School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Manchester M13 9PT, UK
Correspondence
Geoffrey D. Robson
geoff.robson{at}man.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Previously, we reported that the viability of A. fumigatus decreases rapidly when the organism enters stationary phase in liquid culture, and that this is associated with the appearance of an apoptotic-like phenotype (Mousavi & Robson, 2003). Although programmed cell death and the underlying mechanisms are well documented in mammalian cells (Strasser et al., 2000
; Hengartner, 2000
; Kaufmann & Hengartner, 2001
), there have been few studies on the mechanism of cell death in fungi, and in filamentous fungi in particular (Umar & Van Griensven, 1997
; Raju & Perkins, 2000
; Lu et al., 2003
; Cheng et al., 2003
). In the yeast Saccharomyces cerevisiae, expression of the mammalian pro-apoptotic protein Bax induces an apoptotic-like phenotype, which is suppressed by simultaneous overexpression of Bcl-XL, a member of the mammalian anti-apoptotic Bcl-2 family (Ligr et al., 1998
). Moreover, death in S. cerevisiae during the starvation phase, and following treatment with hydrogen peroxide, has also been shown to be associated with an apoptotic-like phenotype and to involve a metacaspase, a protease related to the mammalian caspase family (Madeo et al., 2002
). Treatment of S. cerevisiae with toxic levels of acetic acid is also associated with an apoptotic-like phenotype (Ludovico et al., 2001
), and was subsequently shown to involve the mitochondrion and cytochrome c release (Ludovico et al., 2002
), in a similar manner to that seen in mammalian cells (Hengartner, 2000
). Recently, Cheng et al. (2003)
reported that the antifungal sphingoid long-chain base phytosphingosine induces an apoptotic phenotype in Aspergillus nidulans in a pathway that did not appear to involve metacaspase activity (Cheng et al., 2003
).
In this study, we report that cell death in A. fumigatus, induced either by hydrogen peroxide (oxidative death) or by treatment with the widely used antifungal agent amphotericin B, is associated with the induction of an apoptotic-like phenotype, suggesting that these agents cause death in A. fumigatus by inducing a primitive form of apoptosis. Moreover, unlike entry into the stationary phase, the development of the apoptotic-like phenotype induced by these two fungicidal agents was not blocked by the broad-spectrum caspase inhibitor Z-VAD-fmk, suggesting the presence of two apoptotic-like pathways in A. fumigatus.
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METHODS |
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Effect of H2O2, amphotericin B and itraconazole on mycelial growth.
To study the effect of H2O2, amphotericin B and itraconazole on the growth and viability of A. fumigatus, various concentrations of H2O2 (from a 30 %, v/v, stock solution), amphotericin B (from a 1 mg ml1 stock) or itraconazole (from a 1 mg ml1 stock) were added to early exponential phase cultures (OD 1·5 EEL units), and viability monitored by determining c.f.u. ml1 at 30 min intervals. For H2O2-treated mycelia, 0·25 % (w/v) catalase (Sigma) was added after sampling to remove H2O2 from the sample, whilst for amphotericin B or itraconazole treatments, mycelia were washed briefly in sterile deionized water. To determine if DNA degradation had occurred, mycelium was frozen in liquid nitrogen, ground to a fine powder in a mortar and pestle, and genomic DNA extracted according to Reader & Brody (1985)
. DNA was run on a 1·5 % (w/v) agarose gel in TPE buffer (0·09 M Tris/phosphate, pH 8·0, 2 mM EDTA) and visualized following ethidium bromide staining (0·4 µg ml1 in TPE buffer).
