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Biochemistry |
School of Biochemistry and Molecular Genetics and Cellular Analysis Facility, School of Microbiology and Immunology, University of New South Wales, Kensington, Sydney 2052, Australia1
Microbiology Group, School of Biosciences (BIOSI, Main Building), Cardiff University, Cardiff CF10 3TL, UK2
Author for correspondence: David Lloyd. Tel: +44 29 2087 4772. Fax: +44 29 2087 4305. e-mail: lloydd{at}cf.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Keywords: hydrogen peroxide, oxidative stress, reactive oxygen species
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N’-dicyclohexylcarbodiimide; DiBAC4(3), bis(1,3-dibutylbarbituric acid) trimethine oxonol
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and mortality (Shukry et al., 1986 ). The trophozoite infects the upper jejunum by attachment to the mucosal surface. Waterborne transmission occurs by ingestion of cysts (Craun, 1990 ), which survive for periods of months in freshwater ecosystems at low temperatures. Control of contamination of fresh water and supplies of potable water is achieved only with difficulty, due to resistance of cysts to chemical agents (Winiecka-Krusnell & Linder, 1998 ), and the probable existence of animal reservoirs (Smith et al., 1995 ).
The upper intestinal lining is well supplied with capillaries, and O2 concentration there has been measured at 60 µM (Atkinson, 1980 ). G. intestinalis is usually regarded as an ‘anaerobic’ protozoon, and grown and studied as such in laboratory culture (Meyer, 1976 ). However, several observations indicate that this organism is a microaerophile (Paget et al., 1989 , 1993 ). Measurement of O2 consumption as a function of dissolved O2 indicates that at low levels (0–50 µM), the organism is capable of scavenging O2 (apparent Km for O2 6·4 µM for the trophozoite). Above a threshold of 80 µM O2, O2 inhibits its own consumption. Changes in the balance of major fermentation products occur as O2 concentration is increased. Paget et al. (1990) found that anaerobically, alanine and ethanol were formed in roughly equimolar amounts, ethanol production occurred maximally at 1·0 µM O2, whereas acetate accumulation continued to increase to at least 46 µM O2. The intracellular redox state, as indicated by in vivo measurement of NADH, and the integrity of an EPR-detectable iron–sulphur centre are both highly sensitive to traces (<0·1 µM) of O2 (Paget et al., 1993 ; Ellis et al., 1993 ). Here we aim to define the limits of O2 tolerance of this microaerophilic organism, and to characterize the nature of the structural and functional consequences of accumulation of reactive O2 species.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, was obtained from the Queensland Institute of Medical Research. Trophozoites were grown axenically at 37 °C, using TYI-S-33 medium (Keister, 1983 ; Edwards et al., 1989 ) but with complement-inactivated new-born calf serum at 10% (v/v) Cultures were initiated by inoculating 15 ml medium in polypropylene centrifuge tubes with 4x104 organisms ml-1. A 0·5 ml air headspace was routinely employed. Growth was for 2 d and final cell numbers reached about 107 per tube.
Harvesting.
Tubes were chilled on ice for 20 min and then shaken gently to dislodge adhered trophozoites. Cell numbers were measured by counting on a haemocytometer slide. Cell suspensions were centrifuged at 1000 g (3000 r.p.m.) for 4 min at room temperature in a bench centrifuge at room temperature. After washing once with phosphate-buffered saline (pH 7·1, PBS) and recentrifugation, organisms were finally resuspended in PBS or in 0·31 M mannitol solution (as indicated) and kept at 4 °C.
Measurements of O2 consumption rates.
Cell suspensions (2·2x10-6 organisms ml-1), 2 ml total volume (1:1) in PBS were incubated at 37 °C in a ‘closed’ O2 electrode vessel (Rank) with magnetic stirring (200 r.p.m.). Where incubations were performed at low O2 concentrations, a stainless-steel open O2 electrode system fitted with a Teflon-membrane-covered O2 electrode (Radiometer) (Lloyd et al., 1979 ) was employed. A digital gas mixer (Lundsgaard & Degn, 1973 ) was used to mix 1% or 5% O2 with N2; after humidification by passage over moist filter paper, the gas mixture was passed over the surface of the stirred liquid vortex (stirring at 500 r.p.m.), enabling the O2 tension to be maintained at desired levels. Addition of substrates (e.g. glucose) or of inhibitors were made through a septum. To obtain the transfer constant, k, the value of t1/2 for equilibration of buffer upon switching the gas phase from 1% O2 to N2 was determined, then k was calculated from the relationship k=loge2/t1/2.
