1887

Abstract

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(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 , , , and . Under conditions where they exhibit normal vitality, these organisms exclude DiBAC(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-sensitive organisms and allows DiBAC(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.

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2004-05-01
2020-07-13
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References

  1. Biagini G. A., Suller M. T. E., Finlay B. J., Lloyd D.. 1997a; Oxygen uptake and antioxidant responses of the free-living diplomonad Hexamita sp. J Eukaryot Microbiol44:447–453[CrossRef]
    [Google Scholar]
  2. Biagini G. A., Hayes A. J., Suller M. T. E., Winters C., Finlay B. J., Lloyd D.. 1997b; Hydrogenosomes of Metopus contortus physiologically resemble mitochondria. Microbiology143:1623–1629[CrossRef]
    [Google Scholar]
  3. Biagini G. A., Lloyd D., Kirk K., Edwards M. R.. 2000; The membrane potential of Giardia intestinalis. FEMS Microbiol Lett192:153–157[CrossRef]
    [Google Scholar]
  4. Biagini G. A., Park J. H., Lloyd D., Edwards M. R.. 2001a; The antioxidant potential of pyruvate in the amitochondriate diplomonads Giardia intestinalis and Hexamita inflata. Microbiology147:3359–3365
    [Google Scholar]
  5. Biagini G. A., Knodler L. A., Saliba K. J., Kirk K., Edwards M. R.. 2001b; Na+-dependent pH regulation by the amitochondriate protozoan parasiteGiardia intestinalis. J Biol Chem276:29157–29162[CrossRef]
    [Google Scholar]
  6. Diamond L. S.. 1957; The establishment of various trichomonads of animals and man in axenic cultures. J Parasitol43:488–490
    [Google Scholar]
  7. Dinsdale M. G., Lloyd D., Jarvis B.. 1995; Yeast vitality during cider fermentation: two approaches to the measurement of membrane potential. J Inst Brew101:453–458[CrossRef]
    [Google Scholar]
  8. Ellis J. E., Cole D., Lloyd D.. 1993a; Influence of oxygen on the fermentative metabolism of metronidazole-sensitive and resistant strains of Trichomonas vaginalis. Mol Biochem Parasitol56:79–88
    [Google Scholar]
  9. Ellis J. E., Wingfield J. M., Cole D., Boreham P. F. L., Lloyd D.. 1993b; Oxygen affinities of metronidazole-resistant and -sensitive stocks of Giardia intestinalis. Int J Parasitol23:35–39[CrossRef]
    [Google Scholar]
  10. Emri M., Balkay L., Kraszai Z., Tron L., Martin T.. 1998; Wide applicability of a flow cytometric assay to measure absolute membrane potentials on a millivolt scale. Eur Biophys J28:78–83[CrossRef]
    [Google Scholar]
  11. Fenchel T., Finlay B. J.. 1990; Oxygen toxicity, respiration and behavioural responses to oxygen in free-living anaerobic ciliates. J Gen Microbiol136:1953–1959[CrossRef]
    [Google Scholar]
  12. Fernandes P. D., Assreuy J.. 1997; Role of nitric oxide and superoxide in Giardia lamblia killing. Braz J Med Biol Res30:93–99
    [Google Scholar]
  13. Harold F. M.. 1986; The Vital Force: a Study of Bioenergetics New York: W. H. Freeman;
  14. Humphreys M. J., Allman R., Lloyd D.. 1994; Determination of the viability of Trichomonas vaginalis using flow cytometry. Cytometry15:343–348[CrossRef]
    [Google Scholar]
  15. Kamo N., Muratsugu M., Hongoh R., Kobatake Y.. 1979; Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state. J Membr Biol49:105–121[CrossRef]
    [Google Scholar]
  16. Kayahara M., Felderhoff U., Pocock J., Hughes M. N., Mehmet H.. 1998; Nitric oxide (NO) and the nitrosonium cation (NO+) reduce mitochondrial membrane potential and trigger apoptosis in neuronal PC12 cells. Biochem Soc Trans26:S340
    [Google Scholar]
  17. Keister D. B.. 1983; Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg77:487–488[CrossRef]
    [Google Scholar]
  18. Krasznai Z., Marian T., Balkay L., Emri M., Tron L.. 1995; Flow cytometric determination of absolute membrane potential of cells. J Photochem Photobiol B28:93–99[CrossRef]
    [Google Scholar]