Analysis of apoptotic markers.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL; TdT-FragEL DNA fragmentation detection kit, Oncogene Research Products) and annexin V-FITC (Oncogene Research Products) were used as markers of apoptosis, and uptake of propidium iodide (PI) was used as a marker of cell-membrane integrity, as previously described (Mousavi & Robson, 2003). In order to detect the expression of these apoptotic markers, the cell wall was first removed by digesting mycelium from mid-exponential-phase growth with Novozyme, as previously described (Mousavi & Robson, 2003
), and protoplasts were then treated with various concentrations of H2O2, amphotericin B or itraconazole for up to 6 h. Following treatment, protoplasts were washed twice by centrifugation (1500 g) for 10 min and resuspended in an equal volume of regeneration buffer (0·1 M phosphate buffer, pH 7·0, 0·9 M sorbitol). In the case of H2O2-treated protoplasts, 0·25 % (w/v) catalase was added prior to washing. To determine protoplast viability, protoplasts were regenerated by spreading gently over the surface of modified Vogel's medium solidified with 1·5 % (w/v) agar (supplemented with 0·9 M sorbitol), and plates incubated at 37 °C until colonies became visible. To inhibit metacaspase activity or protein synthesis, respectively, 25 µg ml1 of the broad-spectrum caspase inhibitor Z-VAD-fmk (Calbiochem) or 50 µg ml1 cycloheximide (Sigma) was added to the protoplast suspension, 1 h prior to treatment with H2O2 or amphotericin B. DAPI staining was used to determine the percentage of protoplasts containing nuclei, as previously described (Mousavi & Robson, 2003
).
Caspase activity.
Intracellular caspase activity was determined using a colorimetric assay based on the cleavage of a p-nitroaniline dye from the C-terminal of specific peptide substrates (Caspase Colorimetric Substrate/Inhibitor Quantipak, Calbiochem). Mycelium was ground in liquid nitrogen and the biomass resuspended in ice-cold lysis buffer (50 mM HEPES, pH 7·4, 1 mM DTT, 0·5 mM EDTA and 0·1 % (v/v) CHAPS), centrifuged at 1500 g for 10 min, and the caspase activity of the supernatant against substrates for caspase-1, -3 and -8 determined according to the manufacturer's instructions. Protein concentration was determined according to Bradford (1976).
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RESULTS |
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To test if the apoptotic-like phenotype was induced by agents that inhibited growth, without being fungicidal, we used the fungistatic azole itraconazole, which in the short term inhibits growth, without killing the cell (Lamb et al., 1999). Treatment of protoplasts with 10 µg itraconazole ml1, a concentration that inhibited protoplast regeneration without causing death, did not cause a significant increase in annexin V-FITC, TUNEL or PI-positive staining (data not shown).
Influence of H2O2 on the appearance of apoptotic markers over time
To study the development of the apoptotic phenotype over time, we treated protoplasts with 0·1 mM H2O2 and monitored the proportion of protoplasts staining positive for annexin V-FITC, TUNEL and PI over 6 h (Fig. 6). Initially, prior to treatment, the proportion of protoplasts staining positive for any of the markers was <10 %. Within 1 h of treatment with 0·1 mM H2O2, the proportion of protoplasts staining positive with annexin V-FITC rose to
45 %, and remained approximately constant thereafter. The proportion of TUNEL-positive protoplasts rose slightly to
15 % after 1 h exposure to H2O2, but had risen to 50 % after 2 h and continued to increase to a maximum of
65 % after 4 h, before decreasing to
40 % after 6 h. PI-positive protoplasts increased slightly to
10 % after 1 h, increased to
25 % after 2 h, and thereafter rose steadily to reach a maximum of
90 % after 5 h.
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DISCUSSION |
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A more sensitive test of apoptotic DNA fragmentation is based on labelling the free 3'-OH termini, which are exposed during apoptotic DNA cleavage. The TUNEL assay, which relies on the incorporation of biotinylated or fluorescein-labelled dUTP, catalysed by terminal deoxynucleotidyl transferase (TdT), enables DNA breakage to be visualized in individual cells undergoing apoptosis and has become one of the most widely used indicators of apoptosis (Gavrieli et al., 1992; Frohlich & Madeo, 2000
). Previously, we demonstrated that cell death during the stationary phase in A. fumigatus was associated with a marked increase in the proportion of TUNEL-positive nuclei indicating apoptotic-like cleavage of the DNA (Mousavi & Robson, 2003
), and TUNEL staining was also reported during cell death in A. nidulans treated with phytosphingosine (Cheng et al., 2003
). In this study, lower concentrations of H2O2 and amphotericin B (0·1 mM and 0·5 µg ml1, respectively) increased the proportion of TUNEL-positive protoplasts indicating apoptotic-like DNA fragmentation, whereas higher concentrations (1·8 mM and 1 µg ml1, respectively) caused a lower increase in TUNEL-positive protoplasts (Fig. 5
). This decrease in TUNEL-staining at higher concentrations correlates with a large increase in PI-staining, indicating loss of membrane permeability and necrotic death. The induction of necrosis by high concentrations of numerous cytotoxic substances, and apoptosis at lower concentrations, is a well known phenomenon (Lieberthal & Levine, 1996
), and has also been reported in S. cerevisiae treated with H2O2 and acetic acid (Madeo et al., 1999
; Ludovico et al., 2001
).
