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University of Pennsylvania School of Medicine, Department of Medicine, Division of Hematology/Oncology, 730 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, USA
Correspondence
Simon A. B. Knight
sknight{at}mail.med.upenn.edu
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; McCluskey et al., 2005; Roehm et al., 1991; Scudiero et al., 1988). Live cells reduce the tetrazole ring and a coloured formazan product is formed, which can be assessed visually and quantified spectrophotometrically. Such assays are used to test the efficiency of antifungal drugs in killing or inhibiting growth of Candida albicans and other fungi (Heyn et al., 2005; Kuhn et al., 2003; Meletiadis et al., 2001; Tellier et al., 1992; Wiederhold et al., 2005). The tetrazolium salts used in biology are aromatic derivatives of 1,2,3,4-tetrazole and include monotetrazolium [e.g. 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT), 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1)] or ditetrazolium [e.g. nitro blue tetrazolium (NBT)] variants (reviewed by Berridge et al., 2005). XTT and WST-1 are reduced to form soluble coloured formazan products (Ishiyama et al., 1993; Paull et al., 1988; Peskin & Winterbourn, 2000), and MTT, which can cross cellular membranes, forms water-insoluble formazan crystals upon reduction (Berridge et al., 2005; Mosmann, 1983).
Despite widespread use of these assays, the gene(s) and encoded enzymes that catalyse the reduction of the tetrazolium salts are still not known. Reduced pyridine nucleotides are likely to be the originating electron donors, but the specific reductases involved remain to be identified (Berridge & Tan, 1993; Vistica et al., 1991). In mammalian cells, the plasma membrane is thought to be the site for the extracellular reduction of the soluble tetrazolium salts (e.g. XTT and WST-1), in a process that might involve NADH coenzyme Q-dependent ferricyanide reductases and a ubiquinone-dependent plasma membrane electron transport chain (Berridge & Tan, 2000; Crane et al., 1985; Kim et al., 2002; Morre, 2004; Villalba et al., 1995).
Whole cells of Saccharomyces cerevisiae can also reduce ferricyanide (Crane et al., 1982), and externally directed ferric reductases (FREs), encoded by the genes ScFRE1 and ScFRE2, are responsible for this process (Dancis et al., 1992; Georgatsou & Alexandraki, 1994). The FREs have wide phylogenic distribution (Lara-Ortiz et al., 2003), and, in C. albicans, there are at least 11 FRE family members (Arnaud et al., 2005; http://www.candidagenome.org/). The FREs are, in turn, related to the large NAD(P)H oxidase (NOX) protein family. The archetypical NOX is gp91phox, which is present in human phagocytes, and responsible for superoxide production within the phagocytic vacuole (reviewed by Segal, 2005). Key features of NOX proteins that are conserved in FRE proteins include four conserved histidine residues for coordination of two b-haem groups within an intramembraneous region, and amino acid motifs associated with FAD and NADPH binding (Finegold et al., 1996). This configuration of cofactors permits the energetically favourable transfer of intracellular electrons from intracellular NADPH across the plasma membrane to an extracellular electron acceptor (Vignais, 2002).
In S. cerevisiae and C. albicans, a primary biological role of the FREs is the reduction of ferric iron chelates, thereby releasing ferrous iron and making it available to the high-affinity iron permease system, which consists of a multicopper oxidase (FET) and an iron permease (FTR) (Askwith et al., 1994; Eck et al., 1999; Knight et al., 2002; Kosman, 2003; Ramanan & Wang, 2000; Stearman et al., 1996). This cellular iron-uptake system is critical for growth in iron-limiting conditions, and is homeostatically upregulated at the level of transcription in response to low levels of environmental iron (Blaiseau et al., 2001; Braun et al., 2000; Courel et al., 2005; Knight et al., 2002; Lan et al., 2004; Rutherford et al., 2005; Yamaguchi-Iwai et al., 1996). In studying the reductive iron-uptake system, we observed that C. albicans was able to reduce ferric and take up ferrous iron at a 40–100-fold greater rate than that of S. cerevisiae (Knight et al., 2005). ScFre1p is known to have broad substrate specificity, and can utilize a variety of electron acceptors as substrates, including nitroprusside, cupric chelates, resazurin and azo dyes, as well as ferric chelates (Hassett & Kosman, 1995; Lesuisse & Labbe, 1994; Ramalho et al., 2005). This led us to investigate if CaFre10p, the predominant externally directed ferric reductase of C. albicans when grown in typical laboratory conditions (Knight et al., 2005), was responsible for the reduction of tetrazolium salts used in the viability/biomass assays of C. albicans.
