Dependence of Synergistic Fungicidal Activity of Cu²⁺ and Allicin, an Allyl Sulfur Compound from Garlic, on Selective Accumulation of the Ion in the Plasma Membrane Fraction via Allicin-Mediated Phospholipid Peroxidation

• Abstract
• Introduction
• Materials and Methods
• Measurement of yeast cell growth and viability
• Detection of intracellular ROS production
• Detection and measurement of plasma membrane phospholipid peroxidation
• Measurement of Cu²⁺ content in subcellular fractions
• Assay of plasma membrane permeability change
• Chemicals
• Results and Discussion
• References

Abstract

Allicin was effective in decreasing the lethal concentration of Cu²⁺ against various fungal strains including a plant pathogen, Fusarium oxysporum, so that the minimum fungicidal concentration (MFC) of the ion for the fungus could be reduced to 2 % of that detected without allicin. In Saccharomyces cerevisiae, Cu²⁺ was not apparently taken up by cells when added alone at a non-lethal concentration, whereas the ion was efficiently incorporated into cells in the presence of allicin, as in the case of cells treated with the ion at a lethal concentration. Although allicin likely increased cellular permeability to Cu²⁺ due to its promotive effect on plasma membrane phospholipid peroxidation, these cell-surface events did not result in endogenous reactive oxygen species (ROS) production, a typical toxic effect of the ion. Cu²⁺ was detected in the cytoplasmic fraction of cells that had been treated with the ion at a lethal concentration, whereas the ion was entrapped in the plasma membrane fraction upon their treatment with the ion at a low concentration in combination with allicin. Cu²⁺ could be solubilized from the plasma membrane fraction by a procedure for the extraction of hydrophobic proteins rather than the extraction of phospholipids, suggesting its complexation with a plasma membrane protein as a result of allicin treatment. Such a subcellular localization of Cu²⁺ resulted in the selective leakage of intracellular K⁺, but not in the disruptive damage on the plasma membrane, and was considered to underlie the synergistic fungicidal activity of Cu²⁺ and allicin.

Introduction

Copper is an essential trace element for growth and metabolism in organisms including microorganisms [1]. However, it exerts toxic effects when its intracellular concentration increases to higher than the physiologically required level [2]. The toxic effects of Cu²⁺ have been elucidated by its promotive effect on the endogenous generation of ROS such as hydroxyl radicals, which results in cellular damage caused by the oxidation of proteins, cleavage of DNA and RNA, and plasma membrane disruption [3], [4], [5]. The Fenton reaction additionally contributes to the production of hydroxyl radicals by Cu²⁺ [6]. Toxic events induced by Cu²⁺ also depend on its direct action such as substitution for other metal ions essential for cell viability [4]. However, the precise molecular mechanism(s) underlying this toxicity has yet to be clearly ascertained.

Allicin (diallyl thiosulfinate) is the main allyl sulfur component of garlic (Allium sativum); alliinase (alliin lyase) catalyzes its synthesis from the corresponding amino acid precursor, alliin [7], [8]. Allicin exerts antimicrobial activities depending on its inhibitory effects on certain thiol-containing enzymes via strong SH-modifying properties, as reflected by the production of S-allylmercaptocysteine from L-cysteine [9], [10], [11]. This sulfur compound decreases cellular glutathione content as a result of its direct interaction with the corresponding SH group [10], [11]. Nevertheless, allicin exhibits antioxidant activity because S-allylmercaptoglutathione, the product of allicin with glutathione, possesses antioxidant properties. This suggests that allicin is protective against the lethal effect of Cu²⁺ dependent on oxidative stress induction.

In our previous study, however, we found the amplification of the fungicidal activity of Cu²⁺ against the yeast Saccharomyces cerevisiae in the presence of allicin [12]. Cu²⁺ indeed accelerated ROS generation within the yeast cells when added at a lethal concentration, but such an intracellular oxidative stress induction was not seen during cell death progression upon Cu²⁺ treatment at a low concentration in combination with allicin. Instead, allicin treatment resulted in the elimination of alkyl hydroperoxide reductase 1 (AHP1) from the yeast plasma membrane fraction, which generally protects against the toxic effect of Cu²⁺ [13]. This may indicate that allicin accelerates plasma membrane disruption in cooperation with the external attack of Cu²⁺ on plasma membrane phospholipids at a concentration lower than that required for Cu²⁺ incorporation across the plasma membrane for inducing endogenous ROS production. It remains unclarified whether the synergistic fungicidal activity of Cu²⁺ and allicin depends on plasma membrane disruption as in the case of toxicity induced by Cu²⁺ alone.

