The plasma membrane of microaerophilic protists: oxidative and nitrosative stress.

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 [DiBAC(4)(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 DiBAC(4)(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 O(2)-sensitive organisms and allows DiBAC(4)(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.

Nitrosative stress induced cytotoxicity in Giardia intestinalis.

AIMS: To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. METHODS AND RESULTS: Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. CONCLUSIONS: These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms.

X-ray elemental analysis differentiates blood vessels and lymphatic vessels in the chick choroid.

PURPOSE: Chick choroidal lymphatics swell rapidly during recovery from form-deprivation occlusion, leading to the question of functional significance. To date, analysis of lymph in initial lymphatics has been problematic because of the difficulties of access. However, elemental composition can be determined using scanning electron microscope (SEM) X-ray microanalysis. This study investigated whether cryo-fixation would permit vascular fluids to be analysed in situ. METHODS: Two chicks were raised normally and seven were raised with monocular occlusion for 12 days before varying periods of normal visual experience. The eyes were rapidly frozen, fractured and X-ray spectra obtained from the lumina of lymphatic and blood vessels in a Cambridge S360 SEM. RESULTS: The elemental signatures of Na/Cl/K/P/S distinguished the two vessel types. CONCLUSIONS: These results suggest this bulk-frozen tissue technique can quantify relative changes in elemental species present in various ocular compartments in response to light-induced changes in the retina.