1 Department of Pharmacy Practice, Division of Infectious Diseases; 2 Department of Medicine; 3 Department of Microbiology; 4 Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS 39216; 5 Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Houston, TX 77030, USA
Received 12 August 2002; returned 22 September 2002; revised 29 October 2002; accepted 4 November 2002
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Abstract |
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Keywords: amphotericin B, Candida albicans, calcium
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Introduction |
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Increases in intracellular calcium have been implicated in the toxic effect of amphotericin B on mammalian cells,4 and amphotericin B has been shown to increase intracellular calcium concentrations in Leishmania species.5 Amphotericin B-induced interleukin-1ß (IL-1ß) production in THP-1 cells has been found to be dependent upon the presence of extracellular calcium. In the absence of extracellular calcium, or in the presence of EGTA, amphotericin B was unable to elicit an IL-1ß response in THP-1 cells in vitro. Furthermore, amphotericin B was found to increase intracellular concentrations of calcium in a dose-dependent fashion in these cells. These changes were found to be dependent upon the presence of extracellular calcium.4
Given the importance of calcium in intracellular signalling in eukaryotic cells, including Candida albicans,6 and its role in mediating the effects of other cytotoxic stimuli, it was reasoned that calcium may also be important in the fungicidal actions of amphotericin B. Thus, studies were performed using C. albicans to determine whether amphotericin B affects the influx or efflux of calcium in C. albicans, and whether the antifungal activity of amphotericin B is dependent upon extracellular calcium concentrations.7
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Materials and methods |
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Standardized methods were used for timekill studies with amphotericin B (Sigma, St Louis, MO, USA) in standard growth medium (RPMI-1640 + MOPS pH 7.0) or in growth medium to which 0.1 or 1.0 mM EGTA had been added.8 Briefly, a standardized fungal suspension (0.5 McFarland standard) was prepared by transferring 34 colonies from a 24 h culture of C. albicans ATCC strain 90028 (amphotericin B MIC 1 mg/L) on potato dextrose agar (PDA) to 10 mL sterile water. Aliquots (1 mL) of this suspension were diluted 1:10 with sterile medium (±EGTA) containing serial two-fold concentrations of amphotericin B (016 x MIC) and incubated on an orbital shaker at 35°C. Samples were aseptically removed at 0, 1, 4, 8, 12 and 24 h of incubation, serially diluted and plated on PDA for colony count determination. All plates were incubated for 48 h at 35°C. Experiments were performed in duplicate. Mean colony counts (log10 cfu/mL) were plotted versus time for each isolate and compared with respect to the rate and extent of antifungal activity at each amphotericin B concentration. Fungicidal activity was defined as >99.9% (>3 log10 reduction) in cfu/mL from the starting inoculum; fungistatic activity was defined as <99.9% (<3 log10 reduction) from the starting inoculum. Data were fitted to a sigmoidal Emax model (Hill equation) for comparison of EC50 values.
Measurement of media calcium concentrations
Free calcium and magnesium concentrations of MOPS-buffered (10 mM, pH 7.2) RPMI-1640 media with and without EGTA (0.1 or 1 mM) were predicted using WinMAXC software (version 2.10; http://www.stanford.edu/%7Ecpatton/downloads.htm).
Direct measurements of calcium were made using the fluorescent calcium-sensitive ratiometric dye fura PE3 (hexapotassium) (fura PE3K; Teflabs, Austin, TX, USA). RPMI medium containing 1 mM EGTA was used directly, whereas media without EGTA or with 0.1 mM EGTA were serially diluted in RPMI medium to which calcium had not been added. Fluorescences of media at 510 nm were measured continuously as the excitation wavelength was increased from 250 to 450 nm before, and then after, addition of fura PE3K (0.1 µM). Fluorescences at 340 and 380 nm excitation in the absence of fura PE3K were subtracted from the corresponding fluorescences in the presence of the probe, and the corrected values were used to calculate the ratio of fluorescence (510 nm emission) at 340 and 380 nm excitation. The fluorescence ratio, R, determined for each medium was then used to estimate free calcium concentration, [Ca2+]free, using the following equation:9
[Ca2+]free = Kd(Ff/Fb)(RRmin)/(RmaxR)
where Kd is the affinity of fura PE3K for calcium; Ff and Fb, respectively, are the fluorescence intensities (510 nm) at 380 nm excitation for free and calcium-bound fura PE3K; and Rmin and Rmax are, respectively, the minimum and maximum fluorescence ratios. The Kd was calculated from the fluorescences of a series of buffers containing increasing free calcium concentrations, made by titration of a 10 mM K·EGTA buffer (100 mM KCl, 1 mM MgSO4, 10 mM EGTA, 10 mM MOPS pH 7.2) with a 10 mM Ca·EGTA buffer (100 mM KCl, 1 mM MgSO4, 10 mM CaEGTA, 10 mM MOPS pH 7.2), both of which contained 0.1 µM fura PE3K. Fluorescence ratios for the initial buffers provided values, respectively, for Rmin and Rmax. Rmax was 16.08, Rmin was 0.82, Ff/Fb was 6.96 and Kd was 119 nM. The osmolality of the calibration buffers and RPMI-1640 media varied by no more than 3%, and no corrections were made for differences in ionic strength. All fluorescence measurements were made using a Perkin Elmer LS50B spectrofluorimeter.