In this study, we examined whether allicin can amplify the fungicidal activity of Cu²⁺ on other fungal strains, including a plant pathogen, Fusarium oxysporum, a target of copper-based fungicides. We thus evaluated the role of allicin in the amplification of the lethal effect of Cu²⁺ using Saccharomyces cerevisiae cells by examining its effect on cellular uptake and the subcellular localization of the ion. We hereby show the characteristic localization pattern of Cu²⁺ in the yeast plasma membrane fraction, not in the cytoplasmic fraction, of allicin-treated cells. The subcellular localization of Cu²⁺ was closely related to the modification of yeast plasma membrane ion transport function, and considered to underlie the synergistic fungicidal activity of Cu²⁺ and allicin.

Materials and Methods

Measurement of yeast cell growth and viability

The effects of allicin on the lethal concentration of Cu²⁺ were examined by the turbidity-dependent microplate assay using Candida albicans IFO 1061 (Institute for Fermentation; Osaka, Japan), Aspergillus fumigatus IFO 5840 (Institute for Fermentation; Osaka, Japan), Fusarium oxysporum NBRC 5942 (NITE Resource Center; Tokyo, Japan) and S. cerevisiae W303 - 1A (Dr. T. Nakamura [14]). Cells of yeast strains were grown in YPD medium, which contained (per liter) 10 g of yeast extract, 20 g of peptone and 20 g of glucose, with vigorous shaking at 30 °C. After diluting an overnight culture with distilled water to 10⁶ cells/mL, the cell suspension was incubated with Cu²⁺ and allicin at various concentrations at 30 °C for 24 h. Filamentous fungi were precultivated in 2.5 % (w/v) malt extract medium (Oriental Yeast Co.; Tokyo, Japan) at 30 °C for 24 h and were diluted 100-fold with distilled water for further incubation with Cu²⁺ and allicin at various concentrations at 30 °C for 24 h. An aliquot (10 μL) of each incubation mixture was further mixed with 100 μL of YPD medium (for yeast) or 2.5 % (w/v) malt extract medium (for filamentous fungi) in a microplate and was incubated at 30 °C for 48 h. Under the above conditions, the minimum fungicidal concentration (MFC) of Cu²⁺ was defined as the concentration that absolutely prevented turbidity increase or mycelial growth in each medium after a 48-h incubation, reflecting a significant loss of cell viability during the preceding 24-h incubation with Cu²⁺ and allicin in distilled water.

Overnight cultures of S. cerevisiae W303 - 1A were also diluted in distilled water to 10⁷ cells/mL, in which Cu²⁺ and allicin were mixed at various concentrations, and the cell suspensions were used in the following experiments for the characterization of fungicidal effects of Cu²⁺ and/or allicin. The viable cell number was determined by counting colony-forming units after a 48-h incubation at 30 °C in YPD medium containing 1.8 % (w/v) agar.

Detection of intracellular ROS production

Intracellular ROS production was examined by a method based on intracellular deacylation and the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate to the corresponding fluorescent compound as follows [14]. Cells cultured overnight were harvested by centrifugation, suspended in YPD medium to obtain a density of 10⁷ cells/mL and incubated with 40 μM 2′,7′-dichlorodihydrofluorescein diacetate at 30 °C for 60 min. The cells were collected by centrifugation and suspended in an equal volume of distilled water. The cell suspensions (1.0 mL) were further incubated with Cu²⁺ at a lethal concentration and at a non-lethal concentration in the absence or presence of allicin at 30 °C for 60 min, and then washed and suspended in 100 μL of phosphate-buffered saline (PBS). Cells were then observed under a phase-contrast microscope and a fluorescence microscope with excitation at 480 nm and emission at 530 nm.

