RESUMO
In this study we first evaluated the effects of hydrogen peroxide (H2O2) on growth and selected properties of Porphyromonas gingivalis, and compared them with those obtained by a reducing agent (cysteine). The growth of P. gingivalis was only moderately affected when H2O2 was added at concentrations up to 30 mM in a complex culture medium. However, when a defined basal medium was used, H2O2 at a concentration of 3 mM completely inhibited growth of P. gingivalis. Incorporation of cysteine at concentrations up to 30 mM in both media had no effect on growth. The effects of H2O2 and cysteine on cell-associated hemagglutinating and Arg-gingipain activities were evaluated using bacteria grown in the complex culture medium. Both activities were strongly decreased when H2O2 was added in the assay mixtures. This inhibitory effect of H2O2 was reversible. On the other hand, including cysteine in the assay mixtures increased both activities. H2O2 and cysteine had no effect on the expression of heat shock protein (HSP)-68 and HSP-75 by P. gingivalis, as determined by SDS-PAGE and Western immunoblotting analysis. In the second part of the study, we tested whether growth of selected oral bacterial species may modify the oxidation-reduction potential (Eh) of the environment. It was found that certain species were able to either decrease (P. gingivalis, Fusobacterium nucleatum, Peptostreptococcus micros, Streptococcus mutans) or increase (Streptococcus sanguis) the Eh of the medium. Our study provides evidence that an oxidizing agent such as H2O2 may affect the biology of P. gingivalis. Moreover, growth of some members of the oral microflora can generate oxidizing and reducing conditions, and thus potentially influence the ecology of subgingival sites by affecting strictly anaerobic bacteria such as P. gingivalis.
Assuntos
Peróxido de Hidrogênio/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Adesinas Bacterianas , Cisteína/farmacologia , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases Gingipaínas , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Hemaglutininas/metabolismo , Oxirredução/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimentoRESUMO
A method for the selective recovery of living microfilariae from Onchocerca volvulus nodules is described. The microfilariae migrate through solidified agar gel into overlayering Hanks balanced salt solution (HBSS). The highest recovery rates of the worms were obtained with 0.3 and 0.4% agar. Optimal conditions for in vitro cultures of the larvae in HBSS were established; pH range 7.0-7.5, glucose concentration 2-5 mg/ml for long term cultures, osmolality 200-309 mOsmoles/l, temperature 4 degrees C for prolonged cultures and 24-28 degrees C for overall best yield of excretory/secretory products (ESP). Subculturing of the larvae reduced contamination of ESP with human serum protein to minimal amounts after 9 recultures done within 96 h.