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.

Mast cells/basophils in the peripheral blood of allergic individuals who are HIV-1 susceptible due to their surface expression of CD4 and the chemokine receptors CCR3, CCR5, and CXCR4.

A population of metachromatic cells with mast cell (MC) and basophil features was identified recently in the peripheral blood of patients with several allergic disorders. This study now shows that these metachromatic cells express on their surface the high-affinity IgE receptor (FcepsilonRI), CD4, and the chemokine receptors CCR3, CCR5, and CXCR4, but not the T-cell surface protein CD3 and the monocyte/macrophage surface protein CD68. This population of MCs/basophils can be maintained ex vivo for at least 2 weeks, and a comparable population of cells can be generated in vitro from nongranulated hematopoietic CD3(-)/CD4(+)/CD117(-) progenitors. Both populations of MCs/basophils are susceptible to an M-tropic strain of human immunodeficiency virus 1 (HIV-1). Finally, many patients with acquired immunodeficiency syndrome have HIV-1-infected MCs/basophils in their peripheral blood. Although it is well known that HIV-1 can infect CD4(+) T cells and monocytes, this finding is the first example of a human MC or basophil shown to be susceptible to the retrovirus. (Blood. 2001;97:3484-3490)

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.

Renal actions of dihydroergocristine and of phentolamine in anaesthetized cats.

1. Comparison has been made of the effects of dihydroergocristine (DCS) and phentolamine mesylate (phentolamine) on cardiac and respiratory rates, systemic arterial pressure, renal clearances of creatinine (C(Cr)) and of p-amino-hippuric acid (C(PAH)) and on the secretion of Na and K, in cats under chloralose anaesthesia.2. Phentolamine antagonized vasomotor tone and the pressor effect of circulating noradrenaline to comparable extent. The extent of reduction in urine flow, C(Cr), C(PAH) and Na excretion correlated with the fall in mean arterial pressure. Innervated and denervated kidneys responded similarly. Cardiac and respiratory rates rose slightly as arterial pressure fell.3. DCS, 10 to 20 mug/kg per min, did not reduce vasomotor tone, markedly reduced the pressor effect of exogenous noradrenaline, caused bradycardia and respiratory slowing but had little or no effect on renal function.4. DCS, 30 to 40 mug/kg per min, lowered mean arterial pressure by 15-25 mm Hg, decreased C(PAH) but not C(Cr), so raising the filtration fraction and caused a small reduction in urine flow and in Na excretion. Innervated and acutely denervated kidneys responded similarly.5. DCS, 30 to 40 mug/kg per min, raised mean arterial pressure, decreased C(PAH), urine flow and Na excretion but did not alter C(Cr) in animals pretreated with full alpha-adrenergic blocking doses of phentolamine.6. DCS, 30 to 40 mug/kg per min, increased the rate of secretion of sympathetic amines from the adrenal medulla and increased the concentration of renin in renal venous blood.7. Isolated kidneys perfused at constant pressure from pump-oxygenator circuits and in saline diuresis responded to DCS (15-17 mug/120 ml. blood) by diuresis and natriuresis and by a rise in the rate of secretion of renin. Higher concentrations of DCS (125-250 mug/120 ml.) were without effect on renal function and did not influence renin secretion.8. The renal effects of full alpha-adrenergic blocking doses of DCS and of phentolamine were comparable, in the whole animal.9. The evidence indicates that the release of noradrenaline by DCS 30-40 mug/kg per min from nerve terminals supplying the juxtaglomerular apparatus may have caused the enhancement of renin secretion.