Detection and measurement of plasma membrane phospholipid peroxidation

Plasma membrane phospholipid peroxidation was examined using a fluorescence probe, diphenyl-1-pyrenylphosphine (DPPP), as follows [15]. Cells cultured overnight were preincubated in PBS at 30 °C at a density of 3 × 10⁷ cells/mL for 5 min. After the addition of DPPP (in dimethyl sulfoxide) at a final concentration of 50 μM, the cell suspension was incubated for 10 min in the dark. Cells were washed three times with PBS and finally suspended in distilled water at a density of 10⁷ cells/mL. The cell suspensions were further incubated with Cu²⁺ at a lethal concentration and a non-lethal concentration in the absence or presence of allicin at 30 °C for 60 min. The fluorescence intensities of the cell samples (10⁶ cells) were measured using an FP-1520S fluorescence detector (JASCO; Tokyo, Japan), in which the wavelengths of excitation and emission were set to 351 and 380 nm, respectively. The arbitrary units were based directly on fluorescence intensity. Cells were also observed under a phase-contrast microscope and a fluorescence microscope.

Measurement of Cu²⁺ content in subcellular fractions

Cells cultured overnight were diluted to 10⁷ cells/mL in distilled water, and the cell suspensions were incubated with Cu²⁺ at a lethal concentration and a non-lethal concentration in the absence or presence of allicin at 30 °C for 60 min. Cells were collected from a 1.0 mL suspension by centrifugation at 5000 g for 3 min and the supernatant obtained was used for the measurement of Cu²⁺ that was not incorporated into cells. After washing with distilled water, cells were resuspended in 1.0 mL of distilled water and disrupted by repeated vortexing with glass beads. The supernatant obtained by centrifugation at 5000 g for 3 min was used for the measurement of Cu²⁺ in the cytoplasmic fraction. Insoluble precipitates were further incubated in a mixture of methanol, chloroform and distilled water for the extraction of plasma membrane phospholipids [16]. After allowing the mixture to stand for 1 h, the upper solvent layer was withdrawn and evaporated under vacuum. The residue was dissolved in 1.0 mL of distilled water and used for the measurement of Cu²⁺ in the plasma membrane phospholipid fraction. The insoluble precipitates obtained above were alternatively incubated in 1.0 mL of 2 % (w/v) sodium dodecyl sulfate (SDS) containing 1 mM phenylmethylsulfonyl fluoride at 95 °C for 10 min for the extraction of plasma membrane-associated proteins. The supernatant obtained after centrifugation at 5000 g for 3 min was used for the measurement of the concentration of Cu²⁺ from the plasma membrane protein fraction by a colorimetric method using a cuprous detection reagent PONAL kit according to the supplier’s instructions (Dojindo; Kumamoto, Japan). The presence of SDS did not interfere with this colorimetric measurement of Cu²⁺.

Assay of plasma membrane permeability change

Plasma membrane permeability was assayed by measuring the effluxes of both intracellular K⁺ and UV-absorbing materials such as nucleotides as follows. Cells cultured overnight were harvested by centrifugation, washed with 50 mM succinate buffer (pH 6.0) and suspended in the same buffer to obtain a density of 10⁷ cells/mL. The cell suspensions obtained were shaken with Cu²⁺ at a lethal concentration and a non-lethal concentration in the absence or presence of allicin at 30 °C for 60 min. The supernatants obtained after cell removal by centrifugation were assayed for K⁺ content using a K⁺ assay kit (HACH; Floriffoux, Belgium) based on the tetraphenylborate method [17]. The amount of UV-absorbing materials was expressed as the value of absorption at a wavelength of 260 nm.

Chemicals

Allicin was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). 2′,7′-Dichlorodihydrofluorescein diacetate was obtained from Molecular Probe (Eugene, OR, USA). DPPP was from Dojindo (Kumamoto, Japan). The other chemicals were of analytical reagent grade.

Results and Discussion

We examined the effects of allicin on the lethal concentrations of Cu²⁺ against various yeast and fungal strains by measuring the MFC. As shown in Fig. [1], Cu²⁺ exhibited a lethal effect on S. cerevisiae cells at 50 μM, and the lethal concentration markedly decreased to 3.13 μM with increasing concentrations of allicin up to 60 μM. Cu²⁺ was less effective in killing C. albicans cells but its lethal concentration similarly decreased in the presence of allicin, thus obtaining the same MFC. Although A. fumigatus is highly resistant to Cu²⁺, the presence of allicin could render the fungus extremely sensitive to the ion, suggesting the involvement of the same type of lethal event in the combined fungicidal actions of Cu²⁺ and allicin against filamentous fungi. Allicin could also decrease the MFC of Cu²⁺ against a strain of the plant pathogen Fusarium oxysporum to less than 2 % of that obtained using the ion alone. This indicates a possibility of the application of allicin for the improvement of copper-based fungicides.