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METHODS |
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). Strain BCa15-2 harbours a homozygous deletion of CaFRE10 (also known as RBT2, CFL95) (Cafre10
: : hisG/fre10 : : hisG-URA3-hisG ura3 : : imm434/ura3 : : imm434) and was a gift from Burk Braun and Alexander Johnson, University of California, San Francisco (Braun et al., 2000). Strain CT218.2 (Cafre10 : : hisG/Cafre10 : : hisG : : CaFRE10-URA3 ura3 : : imm434/ura3 : : imm434) harbours a reinserted functional CaFRE10 and CT212.2 (Cafre10 : : hisG/Cafre10 : : hisG : : Cafre10-1-URA3 ura3 : : imm434/ura3 : : imm434) harbours a reinserted mutated Cafre10 allele, and is auxotrophically matched to CT218.2 (Knight et al., 2005).
S. cerevisiae strains used were YPH499 [MATa ura3-52 lys2-801(amber) ade2-101(ochre) trp1-63 his3-200 leu2-1; Sikorski & Boeke, 1991] and the congenic ferric reductase knock-out 49912 [MATa ura3-52 lys2-801(amber) ade2-101(ochre) trp1-63 fre1 : : LEU2 fre2 : : HIS3; Finegold et al., 1996]. Strain YPH250 rho° is congenic with YPH499 (except for the presence of a trp1-1 allele) and was treated with ethidium bromide to ablate mitochondrial DNA (mtDNA) (Fox et al., 1991).
Growth conditions and media.
For all experiments, strains were streaked from –80 °C glycerol stocks onto 1 % (w/v) yeast extract, 2 % (w/v) peptone, 2 % (w/v) D-glucose and 0.01 % (w/v) adenine (YAPD) agar medium, grown for 2 days at 30 °C, then transferred to room temperature where they were maintained for a maximum of 10 days. All experiments were initiated from inoculation of a single colony to a single culture. For biological replicates, multiple single colonies were individually inoculated into multiple cultures. Defined iron-deficient medium was prepared from complete supplement mixture (CSM, 0.79 g l–1; Q-BioGene) supplemented with 0.1 g uridine l–1, yeast nitrogen base (YNB, 6.7 g l–1, USBiologicals) without amino acids, iron, zinc, manganese or copper, and 2 % (w/v) D-glucose. This was supplemented with 0.4 mg zinc sulfate l–1, 0.4 mg manganese sulfate l–1 and 1 µM copper sulfate. The pH of this unbuffered medium was 4.0–4.4. Milli-Q (Millipore) water was used for all growth media and chemical solutions. All iron-deficient media were prepared in plastic containers and filter-sterilized, and cells were grown in polypropylene or polystyrene vessels. The predicted iron concentration of this low-iron medium was of the order 50–100 nM (Eide et al., 1992).
Ferric, XTT, WST-1, and MTT reductase assays.
The protocol for studying the reductase activities could be divided into three distinct stages: stage 1, growth of the cells in the specific medium; stage 2, washing the cells in the assay buffer to remove the growth media; and stage 3, the enzyme assay. Using this protocol, we were able to compare the effects of the growth conditions on the reductase activities under identical enzyme assay conditions.