In mammalian cells, an earlier indicator of apoptosis is the translocation of PS from the inner to the outer leaflet of the cytoplasmic membrane (Champagne et al., 1999), and can be detected with FITC-labelled annexin V, which specifically binds to PS (Martinet et al., 1999
). As with TUNEL staining, treatment with low concentrations of H2O2 or amphotericin B led to a large increase in annexin V-FITC staining. Moreover, this increase occurred prior to the increase in TUNEL staining (Fig. 6
), and this early increase in PS translocation has also been reported in A. nidulans (Cheng et al., 2003
).
Apoptosis requires the active participation of the cell in the synthesis of new proteins that contribute to cell death. Consequently, protein synthesis inhibitors can actively block apoptosis in cells, and this is widely used to demonstrate the participation of the cell during death (Hiraoka et al., 1997; Sanchez et al., 1997
). In S. cerevisiae, cycloheximide has been shown to block the development of an apoptotic phenotype in response to various stimuli (Madeo et al., 1999
), to block TUNEL staining in A. fumigatus following entry into the stationary phase, and to block TUNEL staining in A. nidulans following treatment with phytosphingosine (Cheng et al., 2003
; Mousavi & Robson, 2003
). When added prior to treatment with low concentrations of H2O2 or amphotericin B, cycloheximide blocked the development of a TUNEL-positive phenotype, whilst having no effect on the later increase in PI-positive staining that indicates loss of membrane integrity and cell death (Fig. 7
). Thus, the development of an apoptotic-like phenotype requires protein synthesis and active participation of the cell. However, although entry into an apoptotic pathway can be prevented by blocking protein synthesis, subsequent cell death through a necrotic process is not prevented, as reported previously when A. fumigatus enters stationary phase (Mousavi & Robson, 2003
).
A number of studies in yeast and mammalian cells have demonstrated that accumulation of ROS within the cytoplasm plays a central role in apoptotic-like cell death (Greenlund et al., 1995; Slater et al., 1995
; Ligr et al., 1998
; Madeo et al., 1999
). It is possible that treatment with low concentrations of H2O2 or amphotericin B may trigger an apoptotic-like phenotype through the accumulation of ROS, and that continued accumulation in the cytoplasm ultimately causes physical damage and loss of cell integrity, as indicated by the increase in PI-positive staining. Treatment of protoplasts with low but toxic concentrations of the strong oxidizing agent sodium hypochlorite also induced an apoptotic phenotype similar to that observed with H2O2, suggesting that this is a general response to oxidative stress (results not shown). Previously, we reported an increase in intracellular activity toward caspase substrates as cultures entered the stationary phase. Moreover, the cell-permeant broad-spectrum caspase inhibitor z-VAD-fmk was able to block the increase in TUNEL and annexin V-FITC staining, suggesting a role for an upstream caspase-like activity in nuclear degradation and PS externalization (Mousavi & Robson, 2003
). In S. cerevisiae, a metacaspase with caspase-like activity, YCA1, has been cloned and shown to mediate apoptosis induced by H2O2 and in chronologically aged cells (Madeo et al., 2002
). However, in this study, no significant activity against caspase substrates was induced following H2O2 or amphotericin treatment. Moreover, inclusion of z-VAD-fmk prior to treatment did not block the development of an apoptotic-like phenotype, suggesting an alternative pathway to that involved in stationary-phase-induced cell death. Caspase-independent death has been described in other systems under specific conditions (De Mario et al., 1997
; Brunet et al., 1998
; Kroemer et al., 1998
; Okuno et al., 1998
), and the caspase inhibitor z-VAD-fmk found not to block the development of an apoptotic-like phenotype (Villa et al., 1997
). In A. nidulans, a metacaspase homologue, casA, has been identified, and a
casA disrupted strain shown still to undergo apoptosis in response to phytosphingosine, suggesting death by a caspase-independent pathway (Cheng et al., 2003
). As previously reported, A. fumigatus appears to contain two metacaspase homologues (Mousavi & Robson, 2003
), both with a high level of identity to A. nidulans casA. Interrogation of the A. nidulans genome sequence at the Whitehead Institute also revealed a second metacaspase homologue in A. nidulans (data not shown). It is possible, therefore, that one metacaspase homologue is activated through starvation, upon entry into the stationary phase, and is active against caspase substrates, whereas the second may be activated by H2O2 and amphotericin B, but is inactive against the synthetic substrates tested in this study.