It is therefore important to clarify how Cu²⁺ exerts its toxicity when it is administered to these fungal strains at a low concentration in combination with allicin. Heavy metals, such as copper, nickel, and zinc, become toxic when their intracellular concentrations increase to higher than physiologically required levels [2]. The allicin-dependent fungicidal activity of Cu²⁺ may be simply attributed to the stimulatory effect of allicin on the cellular uptake of the ion. We first examined the relationship between the cellular uptake of Cu²⁺ and its extracellular concentration under the condition with or without allicin. As shown in Fig. [2], Cu²⁺ was efficiently taken up by cells when added alone at 250 μM so that its extracellular concentration decreased to 236 μM after 1 h of incubation. The difference corresponded to the cellular uptake of the ion equivalent to 14 nmol per 10⁷ cells, which agrees with its lethal effect of accelerating endogenous ROS production at this concentration. Cu²⁺ was taken up by cells to a lesser extent when added at 120 μM, but the ion was not incorporated into cells at 30 μM. In the presence of 30 μM allicin, however, cells become permeable to Cu²⁺ even at the non-lethal concentration (30 μM), resulting in the cellular uptake of the ion equivalent to 10 nmol per 10⁷ cells during 1 h of incubation. The allicin-mediated incorporation of Cu²⁺ showed a clear dose response, which agrees with the synergistic fungicidal activity exhibited by a combination of these compounds [12].

We then examined whether Cu²⁺ can accelerate endogenous ROS production when the ion is taken up by cells in the presence of allicin. As shown in Fig. [3] A, Cu²⁺ at 250 μM accelerated ROS production in the entire cytoplasm, resulting in the decrease in viable cell number to 1.5 % of the original level [12]. The result reflected the cellular uptake of the ion across the plasma membrane into the cytoplasm via plasma membrane-associated Cu²⁺ transporters [18]. Cu²⁺ at 250 μM also accelerated plasma membrane phospholipid peroxidation to a lesser extent than that expected from its disruptive effect on the plasma membrane (Fig. [3] B). The hydroperoxide content of plasma membrane phospholipids should increase with accompanying peroxidation of fatty acyl moieties but it is expected to decrease during phospholipid decomposition via the structural modification of hydroperoxide to carboxylic acid [19]. Allicin can absolutely inhibit yeast cell growth, but shows neither fungicidal activity nor the associated disruptive effect on the plasma membrane even at 120 μM [12]. In agreement with the disappearance of AHP1 from the plasma membrane fraction of allicin-treated cells [12], this sulfur-containing compound could selectively and markedly enhance plasma membrane phospholipid peroxidation but did not promote intracellular ROS production (Fig. [3] A, B and C). It should be noted that Cu²⁺ did not promote intracellular ROS production at 30 μM even when the ion was taken up by cells in the presence of allicin (Fig. [3] A). The allicin-mediated uptake of Cu²⁺ did not contribute to the enhancement of plasma membrane phospholipid peroxidation, which was mostly induced by allicin itself (Fig. [3] B and C). Allicin-mediated plasma membrane phospholipid peroxidation seemed to underlie its stimulatory effect on the cellular uptake of Cu²⁺ together with the allicin-induced potentiation of the fungicidal activity of Cu²⁺, which is not dependent on endogenous ROS production.

Metal uptake in yeast cells typically occurs in two main steps, the first involves a non-specific binding of a metal to the cell surface, and the second involves a metabolism-dependent metal uptake into the cytoplasm [20]. Allicin is likely to stimulate the non-specific binding of Cu²⁺ to the cell surface but not its penetration into the cytoplasm. We therefore examined the subcellular localization of Cu²⁺ when cells were treated with the ion at a lethal concentration and a non-lethal concentration in the presence of allicin. As shown in Fig. [4], Cu²⁺ was mostly detected in the cytoplasmic fraction when it was added at 250 μM, whereas the ion was not detected in this fraction when it was taken up by cells in the presence of allicin. Cu²⁺ remained in the plasma membrane fraction and was efficiently solubilized by a procedure generally used for the extraction of plasma membrane proteins rather than that used for phospholipid extraction. This suggests that allicin treatment promotes the complexation between Cu²⁺ and a protein embedded in the plasma membrane phospholipid bilayers.