Respiration rates (Vr) were calculated from Vr=k(TG-TL), where TG is the O2 concentration in the gas phase, and TL that in the liquid phase.
O2 concentrations in PBS were calculated from the air-saturation value for air at 37 °C (250 µM O2).
Measurement of cellular swelling.
Changes in cell volume were monitored by following the time course of absorbance change at 550 nm.
Confocal laser-scanning microscopy.
A Bio-Rad MRC confocal system attached to a research microscope (1024-Leica DMRB) was used with an argon-krypton air-cooled laser at 448 nm. Images were obtained with ax63 oil-immersion objective (NA 1·38). Section thickness was 5·5 µm. The 0·3 W laser was used at 10% power to minimize photobleaching. Unless otherwise stated, organisms were washed and resuspended in 0·31 M mannitol before observation using ‘FITC’ filters. Images were acquired on Zip disc and printed using an Epson 750 colour printer.
Flow cytometry.
Forward narrow-angle light scatter, side-scatter and fluorescence were measured using a flow cytometer (Multi-Laser Sorter, MoFlo Cytomation) fitted with a Cicero or a Cyclops Summit version 2 operating software. Fluorescence of bis(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] was measured using a water-cooled 200 mW 488 nm argon-ion laser (Coherent I-90). An interference filter (D530/540) was used for emitted wavelengths (Chrome Technology), together with a Hammamatsu Hybrid Photodetector (no. 957-06). The characteristics of 20000 organisms were accumulated routinely.
Flow cytometric measurement of plasma membrane potential.
Plasma membrane potential was measured by flow cytometry (Krasznai et al., 1995 ), using the negatively charged fluorescent membrane potential indicator dye DiBAC4(3). This dye distributes across the cytoplasmic membrane, according to the Nernst equation.
A calibration curve of fluorescence intensity (i.e. channel number) measured from stained cells vs extracellular dye concentration allows evaluation of membrane potential in mV using live cells by comparison with those in depolarized state. Rather than employing fixed organisms (Emri et al., 1998 ), heat treatment (>80 °C) for 3 min was employed in order to produce a suspension with zero plasma membrane potential.
Transmission electron microscopy.
Washed G. intestinalis cells were fixed at 4 °C for 1 h in 0·1 M cacodylate buffer (pH 6·9) containing 1% paraformaldehyde and 2% glutaraldehyde. They were post-fixed with phosphate-buffered 1% OsO4 at 4 °C for 1 h, then dehydrated with successive washes of ethanol: 50%, 70%, 90% at 4 °C and two washes of 100% at room temperature. The cells were embedded in Spurr resin. Ultrathin sections were obtained with an LKB Ultratome III and mounted onto 0·5% Pioloform (in chloroform) coated copper grids. The sections were stained with aqueous uranyl acetate and lead citrate. Grids were analysed using a JEOL 1210V transmission electron microscope.
Assay methods.
Thiols were estimated after reaction with Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid) at 412 nm (
412=14700 l mol-1 cm-1) (Ellman, 1959 ). Hydrogen peroxide was measured in a closed O2 electrode using catalase.
Materials.
Toluidine blue and rose bengal (tetraiodotetrachlorofluorescein) were gifts from Mr Till Böcking and Dr Kevin Barrow.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), when dissolved O2 reached the air-saturated value. Accumulation of H2O2 continued for the duration of the experiment (195 min), to reach 4 µmol per 106 organisms. Under 4% O2 in N2, inactivation took 75 min (Fig. 1b), whereas under 2% O2 in N2 it took 140 min; under 1% O2 in N2, O2 uptake was still measurable after 4 h. Fig. 1(c) shows the accumulation of H2O2 and the decrease in total cellular thiols (conversion from thiol to disulphide forms) when the organisms were stirred under 2% O2 in N2. Cessation of O2 consumption under all conditions of exposure to inhibitory O2 concentrations preceded loss of membrane potential (see below), flagellar movement and cell motility. These changes were irreversible; restoration of anaerobiosis did not lead to recovery of cellular function.