Calcium influx
In as much as the normal extracellular concentration of calcium is in the range of 12.5 mM, and that of magnesium is 1.5 mM,10 the effects of amphotericin B on calcium uptake in C. albicans ATCC strain 90028 in the present studies were evaluated under conditions in which the concentrations of calcium and magnesium were equivalent to those occurring in vivo. Briefly, cells were grown overnight at 37°C as a suspension in brainheart infusion (BHI) medium (Difco Laboratories, Detroit, MI, USA) and then collected by centrifugation. Next, cells were washed twice by suspension and incubation on an orbital platform (80 rpm) for 30 min in a modified Hanks balanced salt solution (HBSS) containing 10 mM glucose, 1.5 mM Mg(C2H3O2)2 (magnesium diacetate), 1.8 mM CaCl2 and 10 mM HEPES (pH 7.4). This and all subsequent procedures were performed at room temperature. The final washed cell pellet was suspended in fresh buffer and divided into aliquots containing
6 x 107 cells. Control cells were maintained, after addition of dimethylsulphoxide (DMSO) (0.1% v/v), for an additional 6090 min prior to measurement of calcium influx. Likewise, aliquots of treated cells were maintained in HBSS/HEPES buffer, except that amphotericin B (Sigma) (5 µg/mL in DMSO; 0.1% v/v) was present for either 60 or 15 min immediately prior to addition of isotopic calcium. Measurement of calcium uptake was initiated by addition of 45CaCl2 (20 µCi/mL;
11 mCi/mmol final specific activity). A zero time sample was removed, filtered and washed within 5 s of addition of the isotope. Additional samples were removed at increasing intervals (0.5, 1, 2, 4, 6, 8 and 10 min) thereafter. Cells in all samples were collected immediately on Whatman 25 mm, 4 µm polycarbonate filters and washed by filtration with 25 vols of cold 100 mM Mg(C2H3O2)2, 10 mM LaCl solution (pH 6.0). Filters were transferred to vials containing 10 mL Bio-Safe II (Research Products International, Mount Prospect, IL, USA), and radioactivity was measured by scintillation counting.
Calcium efflux
For measurements of calcium efflux, C. albicans, grown as a suspension overnight in BHI medium, was collected by centrifugation, and washed twice by suspension and incubation for 30 min in a nominal calciummagnesium HBSS/HEPES buffer (pH 7.4) containing 10 mM glucose, but to which no magnesium or calcium was added. Cells were then resuspended in the same buffer and allowed to stand at room temperature until used. Aliquots (2 x 108 cells) of the final yeast suspension were collected by centrifugation, suspended in 0.4 mL fresh nominal buffer and layered on to the lower half of a 0.9 x 3 cm glass cover slip that had been coated (2 µg/cm2) previously with Cell-Tak (Collaborative Research, Inc., Bedford, MA, USA). Cells were allowed to stand for 4560 min, and the cover slip was then rinsed carefully to remove non-adherent cells. Cells that remained attached were incubated for 2 h in HBSS/HEPES buffer replete with magnesium [1.5 mM as the diacetate salt, Mg(C2H3O2)2] and calcium (1.8 mM CaCl2) and containing 45CaCl2 (50 µCi/mL;
28 mCi/mmol final specific activity). Next, the cover slip with adherent cells was placed in a 1 mL flow-through chamber and superfused with replete HBSS/HEPES buffer, but without isotopic calcium, at a rate of
2 mL/min. Superfusate was collected at 1 min intervals. After 10 min, amphotericin B (2.510 mg/L) or an equal volume of vehicle (DMSO, 0.1 µl/mL superfusate) was added to the superfusate. After 30 min, superfusion was stopped, and the cover slip was removed and immersed in 10 mL of Bio-Safe II to which 2 mL of HBSS/HEPES buffer had been added. Superfusion was then continued for another 4 min to ensure recovery of 45Ca2+ from the tubing located between the cover slip chamber and the fraction collector. Total radioactivity of each superfusate sample, after addition to 10 mL of Bio-Safe II, and residual-cell associated 45Ca2+ was measured by liquid scintillation counting. Residual radioactivity of adherent cells/cover slip ranged from 3500 to 7500 dpm, whereas the radioactivity associated with similarly treated coverslips, without adherent cells, was typically
200 dpm. Calcium efflux during each 1 min period is expressed as a percentage of the total 45Ca2+ (dpm) associated with the cells at the beginning of that interval, i.e. the calcium efflux coefficient.11 Visual inspection of attached cells prior to loading with 45Ca2+, or after the superfusion period, indicated that they were of yeast form.