In this study, we have demonstrated that cell death induced by low but toxic concentrations of H2O2 or amphotericin B triggers the development of a protein-synthesis-dependent apoptotic-like phenotype. Thus, cell death in A. fumigatus in infected humans, as a consequence of phagocyte action or of treatment with the antifungal agent amphotericin B, may actively involve the participation of the fungal cells in their own death, through triggering an apoptotic-like pathway. Further work on the role of ROS, metacaspase and the mitochondrion will be needed to understand better the mechanisms underlying the apoptotic-like pathway(s) in A. fumigatus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, M. J., Worthy, S. A., Flint, J. D. A. & Muller, N. L. (1998). Invasive aspergillosis in the immunocompromised host: utility of computed tomography and bronchoalveolar lavage. Clin Radiol 53, 255257.[Medline]
Brunet, C. L., Gunby, R. H., Benson, R. S. P., Hickman, J. A., Watson, A. J. M. & Bardy, G. (1998). Commitment to cell death measured by clonegenicity is separable from the appearance of apoptotic markers. Cell Death Differ 5, 107115.[CrossRef][Medline]
Bustamante, J., Bersier, G., Romero, M., Badin, R. A. & Boveris, A. (2000). Nitric oxide production and mitochondrial dysfunction during rat thymocyte apoptosis. Arch Biochem Biophys 376, 239247.[CrossRef][Medline]
Champagne, M., Pierre, D., Sergei, O., Martin, B., Pavel, H. & Johanne, T. (1999). Protection against necrosis but not apoptosis by heat stress proteins in vascular smooth muscle cells: evidence for distinct modes of cell death. Hypertension 33, 906913.
Cheng, J., Park, T.-S., Chio, L.-C., Fischl, S. & Ye, X. S. (2003). Induction of apoptosis by sphingoid long-chain bases. Mol Cell Biol 23, 163177.
Clements, J. S. & Peacock, J. E. (1990). Amphotericin B revisited: reassessment of toxicity. Am J Med 88, 22N27N.[Medline]
De Mario, R., Lenti, L., Malisan, F., d'Agostino, F., Tomassini, B., Zeuner, A., Rippo, M. R. & Testi, R. (1997). Requirement for GD3 ganglioside in CD95 and ceramide-induced apoptosis. Science 277, 16521655.
Denning, D. W. (1996). Therapeutic outcome in invasive aspergillosis. Clin Infect Dis 23, 608615.[Medline]
Denning, D. W. (1998). Invasive Aspergillosis. Clin Infect Dis 26, 781805.[Medline]
Denning, D. W., Anderson, M. J., Turner, G., Latgé, J.-P. & Bennett, J. W. (2002). Sequencing the Aspergillus fumigatus genome. Lancet Infect Dis 2, 251253.[CrossRef][Medline]
Derouin, F. (1994). Special issue on Aspergillosis. Pathol Biol 42, 625736.
