RESUMO
Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of chitinase activity.
Assuntos
Materiais Biocompatíveis/metabolismo , Bioensaio , Quitina/metabolismo , Quitinases/metabolismo , Corantes Azur/química , Corantes Azur/economia , Quitinases/análise , Quitinases/genética , Coloides , Corantes/química , Corantes/economia , Meios de Cultura/química , Escherichia coli/genética , Escherichia coli/metabolismo , Estudos de Viabilidade , Concentração de Íons de Hidrogênio , Cinética , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade por Substrato , Fatores de TempoRESUMO
Focused ultrasound has been discovered to locally and reversibly increase permeability of the blood-brain barrier (BBB). However, inappropriate sonication of the BBB may cause complications, such as hemorrhage and brain tissue damage. Tissue damage may be controlled by selecting optimal sonication parameters. In this study, we sought to investigate the feasibility of labeling cells with superparamagnetic iron oxide particles to assess the inflammatory response during focused-ultrasound-induced BBB opening. We show that infiltration of phagocytes does not occur using optimal parameters of sonication. Taken together, the results of our study support the usefulness and safety of focused-ultrasound-induced BBB opening for enhancing drug delivery to the brain. These findings may have implications for the optimization of sonication parameters.