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shows a bright-field image of a population of washed G. intestinalis trophozoites in the presence of 1 µM DiBAC4(3). The same field examined for fluorescence (excitation 490 nm, emission 519 nm; Fig. 2b) indicates that almost all the organisms exclude the fluorophore; those that do not are damaged. After exposure of organisms to O2 almost all the cells still excluded the fluorophore; again those that did not were damaged (not shown). Storage at 4 °C for 18 h (Fig. 2c) gave a population that was completely DiBAC4(3) permeable. Organisms stirred under 5% air remained impermeable to DiBAC4(3) for more than 6 h, whereas under 10% air after 4·5 h many organisms became fluorescent. Confocal laser-scanning microscopy (Fig. 2d) indicated that DiBAC4(3) had permeated the plasma membrane and the flagella. Under 100% air, DiBAC4(3) penetration was rapid, occurring in less than 1 h. Heat-killed organisms also became completely DiBAC4(3) permeable (not shown). Exposure to air for 10 min (Fig. 2e, f) or incubation with 60 µM H2O2 for 10 min (Fig. 2g) gave organisms in which 2,7-dichlorodihydrofluorescein became oxidized to give fluorescence in the cytosol. The distribution of the fluorescence suggested that externally applied H2O2 also gave fluorophore oxidation in peripheral vesicles, which was not the case when organisms were oxidatively stressed in air.
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shows two typical experiments. Live cells did not take up the anionic fluorophore, whereas heat-killed organisms were highly DiBAC4(3) permeable and hence were counted in channels indicative of high fluorescence intensities. Other treatments known to affect ion transport processes and hence lead to partial collapse of the potential across the plasma membrane resulted in fluorescence emission values intermediate between those of active organisms and the completely depolarized dead cells. Cells aerated for 1 h (Fig. 3a, panel 5) and those treated with 60 µM H2O2 for 5 min (Fig. 3b, panel 2) also showed increased permeability to DiBAC4(3). Plots of forward light scatter (a measure of cell size) vs side scatter showed that as cells became damaged by O2, they also progressively increased in volume (data not shown). Heat-killed organisms gave lower forward scatter signals than live ones. Pulse-width measurements showed that cell doublets or aggregates were rare for live, damaged or dead organisms.
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; Emri et al., 1998 ). Heat-killed organisms (with zero membrane potential, used as a control) were taken to have an intracellular fluorescence equal to that of dye in the suspending medium. Fig. 4 shows that live organisms when suspended in 0·31 M mannitol had a plasma membrane potential of -134±3 mV (n=6). Exposure to 5% air for 4·5 h decreased this value to -100 mV, whereas under 100% air, G. intestinalis became almost completely depolarized in 4·5 h. Effects of some inhibitors are also shown: the most effective (albeit at high concentration, 100 µM), was the proton-pumping ATPase inhibitor DCCD. Gramicidin (1 µM), an ionphore acting to increase membrane permeability to H+, K+ and Na+, or the protonophore CCCP (10 µM) were both highly efficient at diminishing potentials.
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shows its immediate inhibitory effect on O2 consumption. That 
OH is produced when G. intestinalis is treated with H2O2 is indicated by protective effects of OH radical quenchers (50 mM sodium benzoate or 50 mM mannitol); ferric nitrilotriacetic acid accentuates H2O2 inhibition. Similar results were obtained with a well-washed non-proliferating suspension of G. intestinalis in a reactor open for gas flow, under an atmosphere of defined low O2 value (Fig. 5b).
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Electron microscopy
All of the main distinguishing features of G. intestinalis can be seen in the controls shown in Fig. 6(a, b), i.e. the ventral disc composed of numerous microtubules, dorsal ribbons and cross-links with its rigid lateral crest, overlying ventrolateral flange and supporting marginal plates or striated bodies. The two equivalent nuclei lie lateral to the kinetosomal complex consisting of eight kinetosomes in four pairs. In the horizontal cross-section (Fig. 6b) the ventral and posteriolateral axonemes are visible along with the caudal axonemes. The anterior axonemes cannot be seen at this level but the anterior flagella can be seen external to the cell. All axonemes and flagella show the typical 9+2 microtubule arrangement. The vertical section (Fig. 6a) shows how the anterior axonemes course anteriorly in the cell before crossing and moving posteriorly to emerge at the cell’s widest point. These axonemes are accompanied by the striated bodies. The microtubules of the median bodies can be seen in their disorganized array. Numerous peripheral vacuoles lie below the plasmalemma. The cytoplasm stains dark owing to the presence of numerous stored granules, e.g. glycogen.
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, d). Most of the cells were ‘ghostlike’, with no dark-staining material in the cytoplasm and condensation within the nucleus. Numerous peripheral vacuoles scattered beneath the plasmalemma on the dorsal surface were visible. These appeared to converge and burst the cell. The ventral disc, axonemes and kinetosomes remained intact, indicating that oxidative stress has no evident impact on microtubule structure.
