Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate.
Exp Parasitol
; 122(4): 299-305, 2009 Aug.
Article
em En
| MEDLINE
| ID: mdl-19393240
ABSTRACT
Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non-protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non-protein thiols. Its specificity was confirmed using buthionine sulphoximine (a gamma-glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non-protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non-protein thiols by conventional anti-leishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non-protein thiols in Leishmania promastigotes.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Compostos de Sulfidrila
/
Leishmania donovani
/
Citometria de Fluxo
/
Fluoresceínas
/
Corantes Fluorescentes
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Exp Parasitol
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Índia