatório da doença na América do Sul. Este trabalho teve como objetivo avaliar a ocorrência da infecção natural pelo T. cruzi em cães de uma área rural do estado de Mato Grosso do Sul, Brasil. Foram utilizados os testes de imunofluorescência indireta (IFI) e ensaio imunossorvente ligado a enzima (ELISA) em 75 cães residentes na área. Foram detectados anticorpos em 45,3% (n=34) e 24,0% (n=18) nos testes de IFI e ELISA, respectivamente. A real prevalência da infecção foi confirmada como 22,7% (n=17) pelo critério de positividade em ambos os testes. Os resultados obtidos confirmam a infecção chagásica nos cães dessa região.