Laboratório de Biologia Celular Parasitária, Programa de Biologia Celular e Parasitologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco G, Ilha do Fundão, Rio de Janeiro, 21949-900, Brazil1
Setor de Microscopia Eletrônica e Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Brazil2
Laboratório de Química Biológica, Instituto Venezoelano de Investigaciones Científicas-IVIC, Venezuela3
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil4
Author for correspondence: Marcos A. Vannier-Santos. Tel: +55 21 260 6963. Fax: +55 21 280 8193. e-mail: vannier{at}biof.ufrj.br
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ABSTRACT |
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Keywords: sterol biosynthesis inhibitors, ketoconazole, terbinafine, acidocalcisome, Leishmania
Abbreviations: ER, endoplasmic reticulum; ESI, electron spectroscopic imaging; SBI, sterol biosynthesis inhibitors
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INTRODUCTION |
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Inclusion vesicles were previously described as membrane-bound organelles displaying electron-dense cores associated with the luminal face of the membrane (Bird et al., 1966 ; Vickerman & Preston, 1976
; Vickerman & Tetley, 1977
). This organelle received different designations such as electron-dense granules (Carvalho et al., 1979
) and lysosomes (Macadam & Williamson, 1974a
, b
; Slomianny & Prensier, 1990
; Williamson & McLaren, 1981
) based upon their ultrastructural appearance and the presence of acid phosphatase activity as reported in Trypanosoma gambiense (Seed et al., 1967
), Trypanosoma rhodesiense (Dvorak et al., 1988
) and Herpetomonas samuelpessoai (Carvalho et al., 1979
).
Understanding the physiological role of these so-called inclusion vesicles has been hampered by the difficulty in matching early histochemical data with current electron microscopy observations, particularly whenever techniques such as quick freezing, freeze-substitution and ultracryomicrotomy are employed (Dvorak et al., 1988 ; Scott et al., 1997
). Thus, organelles of distinct origins and chemical composition may be mistaken (Vickerman & Preston, 1976
; Herbert, 1965a
, b
) and the identification of this compartment on electron micrographs has been considered purely arbitrary (Watson & Lee, 1975
). Despite its widespread distribution in diverse organisms, little is known about the functional dynamics of these structures, particularly in protozoa, and most of the knowledge in this field relies on early publications. Similar organelles in H. samuelpessoai were suggested to comprise endosomal/lysosomal compartments based upon the presence of acid phosphatase activity and incorporation of iron as assessed by X-ray spectra (Carvalho & De Souza, 1977
), in addition to the presence of a porphyrin prosthetic group peroxidase activity (Carvalho et al., 1979
). Similarly, they were termed pigment bodies in Trypanosoma cyclops and may correspond to sites of intracellular accumulation of haemoglobin ingested by endocytosis (Heywood et al., 1974
; Weinman, 1971
). Vickerman & Tetley (1977)
suggested that inclusion vesicles and pigment bodies, detected by both light and electron microscopy (Heywood et al., 1974
), are different entities, but the possibility that they comprise distinct stages of a single compartment was not excluded. It must be noted that the former is more frequent in both T. cyclops (Heywood et al., 1974
) and H. samuelpessoai (Carvalho & De Souza, 1977
) cultivated with haemoglobin and haemin, respectively. Similar electron-dense membrane-bound vesicles were regarded as stored metabolic products in Herpetomonas megaseliae (Janovy et al., 1974
). More recently (Scott & Docampo, 1998
; Scott et al., 1997
), structurally identical compartments in Trypanosoma cruzi were ascribed to be the acidocalcisomes (Vercesi et al., 1994
).
In this study we report data on the formation and composition of acidocalcisomes in ketoconazole- and terbinafine-treated and untreated Leishmania amazonensis promastigotes. The possible intersection of this organelle with the endosomal/lysosomal system is suggested.
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METHODS |
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Ultrastructure and biogenesis of acidocalcisomes.