After 1 h aeration, the bulk of the cells maintained the pyriform structure, but some cells became swollen and misshapen (Fig. 6e). In all cells the ventral disc, its rigid lateral crest and ventrolateral flange were intact; no fragmentation of the disc was visible. The nuclei were no longer spheroid but appeared to have several cytoplasmic projections, and some had associated multilamellar bodies (Fig. 6f). The axonemes were intact and were still located centrally within the cell. The misshapen swollen cells had large cytoplasmic vacuoles and the dark granule-filled cytoplasm was now very sparse. They appeared ‘ghostlike’. In these cells the ventral disc was still intact.
After 2 h aeration the number of swollen and misshapen cells had increased, yet many cells still remained pyriform in structure (Fig. 6g, h, i). Many nuclei had associated multilamellar bodies (Fig. 6h). The ventral discs, lateral crests and ventrolateral flanges were still intact. The cytoplasm was less dense and did not stain as darkly as previously. The number of peripheral vacuoles had increased.
After 3 h aeration only a few cells remained in the intact pyriform shape. Many cells had completely broken down and cellular debris abounded. The cytoplasm stained only weakly, with very few dark-staining granules remaining. Most cells were not recognizable as Giardia (Fig. 6j). The nuclei had broken down and their chromatin had condensed. The ventral disc and axonemes were still intact, suggesting again that oxidative stress does not affect the microtubule structure within the cell.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In those experiments where the O2 dependence of O2 uptake were investigated, the concentration of dissolved O2 was increased from an undetectable level to 5% air saturation in a series of steps of 20 min each. Those data provide indications on apparent Km values and of a threshold above which O2 became inhibitory. In the present investigation, we have systematically investigated the time- and concentration-dependence of the O2 effects during long-term maintenance of steady-state levels. Oxidative damage was shown, not only to the electron-transport systems responsible for O2 reduction, but to cell functions across a wide range. Thus damage to the plasma membrane, as indicated by the progressive loss of ability to exclude the anionic voltage-sensitive dye DiBAC4(3) (Lloyd & Hayes, 1995 ), led to swelling of organisms and loss of the regulatory volume control process, described in fully functional organisms (Park et al., 1995 ). Sites known to be O2-inhibited in G. intestinalis include a (4Fe–4S) iron–sulphur cluster (Ellis et al., 1993 ) and redox-active thiols involved in volume regulation (Park et al., 1995 ). The fact that these effects could also be produced almost immediately by 60 µM H2O2 suggests that exposure to inhibitory levels of O2 results in accumulation of this partial reduction product. The Fenton Haber–Weiss reaction is then free to produce OH from H2O2 and 
, especially as this organism contains no detectable superoxide dismutase (Smith et al., 1988 ), peroxidase or catalase (Brown et al., 1995 ). Enhanced loss of functional activity in the presence of ferric nitrotriacetic acid (Aruoma et al., 1989 ) and diminished inactivation in the pressure of the OH radical scavengers, sodium benzoate or mannitol (Halliwell, 1978 ) suggests that OH, a radical known for its extremely damaging reactivity (Halliwell & Gutteridge, 1989 ) may be an important intracellular oxidant in these experiments. The O2-dependent production of photo-emissive species in Giardia lamblia has been demonstrated (T. Paget & D. Lloyd, unpublished data). These species, observed as low-level chemiluminescence in organisms metabolizing glucose, could be greatly enhanced in the presence of the redox-cycling naphthoquinone menadione. The fact that the oxygen-consuming redox chains of organisms can be damaged by 1O2-generating systems, the photodynamically activated dyes toluidine blue and rose bengal (Krinsky, 1979 ), supports the suggestion (Lamberts & Neckers, 1985 ) that the menadione sensitivity of G. lamblia can also be accounted for by free-radical damage by reactive oxygen species. Thus the anti-giardial effects of a redox-cycling naphothquinone are produced by an amplification of the effects of O2 damage. The anti-giardial effects both of menadione, and of H2O2 reported here, warrant further investigation as a means of control of this important human pathogen. |
ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Atkinson, H. J. (1980). Respiration. In Nematodes as Biological Models , pp. 101-138. Edited by B. H. Zuckerman. London:Academic Press.
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Craun, G. F. (1990). Waterborne giardiasis. In Giardiasis. Human Parasitic Disease , pp. 267-290. Edited by E. A. Meyer. Amsterdam:Elsevier.
