Avaliação da atividade inibitória In vitro de bacteriocinas extraídas de Lactobacillus spp. isolados de aves (Gallus gallus, Linnaeus, 1758)

Pós-graduação em Medicina Veterinária - FMVZ

Rhizospheric streptomycetes as potential biocontrol agents of Fusarium and Armillaria pine rot and as PGPR for Pinus taeda

Pinus taeda is one of the main timber trees in Brazil, occupying 1.8 million ha with an annual productivity of 25-30 m(3) ha(-1). Another important species is Araucaria angustifolia, belonging to the fragile Rainforest biome, which for decades has been a major source of timber in Brazil. Some diseases that affect the roots and/or the stem of these trees and cause "damping-off" of the seedlings, with economic and environmental losses for the forest sector, are caused by the plant pathogenic fungi Fusarium sp. or Armillaria sp. This research project intended to isolate actinobacteria from the Araucaria rhizosphere, which present an antagonistic effect against these fungi. After the selection of the best pathogen inhibitors, morphologic characteristics, enzyme production, and their effect on the growth of Pinus taeda were studied. The actinobacteria were tested for their antagonistic capacity against Fusarium sp. in Petri plates with PDA as substrate. The inhibition zone was measured after 3, 5, 7, and 10 days. Of all the isolates tested, only two of them maintained inhibition zones up to 4 mm for 10 days. The inhibition of Armillaria sp. was tested in liquid medium and also in Petri dishes through the evaluation of the number of the fungal rhizomorphs in dual culture with the actinobacteria. It was found that all five isolates were able to inhibit the rhizomorph production, with the best performance of the isolate A43, which was capable of inhibiting both fungi, Fusarium and Armillaria. In a greenhouse experiment, the effect of five isolates on the growth of Pinus taeda seedlings was tested. Plant height, stem diameter, root and shoot dry matter were determined. The Streptomyces isolate A43 doubled plant growth. These results may lead to the development of new technologies in the identification of still unknown bacterial metabolites and new management techniques to control forest plant diseases.

Efeito antimicrobiano e citotóxico do óleo essencial de Cymbopogon citratus sobre células odontoblastóides

The aim of this study was to evaluate the antimicrobial and cytotoxic effect of essential oil (EO) of lemon grass (Cymbopogon citratus). From the agar diffusion method, different concentrations of EO (0.135%, 0.2% and 1%), and control solutions (chlorhexidine (Chx), distilled water (Ad) and cereal alcohol (Ac)) were applied on cultures of Candida albicans (C.a), Streptococcus mutans (S.m), Streptococcus sobrinus (S.sob) and Lactobacillus acidophilus (L.a). For C.a, S.m and S.sob, the largest inhibition zones in descending order were: Chx, Ac and EO 1%, while the latter two were statistically similar (Mann-Whitney, p> 0.05). For L.a, the largest inhibition halo was observed for the Chx, followed by EO at 1%, 0.2%, 0.135% and Ac. For evaluation of cytotoxicity, the following groups were set: G1: 0,1% EO; G2: pure EO; G3 (positive control): H2 O2 ; G4: cereal alcohol; and G5 (negative control): culture medium – DMEM. The solutions were applied on the cultured MDPC-23 cells, which were plated (30,000 cells/cm2 ) in wells of 24 well-dishes. Cell metabolism was evaluated by MTT assay. Considering G5 (negative control) as 100% of cell metabolism, it was observed for G1, G2, G3 and G4 a percentage reduction in cell metabolism of 29.6%, 82%, 81.2% and 33.4%, respectively. It was concluded that the low concentration of 0,1% OE (C. citratus) was able to inhibit the growth of the strains tested as well as caused mild cytotoxicity to the cultured MDPC-23 cells.

Desenvolvimento de um embutido cárneo fermentado, com teores reduzidos de gordura e sais de cura, através da utilização de culturas probióticas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds of Dimorphandra gardneriana and Platymiscium floribundum, native plants from Caatinga biome