For all assays, cells were grown in 96-well plates with 150 µl medium at 30 °C without shaking. After 17 h, cells were resuspended and subinoculated (1 : 20) into fresh medium. The freshly inoculated cultures were incubated at 30 °C for 5–6 h. Cells were pelleted by centrifugation (1000 g, 5 min) at room temperature and washed twice with standard reductase buffer [50 mM citrate, pH 6.6, with 5 % (w/v) D-glucose]. OD720 of the cell pellets resuspended in reductase buffer was measured for cell number normalization, and assays were initiated by the addition of appropriate substrate (ferric ammonium sulfate, XTT, WST-1 or MTT; see details below) in reductase buffer to the cell suspensions. Reactions were carried out at 30 °C in the dark. After 10–15 min, the ferric, XTT and WST-1 reactions were stopped by the addition of 25 % trichloroacetic acid. We have previously determined that the reduction of ferric iron is linear over this time period (Knight et al., 2005). The cells were pelleted by centrifugation (1000 g for 5 min) and the supernatants were transferred to fresh 96-well plates for absorbance measurements (BioTek EL800 plate reader). The MTT reactions were stopped after 1.75 h with acidified (0.04 M HCl) 2-propanol. Cells were pelleted by centrifugation (1000 g, 5 min) and the absorbance of the supernatant was measured. For all assays, reactions containing no cells were used to determine blank values, which were subtracted from values obtained in assays with cells. Absorbance values of products formed were normalized with optical measurements of culture turbidity. Greater than 99.9 % of cells were in the yeast form.
The ferric reductase assays rely on the formation of a red bathophenanthroline disulfonate (BPS)–Fe(II) complex, which is quantified by measuring absorbance at 515 nm (Dancis et al., 1990; Lesuisse et al., 1987). The source of ferric iron was 1 mM ammonium ferric sulfate (Fluka) and the concentration of BPS (Sigma) was 1 mM.
The XTT reductase assay relies on the reduction of XTT to form a red formazan product, which is quantified by measuring absorbance at 515 nm. XTT (Sigma) was prepared in standard reductase buffer and used at a concentration of 0.37 mM with 0.5 µM freshly made menadione (Sigma). Menadione was excluded from the XTT assay when superoxide dismutase (SOD) was included, as menadione can spontaneously yield superoxide following enzymic reduction (Hassan & Fridovich, 1979). The absence of menadione decreased overall XTT activity by 
20 %.
The WST-1 assays rely on reduction of WST-1 to form a yellow water-soluble formazan product, which is quantified by measuring absorbance at 450 nm (Ishiyama et al., 1993). WST-1 (Dojindo) was prepared as a 10 mM stock in Milli-Q water and stored as aliquots at –20 °C. Prior to use, an aliquot was thawed, diluted to 1 mM in standard reductase buffer, and added to cells at a final concentration of 0.5 mM. Initial experiments demonstrated that the electron/hydrogen carrier 1-methoxy-5-methylphenazinium methylsulfate (1-mPMS; Dojindo), at 10 µM, had no effect (Student's t test, P=0.845) on the reduction of WST-1 (data not shown). This recapitulates the lack of effect of 1-mPMS on the reduction of WST-1 by activated neutrophils (Tan & Berridge, 2000). Therefore, 1-mPMS was not included in the WST-1 assays.
The cellular reduction of MTT leads to the formation of insoluble indigo coloured crystals. MTT (Sigma) was prepared as a 1 g l–1 stock solution in reductase buffer and combined with cells at a final concentration of 0.5 g l–1. At the end of the assay, the formazan crystals were dissolved in acidified (0.04 M HCl) 2-propanol and the absorbance of the supernatant measured at 595 nm.
In the reactions with iron-containing SOD (Sigma) from Escherichia coli, SOD was dissolved in 20 mM HEPES and added at a 1 : 10 dilution to the suspended cells, immediately prior to addition of reductase substrate. Addition of HEPES buffer alone was used as control.
Measurement of oxygen consumption.