Thymidine uptake
Effects of amphotericin B on yeast viability during the measurement of calcium fluxes were assessed by measurement of both cell number and subsequent incorporation of radiolabelled thymidine. C. albicans, grown overnight as a suspension in BHI medium, was collected by centrifugation, washed and then resuspended in HBSS/HEPES buffer (as described for calcium flux experiments) to a final density of 13 x 107 cells/mL. The suspension was divided equally into three aliquots. DMSO (0.5 µl/mL) was added to one (control), amphotericin B was added to the other two aliquots in an equal volume of DMSO to a final concentration of 5 mg/L, and all were incubated at room temperature on an orbital shaker (60 rpm). Fifteen (amphotericin B) or 60 min (amphotericin B, control) thereafter, cells were collected by centrifugation and then resuspended to the original volume in BHI medium containing 2 µCi/mL [3H]methyl thymidine (2 Ci/mmol; New England Nuclear, Boston, MA, USA). Duplicate aliquots of each suspension were taken immediately for determination of cell number, and four aliquots were taken for measurement of initial (t = 0) cell-associated radioactivity. Cells were next incubated at 37°C with shaking, and additional aliquots were removed after 1 and 2 h. For measurement of cell density, each suspension was serially diluted, and cells were counted manually using a haemocytometer. The dilution that permitted counting of 100200 cells was used in the final calculation of cell number. For measurement of thymidine uptake, cell aliquots were diluted in two volumes of HBSS/HEPES buffer and centrifuged (10 000g for 5 min). The cell pellet was washed once and then transferred to 20 vols of Bio-Safe II liquid scintillation cocktail.
Replicate measurements were averaged to provide a single value per treatment per time point in any one experiment, and the experiment was repeated three times. ANOVA was used to determine whether significant (P < 0.05) treatment or time effects existed, and means were compared using the NewmanKeul test.
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Results |
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Activity of amphotericin B against C. albicans was concentration dependent (Figure 1). At concentrations >1 x MIC (1 mg/L or 1.082 µM), amphotericin B exhibited rapid fungicidal activity that was not affected by 0.1 mM (data not shown) or 1.0 mM EGTA. Concentrationresponse curves for amphotericin B (at 24 h) without (Figure 1a) or with (Figure 1b) EGTA were indistinguishable (EC50: 0.62 and 0.73 x MIC, respectively).
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Results of both empirical and mathematical estimates of media calcium concentrations were consistent with an EGTA-dependent reduction in extracellular calcium concentration (with minimal predicted effect on extracellular magnesium concentration) (Table 1).
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Acute (10 min) calcium accumulation by C. albicans was biphasic (Figure 2). The initial phase of calcium uptake was faster, but short-lived. Within 1 min, there was a transition to a second, more prolonged phase in which the rate of calcium accumulation was
50 times slower (0.87 ± 0.11 versus 0.018 ± 0.005 nmol/min/108 cells). Incubation with amphotericin B affected both phases of calcium uptake (Figures 2 and 3). For instance, exposure of yeast to 5 mg/L amphotericin B for 60 min decreased the rate of the initial phase by 67% and increased the rate of the secondary phase more than four-fold. Exposure to 5 mg/L amphotericin B for a shorter period (15 min), in contrast, did not significantly affect calcium accumulation. Nonetheless, the rates of accumulation during both the initial and the secondary phases of calcium uptake by cells exposed to amphotericin B for only 15 min were intermediate to those of control cells and cells exposed to amphotericin for 60 min; results were consistent with time-dependent effects of amphotericin B on the plasma membrane.