We considered that such a complexation may interfere with a certain plasma membrane function even though it is not inhibitory to the mechanism underlying the maintenance of plasma membrane integrity. Attention was paid to the effects of Cu²⁺ and/or allicin on the leakage of K⁺ and UV-absorbing materials, which are commonly used as indicators of plasma membrane permeabilization [3], [21]. As shown in Fig. [5], allicin did not induce the plasma membrane permeability change of yeast cells, as determined from the loss of K⁺ efflux and the leakage of UV-absorbing materials. At a lethal concentration, Cu²⁺ enhanced the cellular release of K⁺ and UV-absorbing materials to considerable extents, which agrees with its disruptive effect on plasma membrane phospholipid bilayers [22]. Unexpectedly, the combined fungicidal activities of Cu²⁺ and allicin were characterized by an extremely enhanced efflux of K⁺ to the extent comparable to that observed following amphotericin B (AmB) treatment. UV-absorbing materials were not released from the cells, indicating that the plasma membrane retained its function as a barrier to intracellular metabolites. The polyene macrolide antibiotic AmB exhibits a potent fungicidal activity induced by the acceleration of K⁺ efflux via the formation of K⁺-specific channels across the plasma membrane due to its interaction with ergosterol [23]. The synergistic fungicidal activity of Cu²⁺ and allicin may be related to a similar modification of plasma membrane ion transport function. Allicin is likely to induce the structural modification of a plasma membrane protein in favor of its direct interaction with Cu²⁺ or by indirectly enhancing such an interaction through plasma membrane phospholipid peroxidation.

In this study, we for the first time demonstrate a Cu²⁺-mediated lethal event, which is not accompanied by ROS production or plasma membrane disruption. The ion exhibited a fungicidal activity at a concentration lower than that required for stimulating plasma membrane-associated transporters in the presence of allicin. Under these conditions, the ion was detected in the yeast plasma membrane fraction, possibly due to its stable complexation with a protein embedded in phospholipid bilayers. As deduced from its strong SH-modifying ability, allicin may contribute to a conformational change of a plasma membrane protein that favors an increase in the Cu²⁺-binding ability of the protein. Our study of the detection and identification of such a protein from allicin-treated cells is currently in progress.