Edwards, M. R., Gilroy, F. V., Jiminez, M. B. & O’Sullivan, W. J. (1989). Alanine is a major end product of metabolism by Giardia lamblia: a proton nuclear magnetic resonance study. Mol Biochem Parasitol 37, 19-26.[Medline]
Ellis, J. E., Williams, R., Cole, D., Cammack, R. & Lloyd, D. (1993). Electron transport components of the parasitic protozoon Giardia lamblia. FEBS Lett 325, 196-200.[Medline]
Ellman, G. L. (1959). Method for the modification and quantitative detection of thiol groups. Arch Biochem Biophys 82, 70.[Medline]
Emri, M., Balkay, L., Krasznai, Z., Tron, L. & Martin, T. (1998). Wide applicability of a flow cytometric assay to measure absolute membrane potentials on a millivolt scale. Eur Biophys J 28, 78-83.
Halliwell, B. (1978). Superoxide-dependent formation of hydroxyl radicals in the presence of iron chelates. FEBS Lett 92, 321-326.[Medline]
Halliwell, B. & Gutteridge, J. M. C. (1989). Free Radicals in Biology and Medicine. Oxford: Oxford University Press.
Keister, D. B. (1983). Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77, 487-488.[Medline]
Krasznai, Z., Marian, T., Balkay, L., Emri, M. & Tron, L. (1995). Flow cytometric determination of absolute membrane potential of cells. J Photochem Photobiol B Biol 28, 93-99.[Medline]
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Lamberts, J. J. M. & Neckers, D. C. (1985). Rose Bengal derivatives as singlet oxygen sensitizers. Tetrahedron 41, 2183-2190.
Lloyd, D. & Hayes, A. J. (1995). Vigour, vitality and viability of microorganisms. FEMS Microbiol Lett 133, 1-7.
Lloyd, D., Edwards, S. W., Kristensen, B. & Degn, H. (1979). The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii. Biochem J 182, 11-15.[Medline]
Lundsgaard, J. & Degn, H. (1973). A digital gas mixer. IEEE Trans Biomed Eng 20, 384-387.[Medline]
Meyer, E. A. (1976). Giardia lamblia: isolation and axenic cultivation. Exp Parasitol 39, 301-310.
Ortega, Y. R. & Adam, R. D. (1997). Giardia: overview and update Clin Infect Dis 25, 545-550.[Medline]
Paget, T., Jarroll, E., Manning, P., Lindmark, D. & Lloyd, D. (1989). Respiration in the cysts and trophozoites of Giardia muris. J Gen Microbiol 13, 145-154.
Paget, T., Raynor, M. H., Shipp, D. W. E. & Lloyd, D. (1990). Giardia lamblia produces alanine anaerobically but not in the presence of oxygen. Mol Biochem Parasitol 42, 63-68.[Medline]
Paget, T., Kelly, M., Jarroll, E., Lindmark, D. & Lloyd, D. (1993). The effects of oxygen on fermentation in Giardia lamblia. Mol Biochem Parasitol 57, 65-72.[Medline]
Park, J. H., Schofield, P. J. & Edwards, M. R. (1995). The role of alanine in the acute response of Giardia intestinalis to hypo-osmotic shock. Microbiology 141, 2455-2462.
Shukry, S., Zaki, A. M., Dupont, H. L., Shoukry, I., El Tagi, M. & Hamed, S. (1986). Detection of enteropathogens in fatal and potentially fatal diarrhea in Cairo, Egypt. Clin Microbiol 24, 959-962.
Smith, H. V., Robertson, L. J., Campbell, A. T. & Girdwood, R. W. A. (1995). Giardia and giardiasis: what’s in a name. Microbiol Eur 3, 22-29.
Smith, N. C., Bryant, C. & Boreham, D. F. L. (1988). Possible roles for pyruvate–ferredoxin oxidoreductase and thiol-dependent peroxidase and reductase activities in resistance to nitroheterocyclic drugs in Giardia intestinalis. Int J Parasitol 18, 991-997.[Medline]
Winiecka-Krusnell, J. & Linder, E. (1998). Cysticidal effect of chlorine dioxide on Giardia intestinalis cysts. Acta Trop 70, 369-372.[Medline]
Received 7 June 2000;
revised 7 August 2000;
accepted 21 August 2000.
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) and H2O2 (
) accumulation under air. (b) Dissolved O2 under 4% O2 in N2 (
), 2% O2 in N2% (----) and 1% O2 in N2 (——). (c) H2O2 accumulation (
) under 2% O2 in N2 %.