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Effect of amphotericin B on calcium efflux
Despite its relatively rapid effects on calcium uptake, amphotericin B had no discernible effect on efflux of calcium within a comparable period. As noted in Figure 4, calcium efflux from cells exposed to 5 mg/L amphotericin B for as long as 20 min was indiscernible from calcium efflux from unexposed cells. At 10 mg/L, amphotericin B also failed to affect calcium efflux (data not shown). The absence of an effect of amphotericin B on calcium efflux was also independent of loading conditions. Amphotericin B was without effect in cells loaded with isotopic calcium for up to 20 h, regardless of whether loading took place in the presence or absence of extracellular calcium (data not shown).
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Short-term exposure of C. albicans to amphotericin B under conditions in which calcium fluxes were measured had no apparent effect on cell number. The number of yeast cells determined at the end of either a 15 or 60 min incubation at room temperature in HBSS/HEPES buffer containing 5 mg/L amphotericin B was within 1% of the initial count (13.1 ± 0.4 x 107 cells/mL). The number (density) of control cells also remained constant under these conditions during the same periods. Nonetheless, when yeast cells were subsequently incubated under growth conditions (i.e. in BHI medium at 37°C), prior treatment with amphotericin B affected both cell number and thymidine uptake (Figure 5). Yeast not previously exposed to amphotericin B exhibited a modest (15%) increase in cell number (Figure 5a) within 1 h that was accompanied by accumulation of thymidine (Figure 5b). Exposure to 5 mg/L amphotericin for 15 min, although not affecting the absolute cell count at 2 h, decreased the rate of yeast proliferation. It also substantially decreased the initial rate of thymidine uptake. A longer (60 min) prior exposure to the same concentration of amphotericin B subsequently caused about a 25% loss of cells during a 2 h incubation in BHI medium. Interestingly, surviving cells continued to accumulate thymidine, albeit at a slower rate than cells exposed to amphotericin B for only 15 min (or not at all).
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Discussion |
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Results of the present studies indicate that in C. albicans, activity of amphotericin B is independent of a change in extracellular calcium concentrations. Depletion of extracellular calcium had no effect on amphotericin B-induced cell killing. Furthermore, the present data indicate that changes in transmembrane calcium fluxes do not contribute to the long-term fungicidal activity of amphotericin B. However, the possibility that calcium has a permissive role in the actions of amphotericin B can not be excluded.
Previous measurements of calcium accumulation in yeast forms of Saccharomyces cerevisiae2 and C. albicans21 were made under conditions of nominal (µM) calcium and in the absence of magnesium; the latter has been noted to displace calcium associated with the cell wall of S. cerevisiae22 and to decrease calcium accumulation in both S. cerevisiae and C. albicans.21,22 Results of preliminary studies in this laboratory also indicate that addition of magnesium to the medium decreases the uptake of 45Ca2+ by C. albicans previously maintained in a nominal calcium and magnesium buffer (data not shown). Addition of 1.8 mM unlabelled calcium also caused a decrease in the accumulation of 45Ca2+ in C. albicans expressed in terms of radioactivity, but if isotope dilution is taken into account, the rate of calcium influx (expressed in terms of mass) in the presence of 1.8 mM CaCl2 is 50 times greater than it is in the presence of nominal (6 µM 45CaCl2 only) calcium (563.0 ± 61.1 versus 11.5 ± 0.6 pmol/min/108 cells). Likewise, in the presence of millimolar concentrations of both calcium (1.8 mM CaCl2) and magnesium [1.5 mM Mg(C2H3O2)2], the rate of calcium uptake (235.3 ± 36.1 pmol/min/108 cells), although decreased compared with that in the presence of 1.8 mM calcium alone, was still some 25-fold greater than the rate in the presence of nominal calcium.
The biphasic nature of acute calcium uptake in C. albicans reported here is similar to that reported by Eilam & Chernichovsky23 in S. cerevisiae. Calcium uptake initiated by addition of 1 µM calcium to S. cerevisiae previously maintained in the absence of calcium (and magnesium) exhibited an initial rapid phase that became saturated within 3040 s, and was followed within 6090 s by a second, linear phase of calcium uptake. The initial phase was attributed to influx across the membrane into the cytosol and binding to the cell, whereas the second phase was attributed to accumulation within intracellular vesicles. Calcium uptake by C. albicans yeast reported here, measured in the presence of extracellular calcium and magnesium, plateaued within the same period (60 s), but was not followed by a secondary increase in the rate of calcium accumulation. Rather, calcium uptake continued, but at a substantially slower rate for as long as 910 min. It is presently unclear whether this slower, but apparently sustained, calcium uptake in C. albicans reflects accumulation within intracellular vesicles. Interestingly, amphotericin B decreased the initial rate of calcium uptake in C. albicans and concomitantly increased the second phase. The former effect is consistent with a decrease in membrane integrity due to binding of ergosterol and formation of pores, such that regulated calcium uptake is disrupted. With a loss of membrane integrity, and whatever restriction the membrane imposed on cytosolic calcium concentration, intracellular vesicles may be exposed directly to millimolar concentrations of calcium, with the rate of vesicular calcium uptake increasing accordingly.