  19. Lloyd D.. 1993; Flow Cytometry in Microbiology London: Springer;
  20. Lloyd D., Hayes A. J.. 1995; Vigour, vitality and viability of microorganisms. FEMS Microbiol Lett133:1–7[CrossRef]
    [Google Scholar]
  21. Lloyd D., Williams J., Yarlett N., Williams A. G.. 1982; Oxygen affinities of the hydrogenosome-containing protozoa Tritrichomonas foetus and Dasytricha ruminantium, and two aerobic protozoa, determined by bacterial bioluminescence. J Gen Microbiol128:1019–1022
    [Google Scholar]
  22. Lloyd D., Harris J. C., Maroulis S., Biagini G. A., Wadley R. B., Turner M. P., Edwards M. R.. 2000; The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity. Microbiology146:3109–3118
    [Google Scholar]
  23. Lloyd D., Mason D. J., Suller M. T. E.. 2001; Microbial infections. In Cytometric Analysis of Cell Phenotype and Function pp.2982–3213Edited by McCarthy D. A., Macey M. G.. Cambridge University Press;
    [Google Scholar]
  24. Lloyd D., Williams A. S., James C. J.. 2002a; Nitrite inhibits hydrogen production and kills the cattle parasite Tritrichomonas foetus. J Appl Microbiol93:492–496[CrossRef]
    [Google Scholar]
  25. Lloyd D., Harris J. C., Biagini G. A..11 other authors 2002b; Oxygen homeodynamics in Giardia. In Giardia: the Cosmopolitan Parasite pp.29–43Edited by Olson B. E.. Olson M. E., Wallis P. M.. Wallingford, Oxfordshire: CAB International;
    [Google Scholar]
  26. Lloyd D., Harris J. C., Maroulis S., Mitchell A., Hughes M. N., Wadley R. B., Edwards M. R.. 2003a; Nitrosative stress induced cytotoxicity in Giardia intestinalis. J Appl Microbiol95:576–583[CrossRef]
    [Google Scholar]
  27. Lloyd D., Lemar K. M., Salgado L. E., Gould T. M., Murray D. B.. 2003b; Respiratory oscillations in yeast: mitochondrial reactive oxygen species, apoptosis and time; a hypothesis. FEMS Yeast Res3:333–339[CrossRef]
    [Google Scholar]
  28. Mason D.. 1994; The use of flow cytometry to rapidly estimate bacterial antibiotic sensitivity PhD thesis, University of Wales;
  29. Mason D. J., Allman R., Stark J. M., Lloyd D.. 1994; Rapid estimation of bacterial antibiotic susceptibility with flow cytometry. J Microsc176:8–16[CrossRef]
    [Google Scholar]
  30. Midgley M.. 1986; The phosphonium ion efflux system of Escherichia coli: relationship to the ethidium efflux system and energetic studies. J Gen Microbiol132:3187–3193
    [Google Scholar]
  31. Mitchell P.. 1979; Keilin's respiratory chain concept and its chemiosmotic consequences. Science206:1148–1159[CrossRef]
    [Google Scholar]
  32. Morré D. J., Crane F. L., Sun I. L., Navas P.. 1987; The role of ascorbate in biomembrane energetics. Ann N Y Acad Sci498:153–171[CrossRef]
    [Google Scholar]
  33. Novo D., Perlmutter N. G., Hunt R. H., Shapiro H. M.. 1999; Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique. Cytometry35:55–63[CrossRef]
    [Google Scholar]
  34. Paget T. A., Jarroll E. L., Manning P., Lindmark D. G., Lloyd D.. 1989; Respiration in the cysts and trophozoites of Giardia muris. J Gen Microbiol13:145–154
    [Google Scholar]
  35. Ryu J.-S., Lloyd D.. 1995; Cell cytotoxicity of sodium nitrite, sodium nitroprusside and Roussin's black salt against Trichomonas vaginalis. FEMS Microbiol Lett130:183–188[CrossRef]
    [Google Scholar]
  36. Scott J. A., Rabito C. A.. 1988; Oxygen radicals and plasma membrane potential. Free Radic Biol Med5:237–246[CrossRef]
    [Google Scholar]
  37. Shapiro H. M.. 2000; Membrane potential estimation by flow cytometry. Methods21:271–279[CrossRef]
    [Google Scholar]
  38. Smith J. C.. 1990; Potential-sensitive molecular probes in membranes of bioenergetic relevance. Biochim Biophys Acta1016:1–28[CrossRef]
    [Google Scholar]
  39. Yarlett N., Yarlett N. C., Lloyd D.. 1986; Metronidazole-resistant clinical isolates of Trichomonas vaginalis have lowered oxygen affinities. Mol Biochem Parasitol19:111–116[CrossRef]
    [Google Scholar]
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