Nos últimos anos, o Ministério da Saúde do Brasil e a Organização Mundial da Saúde tem apoiado a investigação de novas tecnologias que possam contribuir para a vigilância, novos tratamentos e controle da leishmaniose visceral no país. Assim, o objetivo deste trabalho foi isolar compostos de plantas do bioma Caatinga, e investigar a toxicidade destes compostos contra as formas promastigotas e amastigotas de Leishmania infantum chagasi, principal parasita responsável pela leishmaniose visceral na América do Sul, e avaliar a sua capacidade para inibir a enzima acetil-colinesterase (AChE). Após a exposição aos compostos em estudo, foram realizados testes utilizando a forma promastigota que expressa luciferase e ELISA in situ para medir a viabilidade das formas promastigotas e amastigota, respectivamente. O ensaio colorimétrico MTT foi realizado para determinar a toxicidade destas substâncias utilizando células monocíticas murina RAW 264.7. Todos os compostos foram testados in vitro para as sua propriedade anti-colinesterásica. Um cumarina, escoparona, foi isolada a partir de hastes de Platymiscium floribundum, e os flavonóides, rutina e quercetina, foram isolados a partir de grãos de Dimorphandra gardneriana. Estes compostos foram purificados, utilizando cromatografia em coluna gel eluída com solventes orgânicos em misturas de polaridade crescente, e identificados por análise espectral. Nos ensaios leishmanicidas, os compostos fenólicos mostraram eficácia contra as formas extracelulares promastigotas, com EC50 para escoporona de 21.4µg/mL e para quercetina e rutina 26 e 30.3µg/mL, respectivamente. Os flavonóides apresentaram resultados comparáveis à droga controle, a anfotericina B, contra as formas amastigotas com EC50 para quercetina e rutina de 10.6 e 43.3µg/mL, respectivamente. Os compostos inibiram a enzima AChE com halos de inibição variando de 0,8 a 0,6cm, indicando um possível mecanismo de ação para a atividade leishmanicida.

A preliminary study of the antibacterial potential of cetylpyridinium chloride in root canals infected by E. faecalis

The aim of this preliminary study was to verify the antibacterial potential of cetylpyridinium chloride (CPC) in root canals infected by Enterococcus faecalis. Forty human maxillary anterior teeth were prepared and inoculated with E. faecalis for 60 days. The teeth were randomly assigned to the following groups: 1: Root canal preparation (RCP) + 0.1% CPC with positive-pressure irrigation (PPI, Conventional, NaviTip®); 2: RCP + 0.2% CPC PPI; 3: RCP + 2.5% NaOCl PPI; 4: RCP + 2.5% NaOCl with negative-pressure irrigation system (NPI, EndoVac®); 5: Positive control; and 6: Negative control. Four teeth of each experimental group were evaluated by culture and 4 by scanning electron microscopy (SEM). In all teeth, the root canals were dried and filled with 17% EDTA (pH 7.2) for 3 min for smear layer removal. Samples from the infected root canals were collected and immersed in 7 mL of Letheen Broth (LB), followed by incubation at 37°C for 48 h. Bacterial growth was analyzed by turbidity of culture medium and then observed with a UV spectrophotometer. The irrigating solutions were further evaluated for antimicrobial effect by an agar diffusion test.The statistical data were treated by means, standard deviation, Kruskal-Wallis test and analysis of variance. Significance level was set at 5%. The results showed the presence of E. faecalis after root canal sanitization. The number of bacteria decreased after the use of CPC. In the agar diffusion test, CPC induced large microbial inhibition zones, similar to 2% chlorhexidine and large than 2.5% NaOCl. In conclusion, cetylpyridinium chloride showed antibacterial potential in endodontic infection with E. faecalis.

A simple inhibition coefficient for quantifying potency of biocontrol agents against plant-pathogenic fungi

Microbial interactions depend on a range of biotic and environmental variables, and are both dynamic and unpredictable. For some purposes, and under defined conditions, it is nevertheless imperative to evaluate the inhibitory efficacy of microbes, such as those with potential as biocontrol agents. We selected six, phylogenetically diverse microbes to determine their ability to inhibit the ascomycete Fusarium
coeruleum, a soil-dwelling pathogen of potato tubers that causes the storage disease dry rot. Interaction assays, where colony development was quantified (for both fungal pathogen and potential control agents), were therefore carried out on solid media. The key parameters that contributed to, and were indicative of, inhibitory efficacy were identified as: fungal growth-rates (i) prior to contact with the biocontrol
agent and (ii) if/once contact with the biocontrol agent was established (i.e. in the zone of mixed
culture), and (iii) the ultimate distance traveled by the fungal mycelium. It was clear that there was no correlation between zones of fungal inhibition and the overall reduction in the extent of fungal colony development. An inhibition coefficient was devised which incorporated the potential contributions of distal inhibition of fungal growth-rate; prevention of mycelium development in the vicinity of the biocontrol
agent; and ability to inhibit plant-pathogen growth-rate in the zone of mixed culture (in a ratio of 2:2:1). The values derived were 84.2 for Bacillus subtilis (QST 713), 74.0 for Bacillus sp. (JC12GB42), 30.7 for Pichia anomala (J121), 19.3 for Pantoea agglomerans (JC12GB34), 13.9 for Pantoea sp. (S09:T:12), and
21.9 (indicating a promotion of fungal growth) for bacterial strain (JC12GB54). This inhibition coefficient, with a theoretical maximum of 100, was consistent with the extent of F. coeruleum-colony development (i.e. area, in cm2) and assays of these biocontrol agents carried out previously against Fusarium
spp., and other fungi. These findings are discussed in relation to the dynamics and inherent complexity of natural ecosystems, and the need to adapt models for use under specific sets of conditions.