Promastigotes and amastigotes were incubated with different concentrations of either terbinafine or ketoconazole (0·0011 µM) in Warren medium, fixed in 2·5% glutaraldehyde and 0·2% picric acid in 0·1 M sodium cacodylate buffer, pH 7·2, after 72-and 96-h treatments. Cells were washed, post-fixed in 1% OsO4, 0·8% potassium ferricyanide in the same buffer, dehydrated in acetone and embedded in Epon. Thin sections were collected on 400 mesh copper grids, stained with uranyl acetate and lead citrate and observed with a Zeiss CEM 902 transmission electron microscope.
Endosomal/lysosomal origin of acidocalcisomes.
Treated and untreated parasites were incubated for 60120 min in the presence of horseradish peroxidase (Graham & Karnovsky, 1966 ) or 10 nm gold-labelled human transferrin as described by Borges et al. (1998)
. Purified human cystatin C (kindly provided by Dr Julio Scharfstein, Universidade Federal do Rio de Janeiro, Brazil) was conjugated to 10 nm gold particles as described by Roth (1983)
. For acid phosphatase activity detection, treated and untreated parasites were washed in PBS, pH 7·2, briefly fixed in cold 1% Grade I glutaraldehyde in 0·1 M cacodylate buffer, pH 7·2, rinsed in 10 mM sodium acetate buffer, pH 5·0, at 4 °C and incubated for 45 min at 37 °C in the same buffer containing 1 mM CeCl3, 1 mM sodium-ß-glycerophosphate, 5% sucrose (Robinson & Karnovsky, 1983
). Cells were then washed, fixed again in 2·5% glutaraldehyde in cacodylate buffer for 1 h at room temperature and processed as described above. Sections were stained with lead citrate and observed under a Zeiss 900 electron microscope.
Composition of the acidocalcisomes.
Electron spectroscopic imaging (ESI) of acidocalcisomes was carried out in ultrathin sections (3050 nm) of treated specimens collected on 400 mesh nickel grids. The unstained material was analysed on a Zeiss CEM 902 electron microscope equipped with an integrated magnetic prism and attached to a digital image analysis system (IBAS, Kontron). The microscope was operated in image mode with an energy selecting slit aperture of 20 eV. The accelerating voltage was 80 kV and the condenser and objective apertures were 100 and 90 µm, respectively. Elemental distribution images of oxygen and phosphorus were calculated using the three windows method (Reimer, 1991 ). This method uses two images above and one below the absorption edge of an element to remove background electrons and obtain a distribution map of that element. For oxygen, the energy windows below the edge were centred at 490 and 515 eV and the one above centred at 545 eV. For phosphorus, the two energy windows below were 100 and 110 eV and the one above was 150 eV. For calcium, the two-window method was used with an energy window below the edge at 330 eV and another above the edge at 360 eV. Elemental maps were calculated by digital subtraction of these two images. For electron energy loss spectroscopy (EELS), spectra were recorded by an integrated photomultiplier operated by dedicated software via an IEEE 488 interface (Kontron). The microscope was operated in spectrum mode at 80 kV using an objective aperture of 30 µm. The energy selecting slit aperture was approximately 2 eV. Intensities of energy losses during acquisition were measured at each 1 eV by a digital multimeter.
Drugs.
Terbinafine [SF-86327; (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalene methanamine] was provided by A. Lindenmann and H. Stähelin (Sandoz, Switzerland) through Luís Rodrigues (Sandoz, Venezuela). It was added in cultures as a DMSO solution. The final DMSO concentrations never exceeded 1% (v/v) and had no effect on the proliferation or morphology of parasites. John Russe (Janssen Pharmaceutica, Caracas, Venezuela) provided ketoconazole {cis-1-acetyl-4-[4-([2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]-methoxy)phenyl]piperazine}. The drug was added as an aqueous solution titrated to pH 2·4 with HCl and sterilized by filtration.