Frohlich, K. U. & Madeo, F. (2000). Apoptosis in yeast, a monocellular organism exhibits altruistic behaviour. FEBS Lett 473, 69.[CrossRef][Medline]
Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. (1992). Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 119, 493501.[Abstract]
Greenlund, L. J., Deckwerth, T. L. & Johnson, E. M. (1995). Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 14, 303315.[Medline]
Groll, A. H., Shah, P. M., Mentzel, C., Schneider, M., Just-Nuebling, G. & Huebner, K. (1996). Trends in post-mortem epidemiology of invasive fungal infections at a university hospital. J Infect 33, 2332.[Medline]
Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770776.[CrossRef][Medline]
Hiraoka, W., Fuma, K. & Kuwabara, M. (1997). Concentration-dependent modes of cell death in Chinese hamster V-79 cells after treatments with H2O2. J Radiat Res 38, 95102.[Medline]
Hofmann, F., Ohnimus, H., Strupp, W., Zimmerman, U. & Jassoy, G. (1999). Electric field pulses can induce apoptosis. J Membr Biol 169, 103109.[CrossRef][Medline]
Hospenthal, D. R., Kwon-Chung, K. J. & Bennet, J. E. (1998). Concentrations of airborne Aspergillus compared to the incidence of invasive aspergillosis: lack of correlation. Med Mycol 36, 165168.[CrossRef][Medline]
Kaizer, L., Huguenint, T., Lew, P. D., Chapuis, B. & Pittet, D. (1998). Invasive aspergillosis. Clinical features of 35 proven cases at a single institution. Medicine 77, 188194.[CrossRef][Medline]
Karim, M., Alam, M., Shah, A. A., Ahmed, R. & Sheikh, H. (1997). Chronic invasive aspergillosis in apparently immunocompetent hosts. Clin Infect Dis 24, 723733.[Medline]
Kaufmann, S. H. & Hengartner, M. O. (2001). Programmed cell death: alive and well in the new millennium. Trends Cell Biol 11, 526534.[CrossRef][Medline]
Knapp, P. E., Bartlett, W. P., Williams, L. A., Yamada, M., Ikenaka, K. & Scoff, R. P. (1999). Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival. Cell Death Differ 6, 136145.[CrossRef][Medline]
Kroemer, G., Dallaporter, B. & Rosche-Rigon, M. (1998). The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60, 619647.[CrossRef][Medline]
Lamb, D., Kelly, D. & Kelly, S. (1999). Molecular aspects of azole antifungal action and resistance. Drug Resist Updat 2, 390402.[CrossRef][Medline]
Latgé, J.-P. (1999). Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12, 310350.
Latge, J.-P. (2001). The pathobiology of Aspergillus fumigatus. Trends Microbiol 9, 382389.[CrossRef][Medline]
Levitz, S. M., Selsted, M. E., Ganz, T., Lehrer, R. I. & Diamond, R. D. (1986). In vitro killing of spores and hyphae of Aspergillus fumigatus and Rhizopus oryzae by rabbit neutrophil cationic peptides and bronchoalveolar macrophages. J Infect Dis 154, 483489.[Medline]
Lieberthal, W. & Levine, J. S. (1996). Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury. Am J Physiol 271, F477F488.[Medline]
Ligr, M., Madeo, F., Frohlich, E., Hilt, W., Frohlich, K. & Wolf, D. H. (1998). Mammalian Bax triggers apoptotic changes in yeast. FEBS Lett 438, 6165.[CrossRef][Medline]
Lu, B. C., Gallo, N. & Kijes, U. (2003). White-cap mutants and meiotic apoptosis in the basidiomycete Coprinus cinereus. Fungal Genet Biol 39, 8293.[CrossRef][Medline]
Ludovico, P., Sousa, M. J., Silva, M. T., Leao, C. & Côrte-Real, M. (2001). Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology 147, 24092415.
Ludovico, P., Rodrigues, F., Almeida, A., Silva, M. T., Barrientos, A., Leao, C. & Côrte-Real, M. (2002). Cytochrome C release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Mol Biol Cell 13, 25982606.
Madeo, F., Fröhlich, E., Ligr, M., Grey, M., Sigrist, S. J. & Wolf, D. H. (1999). Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145, 757767.
Madeo, F., Herker, E., Maldener, C. & 8 other authors (2002). A caspase-related protease regulates apoptosis in yeast. Mol Cell 9, 911917.[Medline]
Martinet, W., van den Plas, D., Raes, H., Reekmans, R. & Contreras, R. (1999). Bax-induced cell death in Pichia pastoris. Biotechnol Lett 21, 821829.[CrossRef]
Morgenstern, D. E., Gifford, M. A. C., Li, L. L., Doerschuk, C. M. & Dinauer, M. C. (1997). Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to Aspergillus fumigatus. J Exp Med 185, 207218.