The rate of cellular oxygen consumption was measured using a Clark-style electrode and a temperature-controlled chamber at 30 °C (System 203; Instech). The analogue signal was converted to a digital signal for data collection and analysis (Powerlab 2/25 and software; ADInstruments). One million cells in exponential phase were diluted into prewarmed fresh media and placed into the sealed chamber. After a 30 s equilibration, recording was started and continued until dissolved oxygen was completely consumed. Oxygen consumption was linear with respect to time during this period.
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RESULTS |
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). Direct comparison of whole-cell ferric reductase activity of C. albicans (wild-type strain SC5314) to a well-characterized S. cerevisiae strain (YPH499) after growth on low-iron defined media, indicated that the cell surface reductase activity of C. albicans was at least 10-fold higher than that of S. cerevisiae [12.2±2.3 compared to 1.1±0.3 nmol Fe(II) (106 cells)–1 h–1, respectively, n=5]. Likewise XTT reduction was also considerably greater in C. albicans than in S. cerevisiae [0.117±0.019 compared to 0.003±0.001 (A515)–1 (A720)–1 min–1, respectively; values are means±SD for n=5].
C. albicans XTT surface reductase activities: roles for CaFRE10, iron and pH regulation
The activity of C. albicans responsible for the reduction of XTT to the formazan product was regulated by the level of iron in the growth medium prior to the assay (Fig. 1). This iron-regulated activity was reminiscent of the iron-regulated reduction of ferric compounds catalysed by CaFre10p (Knight et al., 2005). To determine if CaFre10p was involved in XTT reduction, a Cafre10–/– homozygous deletion strain (strain BCa15-2; ) was compared to SC5314 (WT) (Fig. 1a). When cells were grown in low-iron defined medium and then washed and assayed, wild-type C. albicans had 10-fold higher XTT activity than the Cafre10–/– mutant. C. albicans CT281.2, harbouring a reinserted functional CaFRE10 (R), restored XTT activity to 73 % of that of SC5314, whereas reinsertion of a non-functional Cafre10-1 allele (strain CT212.2; m) had no effect. This genetic complementation of XTT activity was similar to that observed for ferric reductase activity (Knight et al., 2005). When grown in low-iron conditions, the Cafre10–/– mutant retained some XTT activity. However, when the Cafre10–/– mutant was grown in the presence of iron, the XTT activity was repressed to virtually zero (Fig. 1a). Overall, C. albicans XTT activity was regulated by the level of iron exposure during growth. Furthermore, for cells grown in unbuffered medium, in low or iron-supplemented conditions, the majority of the XTT activity was dependent on CaFRE10.
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). We had also previously observed that, for C. albicans grown at near-neutral pH, CaFRE10 contributed very little to the total cell surface ferric reductase activity. In contrast, for cells grown in unbuffered (pH 4.0–4.4) medium, CaFRE10 encodes the majority of the activity (Knight et al., 2005; and data not shown). To examine how the pH of the growth medium might affect the ability of C. albicans to reduce XTT, wild-type and CaFRE10 mutant strains were grown in defined medium with 50 mM HEPES to maintain pH between 6.7 and 6.8 (Fig. 1b
). At this near-neutral pH, XTT activity was independent of CaFRE10 (Fig. 1b), yet was still regulated by the amount of iron in the growth medium. In addition, the XTT activity of cells grown in buffered medium was two- to threefold greater than for cells grown in unbuffered medium (note different scale of Fig. 1a, b). The identity of the C. albicans gene(s) encoding the iron-regulated reductase activity expressed in buffered growth medium is unknown.