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RESULTS |
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Phosphorus, oxygen and calcium content of acidocalcisomes
Inelastically scattered electrons with element-specific energy losses were used to determine the distribution of several elements in promastigotes before (not shown) and after terbinafine treatment. Both the electron energy loss spectra and elemental maps of the acidocalcisomes revealed the presence of phosphorus, oxygen and calcium. Elemental mapping of acidocalcisomes showed a homogeneous distribution of phosphorus (Fig. 4b), calcium (Fig. 4c
) and oxygen (Fig. 4d
) all over the acidocalcisome cores (Fig. 4a
). Electron energy loss representative spectra confirmed the presence of these elements by demonstrating the calcium L2,3 (346 eV), oxygen K (532 eV), nitrogen (402 eV) edges (Fig. 5a
). The L2,3 edge for phosphorus (132 eV; Fig. 5b
) and occasionally zinc (not shown) were also detected in this organelle. We cannot exclude the possibility that other elements were washed out during sample processing and/or sectioning.
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DISCUSSION |
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Studies carried out in T. cruzi have shown the presence of a vacuolar-type H+-ATPase (Scott & Docampo, 1998 ), a Ca2+-ATPase (Lu et al., 1998
) and a pyrophosphatase (Scott et al., 1998
) in the membrane of the acidocalcisome, identified as a membrane-bound organelle containing an electron-dense inclusion which occupies the whole organelle as seen in cryosections (Scott et al., 1997
) or part of it as seen in transmission electron microscopy of routinely processed cells. The available data suggest that this organelle plays an important role in the homeostasis of Ca2+, whose mobilization was shown to influence host cell infection by L. amazonensis amastigotes (Lu et al., 1997
). Ca2+ present in these organelles may also be involved in the stacking of parasite membranes, since it can promote a rapid interaction between multilamellar phospholipid vesicles (Rand et al., 1985
), resembling the myelin-like figures observed in SBI-treated L. amazonensis.
Previous studies have shown that drugs such as acriflavine and suramin (Macadam & Williamson, 1974a , b
) also induce the appearance of electron-dense inclusion vesicles. However, further studies are necessary to determine whether they are of the same nature. Our observations on SBI-treated cells showed the presence of a large number of normal and polymorphic electron-dense acidocalcisome-like organelles surrounded by profiles of the ER. The images obtained were suggestive of an autophagic process, as characterized in detail in other cell systems (reviewed by Dunn, 1990
, 1994
). It is well known that drugs such as suramin (Macadam & Williamson, 1974b
) can induce autophagy but the mechanisms underlying this process remain to be clarified. ER breakdown and phospholipid accumulation have been reported in drug-treated T. rhodesiense (Macadam & Williamson, 1974b
). The phosphorus content of these organelles might be derived from rRNA autophagic hydrolysis since ribosome degradation has been reported (Macadam & Williamson, 1974b
). This seems unlikely in ketoconazole- and terbinafine-treated L. amazonensis since, contrary to membrane whorls, ribosome aggregates were not observed in forming autophagic vacuoles. It is reasonable to suppose that the enhanced membranous content of the autophagic vacuoles indicates an altered phospholipid turnover rate. The antifungal effects of SBI are believed to involve the accumulation of aberrant and toxic sterols in the plasma membrane (Groll et al., 1998
) but cytoplasmic lipid deposits were also observed in SBI-treated Leishmania, possibly resulting from a dysfunction in autophagy regulation (Vannier-Santos et al., 1995
).
Enzyme cytochemistry demonstrated the presence of reaction products indicative of acid phosphatase activity associated with some acidocalcisomes, thus suggesting their relation with lysosomes. Plasmodium acid phosphatase- and hydrolase-positive compartments, rather similar to the ones observed here, have been reported to be localized near digestive vacuoles in close contact with residual bodies (Slomianny & Prensier, 1990 ). The cytochemical detection of hydrolytic enzymes does not necessarily imply their activity in these compartments under natural conditions, but the intergradation of the acidocalcisome contents strongly suggests a digestive function.
