Related Experiment Videos
Action of DL-alpha-difluoromethyl ornithine on Acanthamoeba culbertsoni
1Division of Biochemistry, Central Drug Research Institute, Lucknow, India.
Abstract:
The multiplication of A. culbertsoni in the peptone medium was not inhibited by 10-20 mM concentration of alpha-difluoromethyl ornithine (DMFO) while a partial and transient inhibition of cell multiplication was observed by 10-20 mM DFMO in proteose peptone, yeast extract, glucose (PYG) medium. Ornithine decarboxylase (ODC) activity in the cells and cell free extracts was strongly inhibited by DFMO, excluding enzyme refractoriness and impermeability of cells for DFMO as the possible causes of DFMO resistance. The presence of polyamines in the peptone and PYG media as well as uptake of polyamines by the amoebae has been demonstrated. The growth and multiplication of A. culbertsoni in chemically defined medium was not affected by 1-5 mM DFMO while 10-20 mM DMFO yielded partial inhibition. A lowering of diaminopropane levels and enhancement of spermidine levels was observed in DFMO inhibited cells and level of ODC was drastically reduced in the inhibited cultures. Uptake of polyamines from the growth media may partly account for DFMO resistance of A. culbertsoni. Alternative mechanisms for DFMO resistance are indicated.
Insights
Alpha-difluoromethyl ornithine (DFMO) showed resistance in Acanthamoeba culbertsoni multiplication. Polyamines in growth media may contribute to this resistance, suggesting alternative mechanisms beyond enzyme inhibition.
Area of Science:
- Biochemistry
- Cell Biology
- Parasitology
Background:
- Acanthamoeba culbertsoni is an opportunistic protozoan parasite.
- Alpha-difluoromethyl ornithine (DFMO) is a known inhibitor of ornithine decarboxylase (ODC).
- Understanding drug resistance mechanisms in A. culbertsoni is crucial for potential therapeutic strategies.
Purpose of the Study:
- To investigate the resistance of A. culbertsoni to DFMO.
- To elucidate the role of polyamines and ODC activity in DFMO resistance.
- To explore potential alternative mechanisms of DFMO resistance in A. culbertsoni.
Main Methods:
- Culturing A. culbertsoni in various media (peptone, PYG, chemically defined).
- Assessing cell multiplication inhibition by different DFMO concentrations.
- Measuring ODC activity in whole cells and cell-free extracts.
- Quantifying polyamine levels (diaminopropane, spermidine) in inhibited cells.
Main Results:
- DFMO did not inhibit A. culbertsoni multiplication in peptone medium but showed partial, transient inhibition in PYG medium.
- ODC activity was strongly inhibited by DFMO, ruling out enzyme refractoriness or impermeability.
- Polyamines were present in media and taken up by amoebae; their uptake may partly explain DFMO resistance.
- DFMO-inhibited cells showed reduced diaminopropane and increased spermidine levels, with drastically reduced ODC.
Conclusions:
- A. culbertsoni exhibits resistance to DFMO, particularly in media rich in polyamines.
- Uptake of exogenous polyamines is a significant factor contributing to DFMO resistance.
- Alternative resistance mechanisms, besides ODC inhibition, are likely involved in A. culbertsoni's response to DFMO.