Partial inhibition of WST-1 and XTT reduction by SOD
WST-1 is a water-soluble tetrazolium salt that is membrane-impermeable and is reduced to a water-soluble formazan. It can be reduced by superoxide anion, and inhibition of this reaction by SOD can be used to assess superoxide production (Tan & Berridge, 2000). Frequently, with assays that involve reduction of WST-1, an intermediate electron/hydrogen carrier is employed. For WST-1, the electron/hydrogen carrier is 1-mPMS. We observed no effect on the ability of C. albicans to reduce WST-1 in the presence or absence of 1-mPMS (data not shown), and therefore did not include 1-mPMS in the assays reported here. To investigate if superoxide was involved in WST-1 and XTT reduction by C. albicans, SOD was added to the reactions immediately prior to the addition of WST-1 or XTT (Fig. 2a). In the presence of 30 µg SOD ml–1, WST-1 reductase activity was decreased to 74 % of control values (no SOD). This suggests that the majority of WST-1 reduction was not mediated by superoxide. The presence of SOD (30 µg ml–1) had a greater inhibitory effect on XTT reduction, but most XTT reduction (64 % of control activity) was SOD-independent.
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; Lesuisse et al., 1987), and is based on earlier assays used to measure ferricyanide reduction (Avron & Shavit, 1963). Citrate can function as a chemical reductant and is required for optimal ferric reductase activity of intact yeast cells. To test if citrate might be required to mediate reduction of WST-1, we compared the ability of wild-type and Cafre10–/– C. albicans to reduce WST-1 in our standard assay buffer and in Dulbecco's PBS, pH 7.2, supplemented with 5 % (w/v) D-glucose (PBSG) (Fig. 2b). As observed previously (Fig. 1a), there were clear effects of the presence of iron in the growth medium in down-regulating WST-1 activity, and the majority of the activity was CaFRE10-dependent under both assay conditions. However, there was no difference in the rate of WST-1 reduction between cells assayed in standard assay buffer compared to those assayed in PBSG. Addition of 50 mM citrate to PBSG shifted the pH to 7.3 and decreased the rate of WST-1 reduction by three- to fourfold (Fig. 2b). The reason for this decrease is not known. Taken together, these results suggest that citrate does not mediate the cellular reduction of WST-1.
C. albicans MTT intracellular reductase activities: iron regulation but no role for CaFRE10
MTT has greater lipophilicity than XTT and WST-1, and is likely reduced by intracellular reductants (Berridge et al., 2005). Examination of MTT reduction with whole cells revealed little to no difference in MTT reductase activity between wild-type (Fig. 3a, W) and the Cafre10–/– mutant (Fig. 3a, ), indicating that the reduction of MTT was not dependent on CaFre10p. Light microscopy of cells indicated that the reduced MTT product was associated with intracellular membrane structures, following the reductase assay (data not shown). Despite the lack of dependence on CaFRE10, MTT reduction was still iron-regulated. Three independently isolated C. albicans strains, SC5314 (W), WO-1 [White (Wh) and Opaque (Op) morphologies], and CBS5736 (W2), showed 5–10-fold more MTT reductase activity in iron-deprived compared with iron-replete cells. Both the MTT and the WST-1 reductase activities of the high-frequency switching strain WO-1 were less than those of strains SC5314 and CBS5736. Strain WO-1 was examined further after confirming the morphology of subtypes by microscopic examination of 200–500 cells from representative cultures. Under these mild conditions of iron limitation, there was little effect of iron on the switching process. The opaque cells had slightly, but consistently, higher MTT and WST-1 reductase activities.