The detection of both fluid-phase and receptor-mediated endocytic tracers within acidocalcisome-like compartments further supports the association of these organelles with the endosomal/lysosomal pathway. Interestingly, we have previously observed that gold-labelled transferrin endocytosed by L. amazonensis amastigotes is delivered to lysosomes rather than recycled (Borges et al., 1998 ) as in African trypanosomes (Grab et al., 1992
). The detection of nitrogen by elemental mapping may be indicative of the presence of proteins in these compartments. In this regard it is important to point out that the concentration of polyphosphate synthesized in mammalian cells is much higher than that of Pi plus ATP (Kumble & Kornberg, 1995
). Therefore, the participation of membrane components is suggested (Kornberg, 1995
). Interestingly both phosphate (Rand et al., 1985
; Pisoni & Lindley, 1992
) and calcium (Haller et al., 1996
; Kempler, 1985
; Lemons & Thoene, 1991
) are incorporated into lysosomes of mammalian cells. The function of the phosphate environment in the autophagic pathway may be especially relevant in micro-organisms that accumulate polyphosphate. In yeast cells up to 99% of the polyphosphate is found in lysosome-related vacuoles (Kornberg, 1995
). The lysosomal participation in the formation of the compartment is further supported by sialic acid detection using gold-labelled Limax flavus agglutinin (Vannier-Santos et al., 1991
). It is noteworthy that pyrophosphate is the most abundant phosphate compound in trypanosomatid and apicomplexan parasites (Urbina et al., 1998
).
Based upon the inability to accumulate gold-labelled transferrin (Scott et al., 1997 ), T. cruzi acidocalcisomes have been reported to not be involved in the endosomal pathway. Nevertheless, gold-labelled transferrin, unlike BSA, was only detected in a small part of the T. congolense endosomal system and therefore may not be a good endocytic tracer. It cannot be ruled out that among different species and under distinct experimental conditions these compartments may display discrete chemical compositions (Scott et al., 1997
) and therefore play different roles. It has been pointed out that there is no evidence that Trypanosoma brucei acidocalcisomes are separate organelles and the acidic calcium pools may be related to lysosomes or endocytic vesicles (Xiong et al., 1997
) as in mammalian cells. The detection of acid phosphatase and endocytic tracers in Leishmania Ca2+-and phosphorus-rich compartments strongly suggests that they may intersect lysosomes or residual bodies of the endocytic/autophagic pathway which are found in normal parasites but may be enhanced by chemotherapy-induced metabolic dysfunction. These observations suggest a close association of the endosomal/lysosomal system with acidocalcisomes during the autophagic process in normal parasites or induced by antimicrobial agents in Leishmania.
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ACKNOWLEDGEMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berger, B. J. & Fairlamb, A. H. (1992). Interactions between immunity and chemotherapy in the treatment of trypanosomiasis and leishmaniasis. Parasitology 105, S71-S78.[Medline]
Berman, J. D. (1988). Chemotherapy for leishmaniasis: biochemical mechanisms, clinical efficacy and future strategies. Rev Infect Dis 10, 560-589.[Medline]
Bird, R. G., Molloy, J. O. & Ormerod, W. E. (1966). Granules and tubules in the cytoplasm of the sleeping sickness trypanosome: an electron microscope study. Trans R Soc Trop Med Hyg 60, 753-760.[Medline]
Borges, V. M., Vannier-Santos, M. A. & De Souza, W. (1998). Subverted transferrin trafficking in Leishmania-infected macrophages. Parasitol Res 84, 811-822.[Medline]
Carvalho, T. U. & De Souza, W. (1977). Fine structure and X-ray microanalysis of electron-dense granules in Herpetomonas samuelpessoai. J Parasitol 63, 1116-1117.[Medline]
Carvalho, T. U., Souto-Padón, T. & De Souza, W. (1979). Herpetomonas samuelpessoai: electron microscopy and cytochemistry of electron-dense granules. Exp Parasitol 47, 297-304.[Medline]
Chance, M. L. (1995). New developments in the chemotherapy of leishmaniasis. Ann Trop Med Parasitol 89, 37-43.[Medline]
Dunn, W. A.Jr (1990). Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J Cell Biol 110, 1923-1933.[Abstract]
Dunn, W. A.Jr (1994). Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol 4, 139-143.
Dvorak, J. A., Engel, J. C., Leapmam, R. D., Swyt, C. R. & Pella, P. A. (1988). Trypanosoma cruzi: elemental composition heterogeneity of cloned stocks. Mol Biochem Parasitol 31, 14-26.