The possibility that changes in cell viability contributed to the effects of amphotericin B on calcium uptake can not be excluded. This consideration takes on greater importance as the concentration and duration of exposure to amphotericin B increase. Based on the mechanism of action of amphotericin B, effects on the plasma membrane would be expected even after a brief (1560 min) exposure. Consequent effects on cell function would be expected to be more subtle with shorter exposures, become more pronounced with longer exposures and to vary with the endpoint measured. Moreover, a direct, progressive relationship would be expected between loss of membrane integrity, metabolic function and cell viability, whether the latter is considered in terms of lifedeath or inability to maintain homeostasis. Given these considerations, it may be impossible within a cell population to separate effects of amphotericin B on cell viability from those on calcium influxes.
In that regard, results of the present studies indicate clearly that cell function was affected by amphotericin B. Yet, the relationship between function (thymidine uptake or replication) and viability in the context of calcium fluxes is not as obvious. An immediate effect of amphotericin B action on the plasma membrane would be a change in membrane potential with concomitant effects on transmembrane calcium fluxes, and the present results are consistent with that premise. Nonetheless, even a short-term exposure to amphotericin B, for as short as 15 min, had more global cellular consequences. In addition, durations of exposure to amphotericin B of 15 or 60 min subsequently caused comparable reductions in thymidine uptake, but had opposing effects on cell number. Also, yeast exposed to amphotericin B for 15 min, although functionally compromised, remained viable in as much as they were capable of replicating under appropriate conditions. Although that conclusion may not apply for every individual cell, it does hold for the population as a whole. Likewise, a greater number of yeast were non-viable after exposure to amphotericin B for 60 min, as indicated by a subsequent reduction in cell number when placed into growth-promoting conditions. Yet, remaining cells retained some function and were capable of accumulating thymidine.
Caution is also warranted in evaluation of cell viability because methods used here to examine the short-term effects of amphotericin B provide indices that are only indirectly related to measurements of calcium fluxes. For instance, amphotericin B did not affect cell number when yeasts were kept under non-growth (experimental) conditions (i.e. in HBSS/HEPES buffer). This observation, together with the fact that cell density on cover slips superfused with amphotericin B in the same buffer for as long as 2 h remained constant, suggests that cell viability was not appreciably compromised. However, the presence of a cell wall may have artefactually maintained apparent cell number, even if the integrity of the plasma membrane was lost. Yet, this argument does not appear to apply to long-term (60 min) exposure to amphotericin B. Nor did amphotericin B (even at 10 mg/L) affect calcium efflux, as would be expected if it acutely decreased cell viability. It might also be that the relationship between effects of amphotericin B on function and viability (cell number) reflects the specific experimental conditions; the loss of viability, for example, being more pronounced if the cells are stimulated to grow (i.e. in BHI medium) than it is if the cells are quiescent, but metabolically active (i.e. in HBSS/HEPES buffer). These and other possibilities require further evaluation.
The lack of effect of amphotericin B (from 2.5 to 10 mg/L; although only 5 mg/L is reported here) on calcium efflux indicates that amphotericin B does not mobilize intracellular calcium pools. These data argue against a significant role of calcium in the acute effects of amphotericin B on C. albicans. Yet, the possibility that calcium is required for, or contributes to, the regulation of some cellular processes can not be excluded. An effect of amphotericin B on such a process secondary to alterations in calcium handling by the cell could conceivably contribute to its fungicidal activity. Even so, it would appear to be a long-term effect, and contrasts with the role of an increase in calcium in the onset of apoptosis or necrosis in other cell types.
In conclusion, the antifungal activity of amphotericin B in C. albicans is not directly dependent upon extracellular calcium concentration. Furthermore, amphotericin B had no effect on calcium efflux, and its effect on acute calcium influx had no apparent consequence on long-term calcium accumulation. Studies to further clarify the mechanisms of antifungal action of the polyene antibiotics are warranted.
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Acknowledgements |
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This work was presented at the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1720 September 2000.7
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Footnotes |
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