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The plasma membrane of microaerophilic protists: oxidative and nitrosative stress

G. A. Biagini^2,

¹ Microbiology (BIOSI 1) Main Building, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK
² School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052, Australia
³ Department of Chemistry, King's College University of London, Strand, London WC2R 2LS, UK
⁴ School of Science, Parramatta Campus, University of Western Sydney, Australia

Correspondence
D. Lloyd
Lloydd{at}cf.ac.uk

The trans-plasma-membrane electrochemical potential of microaerophilic protists was monitored by the use of voltage-sensitive charged lipophilic fluorophores; of the many available probes, the anionic oxonol dye bis(1,3-dibarbituric acid)-trimethine oxonol [DiBAC₄(3)] is an example of one which has been successfully employed using fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. Several microaerophilic protists have been investigated with this dye; these were Giardia intestinalis, Trichomonas vaginalis, Tritrichomonas foetus, Hexamita inflata and Mastigamoeba punctachora. Under conditions where they exhibit normal vitality, these organisms exclude DiBAC₄(3) by virtue of their maintenance of a plasma-membrane potential (negative inside). Uptake of the fluorophore is indicative of disturbance to this membrane (i.e. by inhibition of pump/leak balance, blockage of channels or generation of ionic leaks), and is indicative of metabolic perturbation or environmental stress. Here, it is shown that oxidative or nitrosative stress depolarizes the plasma membranes of the aforementioned O₂-sensitive organisms and allows DiBAC₄(3) influx. Oxonol uptake thereby provides a sensitive and early indication of plasma-membrane perturbation by agents that may lead to cytotoxicity and eventually to cell death by necrotic or apoptotic pathways.

Present address: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L35 QA, UK.

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