Grab, D. J., Wells, C. W., Shaw, M. K., Webster, P. & Russo, D. C. W. (1992). Endocytosed transferrin in African trypanosomes is delivered to lysosomes and may not be recycled. Eur J Cell Biol 59, 398-404.[Medline]
Graham, R. C. & Karnovsky, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructure cytochemistry by a new technique. J Histochem Cytochem 14, 291-302.[Medline]
Groll, A. H., De Lucca, A. J. & Walsh, T. J. (1998). Emerging targets for the development of novel antifungal therapeutics. Trends Microbiol 6, 117-124.[Medline]
Haller, T., Dietl, P. & Deetjen, P. (1996). The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release. Cell Calcium 19, 157-165.[Medline]
Herbert, I. V. (1965a). Cytochemistry of in vitro cultured Trypanosoma theileri. Exp Parasitol 16, 348-362.[Medline]
Herbert, I. V. (1965b). Cytoplasmic inclusions and organelles of in vitro cultured Trypanosoma theileri and Trypanosoma melophagium and some speculations on their function. Exp Parasitol 17, 24-40.[Medline]
Heywood, P., Weinman, D. & Lipman, M. (1974). Fine structure of Trypanosoma cyclops in noncellular cultures. J Protozool 21, 232-238.[Medline]
Janovy, J.Jr, Lee, K. W. & Brumbaugh, J. A. (1974). The differentiation of Herpetomonas megaseliae: ultrastructural observations. J Protozool 21, 53-59.[Medline]
Kempler, M. S. (1985). An adenosine triphosphate-dependent calcium uptake pump in human neutrophil lysosomes. J Clin Invest 76, 303-310.[Medline]
Kornberg, A. (1995). Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J Bacteriol 177, 491-496.[Abstract]
Kumble, K. D. & Kornberg, A. (1995). Inorganic polyphosphate in mammalian cells and tissues. J Biol Chem 270, 5818-5822.
Lemons, R. M. & Thoene, J. G. (1991). Mediated calcium transport by isolated human fibroblast lysosomes. J Biol Chem 266, 14378-14382.
Lu, H.-G., Zhong, L., Chang, K.-P. & Docampo, R. (1997). Intracellular Ca2+ pool content and signaling and expression of a calcium pump are linked to virulence in Leishmania mexicana amazonensis amastigotes. J Biol Chem 272, 9464-9473.
Lu, H.-G., Zhong, L., De Souza, W., Benchimol, M., Moreno, S. & Docampo, R. (1998). Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi. Mol Cell Biol 18, 2309-2323.
Macadam, R. F. & Williamson, J. (1974a). Drug effects on the fine structure of Trypanosoma rhodesiense: acriflavine, ethidium and antrycide. Ann Trop Med Parasitol 68, 291-299.[Medline]
Macadam, R. F. & Williamson, J. (1974b). Drug effects on the fine structure of Trypanosoma rhodesiense: suramin, tryparsamide and mapharside. Ann Trop Med Parasitol 68, 301-306.[Medline]
Olliaro, P. L. & Bryceson, A. D. M. (1993). Practical progress and new drugs for changing patterns of leishmaniasis. Parasitol Today 9, 323-328.
Pisoni, R. L. & Lindley, E. R. (1992). Incorporation of [32P]orthophosphate into long chains of inorganic polyphosphate within lysosomes of human fibroblasts. J Biol Chem 267, 3626-3631.
Rand, R. P., Kachar, B. & Reese, T. S. (1985). Dynamic morphology of calcium-induced interactions between phosphatidylserine vesicles. Biophys J 47, 483-489.[Abstract]
Rangel, H., Dagger, F., Hernandez, A., Liendo, A. & Urbina, J. A. (1996). Naturally azole-resistant Leishmania braziliensis promastigotes are rendered susceptible in the presence of terbinafine: comparative study with azole-susceptible Leishmania mexicana promastigotes. Antimicrob Agents Chemother 40, 2785-2791.[Abstract]
Reimer, L. (1991). Energy-filtering transmission electron microscopy. Adv Electron Phys 81, 43-126.