Geological, geochemical and biogeochemical data on boundary zones of the Atlantic Ocean

Results of studies in two biogeochemically active zones of the Atlantic Ocean (the Benguela upwelling waters and the region influenced by the Congo River run-off) are reported in the book. A multidisciplinary approach included studies of the major elements of the ocean ecosystem: sea water, plankton, suspended matter, bottom sediments, interstitial waters, aerosols, as well as a wide complex of oceanographic studies carried out under a common program. Such an approach, as well as a use of new methodical solutions led to obtaining principally new information on different aspects of oceanology.

Inhibition of cell-wall autolysis and pectin degradation by cations

Modification of cell wall components such as cellulose, hemicellulose and pectin plays an important role in cell expansion. Cell expansion is known to be diminished by cations but it is unknown if this results from cations reacting with pectin or other cell wall components. Autolysis of cell wall material purified from bean root (Phaseolus vulgaris L.) occurred optimally at pH 5.0 and released mainly neutral sugars but very little uronic acid. Autolytic release of neutral sugars and uronic acid was decreased when cell wall material was loaded with Ca, Cu, Sr, Zn, Al or La cations. Results were also extended to a metal-pectate model system, which behaved similarly to cell walls and these cations also inhibited the enzymatic degradation by added polygalacturonase (EC 3.2.1.15). The extent of sugar release from cation-loaded cell wall material and pectate gels was related to the degree of cation saturation of the substrate, but not to the type of cation. The binding strength of the cations was assessed by their influence on the buffer capacity of the cell wall and pectate. The strongly bound cations (Cu, Al or La) resulted in higher cation saturation of the substrate and decreased enzymatic degradability than the weakly held cations (Ca, Sr and Zn). The results indicate that the junction zones between pectin molecules can peel open with weakly held cations, allowing polygalacturonase to cleave the hairy region of pectin, while strongly bound cations or high concentrations of cations force the junction zone closed, minimising enzymatic attack on the pectin backbone. (C) 2004 Elsevier SAS. All rights reserved.

Tissue engineering of articular cartilage with biomimetic zones

Articular cartilage damage is a persistent and increasing problem with the aging population, and treatments to achieve biological repair or restoration remain a challenge. Cartilage tissue engineering approaches have been investigated for over 20 years, but have yet to achieve the consistency and effectiveness for widespread clinical use. One of the potential reasons for this is that the engineered tissues do not have or establish the normal zonal organization of cells and extracellular matrix that appears critical for normal tissue function. A number of approaches are being taken currently to engineer tissue that more closely mimics the organization of native articular cartilage. This review focuses on the zonal organization of native articular cartilage, strategies being used to develop such organization, the reorganization that occurs after culture or implantation, and future prospects for the tissue engineering of articular cartilage with biomimetic zones.

Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.

Effect of poly(acrylic acid) end-group functionality on inhibition of calcium oxalate crystal growth

A number of series of poly(acrylic acids) (PAA) of differing end-groups and molecular weights prepared using atom transfer radical polymerization were used as inhibitors for the crystallization of calcium oxalate at 23 and 80°C. As measured by turbidimetry and conductivity and as expected from previous reports, all PAA series were most effective for inhibition of crystallization at molecular weights of 1500–4000. However, the extent of inhibition was in general strongly dependent on the hydrophobicity and molecular weight of the end-group. These results may be explicable in terms of adsorption/desorption of PAA to growth sites on crystallites. The overall effectiveness of the series didn't follow a simple trend with end-group hydrophobicity, suggesting self-assembly behavior or a balance between adsorption and desorption rates to crystallite surfaces may be critical in the mechanism of inhibition of calcium oxalate crystallization.

In vivo knockdown of the androgen receptor results in growth inhibition and regression of well-established, castration-resistant prostate tumours

Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm3. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.