References

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Prof. Dr. Toshio Tanaka

Department of Biology and Geosciences

Graduate School of Science

Osaka City University

3-3-138 Sugimoto

Sumiyoshi-ku

Osaka 558-8585

Japan

Phone: +81-6-6605-3163

Fax: +81-6-6605-3164

Email: tanakato@sci.osaka-cu.ac.jp

References

• 1 Borst-Pauwels G WFH. Ion transport in yeast.  Biochim Biophys Acta. 1981;  650 88-127
  PubMedSearch in Google Scholar
• 2 Gadd G M. Interactions of fungi with toxic metals.  New Phytol. 1993;  124 25-60
  CrossrefPubMedSearch in Google Scholar
• 3 Avery S V, Howlett N G, Radice S. Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition.  Appl Environ Microbiol. 1996;  62 3960-6
  PubMedSearch in Google Scholar
• 4 Halliwell B, Gutteridge J MC. Oxygen toxicity, oxygen radicals, transition metals and diseases.  Biochem J. 1984;  219 1-4
  PubMedSearch in Google Scholar
• 5 Howlett N G, Avery S V. Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation.  Appl Environ Microbiol. 1997;  63 2971-6
  PubMedSearch in Google Scholar
• 6 Jamieson D J. Oxidative stress response of the yeast Saccharomyces cerevisiae .  Yeast. 1998;  14 1511-27
  CrossrefPubMedSearch in Google Scholar
• 7 Miron T, Shin I, Feigenblat G, Weiner L, Mirelman D, Wilchek M. et al . A spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates.  Anal Biochem. 2002;  307 76-83
  CrossrefPubMedSearch in Google Scholar
• 8 Stoll A, Seebeck E. Chemical investigation on alliin, the specific principle of garlic.  Adv Enzymol. 1951;  11 377-400
  PubMedSearch in Google Scholar
• 9 Ankri S, Mirelman D. Antimicrobial properties of allicin from garlic.  Microbes Infect. 1999;  2 125-9
  PubMedSearch in Google Scholar
• 10 Miron T, Rabinkov A, Mirelman D, Wilchek M, Weiner L. The mode of action of allicin: its ready permeability through phospholipids membranes may contribute to its biological activity.  Biochim Biophys Acta. 2000;  1463 20-30
  PubMedSearch in Google Scholar
• 11 Rabinkov A, Miron T, Mirelman D, Wilchek M, Glozman S, Yavin E. et al . S-Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties.  Biochim Biophys Acta. 2000;  1499 144-53
  PubMedSearch in Google Scholar
• 12 Ogita A, Hirooka K, Yamamoto Y, Tsutsui N, Fujita K, Taniguchi M. et al . Synergistic fungicidal activity of Cu²⁺ and allicin, an allyl sulfur compound from garlic, and its relation to the role of alkyl hydroperoxide reductase 1 as a cell surface defense in Saccharomyces cerevisiae .  Toxicology. 2005;  215 205-13
  CrossrefPubMedSearch in Google Scholar
• 13 Nguyên-nhu N T, Knoops B. Alkyl hydroperoxide reductase 1 protects Saccharomyces cerevisiae against metal ion toxicity and glutathione depletion.  Toxicol Lett. 2002;  135 219-28
  CrossrefPubMedSearch in Google Scholar
• 14 Machida K, Tanaka T, Taniguchi M. Depletion of glutathione as a cause of the promotive effects of polygodial, a susquiterpene on the production of reactive oxygen species in Saccharomyces cerevisiae .  J Biosci Bioeng. 1999;  88 526-30
  CrossrefPubMedSearch in Google Scholar
• 15 Takahashi M, Shibata M, Niki E. Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine.  Free Radic Biol Med. 2001;  31 164-74
  CrossrefPubMedSearch in Google Scholar
• 16 Bligh E G, Dyer W J. A rapid method of total lipid extraction and purification.  Can J Med Sci. 1959;  37 911-7
  CrossrefPubMedSearch in Google Scholar
• 17 Ramotowski S, Szczesniak M. Determination of potassium salt content in pharmaceutical preparations by means of sodium tetraphenylborate.  Acta Pol Pharm. 1967;  24 605-13
  PubMedSearch in Google Scholar
• 18 Peña M MO, Koch K A, Thiele D J. Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae .  Mol Cell Biol. 1998;  18 2514-23
  PubMedSearch in Google Scholar
• 19 Santrock J, Gorski R A, O’Gara J F. Products and mechanism of the reaction of ozone with phospholipids in unilamellar phospholipid vesicles.  Chem Res Toxicol. 1992;  5 134-41
  CrossrefPubMedSearch in Google Scholar
• 20 Blackwell K J, Singleton I, Tobin J M. Metal cation uptake by yeast: a review.  Appl Microbiol Biotechnol. 1995;  43 579-84
  PubMedSearch in Google Scholar
• 21 Nakayama K, Yamaguchi T, Doi T, Usuki Y, Taniguchi M, Tanaka T. Synergistic combination of direct plasma membrane damage and oxidative stress as a cause of antifungal activity of polyol macrolide antibiotic niphimycin.  J Biosci Bioeng. 2002;  94 207-11
  CrossrefPubMedSearch in Google Scholar
• 22 Soares E V, Hebbelinck K, Soares H MVM. Toxic effects caused by heavy metals in the yeast Saccharomyces cerevisiae: a comparative study.  Can J Microbiol. 2003;  49 336-43
  CrossrefPubMedSearch in Google Scholar
• 23 Ghannoum M A, Rice L B. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance.  Clin Microbiol Rev. 1999;  12 501-17
  PubMedSearch in Google Scholar

Prof. Dr. Toshio Tanaka

Department of Biology and Geosciences

Graduate School of Science

Osaka City University

3-3-138 Sugimoto

Sumiyoshi-ku

Osaka 558-8585

Japan

Phone: +81-6-6605-3163

Fax: +81-6-6605-3164

Email: tanakato@sci.osaka-cu.ac.jp