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, Sc) was considerably less than that in C. albicans, and, like C. albicans, MTT reduction in S. cerevisiae was independent of the externally directed ferric reductases, encoded by ScFRE1 and ScFRE2 (data not shown). In contrast to C. albicans, there was no evidence that the low amount of MTT reduction observed in S. cerevisiae was iron-regulated. Deletion of mtDNA in S. cerevisiae (
°) caused a small but statistically significant (Student's t test, P=0.0002) increase in the rate of MTT reduction, which was diminished when the cells were grown in the presence of iron (Fig. 3a). WST-1 reduction rates observed in S. cerevisiae were unaffected by mtDNA status; however, the low values may preclude firm conclusions from being made (Fig. 3b). When we measured respiration directly in C. albicans, we did not observe any difference in the rate of oxygen consumption between the wild-type and the Cafre10–/– mutant strains (data not shown). These data suggest that the reductase activity encoded by CaFRE10 is independent of mitochondrial respiration. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The ability of C. albicans CaFre10p to reduce such structurally different substrates raises the possibility that an intermediate electron carrier performs reduction of ferric iron and tetrazolium salts. The standard XTT assay utilizes menadione, and protocols using WST-1 include 1-mPMS (Meletiadis et al., 2001; Scarlett et al., 2005). In our assays, omission of these two electron carrier intermediates from their respective assays led to only small decreases in tetrazolium reduction rates (20 % or less). Citrate is a major component of the reductase assay buffer (Avron & Shavit, 1963; Dancis et al., 1990; Lesuisse et al., 1987); however, it was not required for reduction of WST-1. The presence of SOD did decrease the rate of tetrazolium reduction. However, most XTT and WST-1 reductase activity was SOD-independent. C. albicans is flavinogenic, and flavin secretion is increased when C. albicans is grown under iron-limiting conditions (Burkholder, 1943; Knight et al., 2002). In our study, the cells were washed before the enzyme assays; however, it cannot be excluded that sufficient flavin was secreted by C. albicans, during the 10–15 min assay, to mediate electron transfer from the reductase enzyme to the tetrazolium salt. The possibility that the ferric reductases can transfer electrons directly to the tetrazolium salts remains to be tested.
The expression of CaFRE10 is highly regulated by environmental conditions. Under iron-limiting conditions, CaFRE10 mRNA levels are substantially elevated (Knight et al., 2002). The increase in CaFRE10 expression under iron-limiting conditions is reflected in an increase in cell surface ferric reductase activity and, as shown in this study, an increase in the rate of reduction of XTT and WST-1. However, iron is not the only environmental factor that influences cell surface reductase activity. Changing the pH to near-neutral conditions increased cell surface reductase activity when ferric iron and XTT were electron acceptors, but the activity was independent of CaFRE10. Nonetheless, at near-neutral pH, cell surface reductase activity was still repressed by environmental iron. There are a number of FRE homologues in C. albicans. Microarray data demonstrate that CaFRE10 is down-regulated at alkaline pH and that the expression of four other CaFRE homologues [CaFRE1 (orf19.8848), CaFRE2 (orf19.8849), CaFRE7 (orf19.6139), CaFRE9 (orf19.3538)] are upregulated at alkaline pH (Bensen et al., 2004). Although the gene(s) responsible for encoding the cell surface reductase at neutral and alkaline pH still needs to be identified, the regulated expression of these genes encoding ferric reductase homologues may have strong effects on tetrazolium reduction activity.
The tetrazolium salt MTT differs from XTT and WST-1 in that it is cell-permeable and can cross the plasma membrane (Berridge et al., 2005). Here, we have shown that the reduction of MTT in C. albicans was independent of the cell surface reductase encoded by CaFRE10. This observation is in agreement with the intracellular site of MTT reduction demonstrated in the rat B12 neural cell line (Liu et al., 1997). However, MTT reductase activity in C. albicans was iron-regulated. As stated above, there are a number of FRE homologues in C. albicans of unknown cellular location, and it is possible that one or more of these is responsible for the intracellular reduction of MTT. MTT reductase of C. albicans was much more active than that of S. cerevisiae, and only the C. albicans MTT reductase was iron-regulated. Perhaps C. albicans possesses a distinct regulatory circuit that links iron to NAD(P)H levels. The high reductase activities of C. albicans may distinguish it from non-pathogenic S. cerevisiae. Finally, the use of tetrazolium salts as indicators of C. albicans viability/biomass must be re-evaluated in view of the fact that the enzymes that catalyse the reduction of these compounds are highly regulated by growth conditions.
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ACKNOWLEDGEMENTS |
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Received 13 January 2006;
revised 28 March 2006;
accepted 12 April 2006.
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) and WST-1 reductase activity (
) in the presence of SOD. (b) Wild-type (WT) and Cafre10–/– (