Robinson, J. M. & Karnovsky, M. J. (1983). Ultrastructural localization of several phosphatases with cerium. J Histochem Cytochem 31,11971208.[Abstract]
Roth, J. (1983). The colloidal gold marker system for light and electron microscopic cytochemistry. In Techniques in Immunocytochemistry, Vol. 2, pp. 217284. Edited by G. R. Bullock & P. Petrusz. New York: Academic Press.
Scott, D. A. & Docampo, R. (1998). Two types of H+-ATPases are involved in the acidification of internal compartments in Trypanosoma cruzi. Biochem J 15, 583-589.
Scott, D. A., Docampo, R., Dvorak, J. A., Shi, S. & Leapman, R. D. (1997). In situ compositional analysis of acidocalcisomes in Trypanosoma cruzi. J Biol Chem 272, 28020-28029.
Scott, D. A., De Souza, W., Benchimol, M., Zhong, L., Lu, H. G., Moreno, S. & Docampo, R. (1998). Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi. J Biol Chem 273, 22151-22158.
Seed, J. R., Byram, J.III & Gam, A. A. (1967). Characterization and localization of acid phosphatase activity of Trypanosoma gambiense. J Parasitol 14, 117-125.
Slomianny, C. & Prensier, G. (1990). A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites. J Protozool 37, 465-470.[Medline]
Urbina, J. A., Payares, G., Molina, J. & 8 other authors (1996). Cure of short- and long-term experimental Chagas disease using D0870. Science 273, 969971.[Abstract]
Urbina, J. A., Moreno, B., Vierkotter, S., Oldfield, E., Moreno, S. N. J., Bailey, B. N., Yan, W., Scott, D. A. & Docampo, R. (1998). Pyrophosphate is the most abundant high-energy phosphate-compound in trypanosomatid and apicomplexan parasites and a non-metabolizable analog. Mem Inst Oswaldo Cruz 93 (suppl. II), 72.
Vannier-Santos, M. A., Saraiva, E. M. B. & De Souza, W. (1991). Nuclear and cytoplasmic lectin binding sites in promastigotes of Leishmania. J Histochem Cytochem 39, 793-800.[Abstract]
Vannier-Santos, M. A., Urbina, J. A., Martiny, A., Neves, A. & De Souza, W. (1995). Alterations induced by the antifungal compounds ketoconazole and terbinafine in Leishmania. J Eukaryot Microbiol 42, 337-346.[Medline]
Vercesi, A. E., Moreno, S. J. N. & Docampo, R. (1994). Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei. Biochem J 304, 227-233.[Medline]
Vickerman, K. & Preston, T. M. (1976). Comparative cell biology of the kinetoplastid flagellates. In Biology of the Kinetoplastida, Vol. 1, pp. 35130. Edited by W. H. R. Lumsden & D. A. Evans. London: Academic Press.
Vickerman, K. & Tetley, L. (1977). Recent ultrastructural studies on trypanosomes. Ann Soc Belge Med Trop 57, 441-455.[Medline]
Watson, L. P. & Lee, C. M. (1975). Ultrastructure of Trypanosoma duttoni. Z Parasitenkd 46, 133-140.[Medline]
Werbovetz, K. A., Jeronimo, S. M. B., McDonald, T. L. & Pearson, R. D. (1992). Treatment of leishmaniasis and trypanosomiasis. Curr Opin Infect Dis 5, 840-848.
Weinman, D. (1971). Malaysian primate trypanosomes: intra-cytoplasmic pigment. Southeast Asian J Trop Med Public Health 2, 87.
Williamson, J. & McLaren, D. J. (1981). Localization of phosphatases in Trypanosoma rhodesiense. J Protozool 28, 460-467.[Medline]
Xiong, Z. H., Ridgley, E. L., Enis, D., Olness, F. & Ruben, L. (1997). Selective transfer of calcium from an acidic compartment to the mitochondrion of Trypanosoma brucei. J Biol Chem 272, 31022-31028.
Received 24 August 1998;
revised 10 June 1999;
accepted 6 July 1999.