Mousavi, S. A. A. & Robson, G. D. (2003). Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus. Fungal Genet Biol 39, 221229.[CrossRef][Medline]
Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakeling, A. E., Walker, P. R. & Sikorska, M. (1993). Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 12, 36793684.[Abstract]
Okuno, S., Shigeomi, S., Ito, T., Nomura, M., Hamada, E., Ysujimoto, Y. & Matsuda, H. (1998). Bcl-2 prevents caspase-independent cell death. J Biol Chem 273, 3427234277.
Patel, R. & Paya, C. V. (1997). Infections in solid-organ transplant recipients. Clin Microbiol Rev 10, 86124.[Abstract]
Pathak, A., Pien, F. D. & Carvalho, L. (1998). Amphotericin B use in a community hospital, with special emphasis on side effects. Clin Infect Dis 26, 334338.[Medline]
Patterson, T. F., Kirkpatrick, W. R., White, M., Hiemenz, J. W., Wingard, J. R., Dupont, B., Rinaldi, M. G., Stevens, D. A. & Graybill, J. R. (2000). Invasive aspergillosis. Disease spectrum, treatment practices, and outcomes. Medicine 79, 250260.[CrossRef][Medline]
Raju, N. B. & Perkins, D. D. (2000). Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora. Fungal Genet Biol 30, 213221.[CrossRef][Medline]
Reader, U. & Brody, P. (1985). Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1, 1720.
Roilides, E., Katsifa, H. & Walsh, T. J. (1998). Pulmonary host defences against Aspergillus fumigatus. Res Immunol 149, 454465.[CrossRef][Medline]
Sanchez, A., Álvarez, A. M., Benito, M. & Fabregat, I. (1997). Cycloheximide prevents apoptosis, reactive oxygen species production, and glutathione depletion induced by transforming growth factor in fetal rat hepatocytes in primary culture. Hepatology 26, 935943.[CrossRef][Medline]
Schaffner, A., Davis, C. E., Schaffner, T., Markert, M., Douglas, H. & Braude, A. I. (1986). In vitro susceptibility of fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and opportunism. J Clin Invest 78, 511524.[Medline]
Slater, A. F. C., Stefan, C., Nobel, I., Van den Dobbelsteen, D. J. & Orrenius, S. (1995). Signalling mechanisms and oxidative stress in apoptosis. Toxicol Lett 83, 149153.[CrossRef]
Strasser, A. O'Connor L. & Dixit, V. M. (2000). Apoptosis signalling. Annu Rev Biochem 69, 217245.[CrossRef][Medline]
Thornberry, N. A. (1998). Caspases: key mediators of apoptosis. Chem Biol 5, 97103.
Trinci, A. P. J. (1972). Culture turbidity as a measure of mould growth. Trans Br Mycol Soc 58, 467473.
Trinci, A. P. J. (1983). The effect of Junlon on the morphology of Aspergillus niger and its use in making turbidity measurements of fungal growth. Trans Br Mycol Soc 81, 408412.
Umar, M. H. & Van Griensven, L. J. L. D. (1997). Morphologic cell death in developing primordia of Agaricus bisporus. Mycologia 89, 274277.
Verweij, P. E. & Denning, D. W. (1997). Diagnostic and therapeutic strategies for invasive aspergillosis. Respir Crit Care Med 18, 203215.
Villa, P., Kaufmann, S. H. & Earnshaw, W. C. (1997). Caspases and caspase inhibitors. Trends Biochem Sci 22, 388393.[CrossRef][Medline]
Vogel, H. J. (1956). A convenient growth medium for Neurospora (medium N). Microb Genet Bull 13, 4244.
Vogeser, M., Haas, A., Aust, D. & Ruckdeschel, G. (1997). Post-mortem analysis of invasive aspergillosis in a tertiary care hospital. Eur J Clin Microbiol Infect Dis 16, 16.[Medline]
Wingard, J. R., Kubilis, P., Lee, L. & 7 other authors (1999). Clinical significance of nephrotoxicity in patients treated with amphotericin B for suspected or proven aspergillosis. Clin Infect Dis 29, 14021407.[CrossRef][Medline]
Received 14 October 2003;
revised 6 February 2004;
accepted 25 February 2004.
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