a Department of Biochemistry and Bioproducts Research Center, Yonsei University, 134 Shinchon-dong, Sudemoon-ku, Seoul 120-749, South Korea; b Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK; c Han Wha Group Research and Engineering Center, Taejeon 305-345, South Korea
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In the course of screening for novel antifungal agents from Korean medicinal plants, several extracts were identified, which caused significant inhibition of growth of a range of Candida spp. The active agents were purified from Coptis rhizoma and Phellodendron amurense and both berberine and palmatine were identified. The modes of action of these compounds were investigated by determining their effects on enzymes with important roles in membrane and cell wall biosynthesis of C. albicans.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fungal isolates used were C. albicans (ATCC10231, -11651, -28838), and clinical isolates of Candida krusei, Candida parapsilosis, C. glabrata and Candida guilliermondii (the culture collection of the Department of Microbiology, University of Leeds, UK). Cells were maintained on slopes of Sabourauds dextrose agar (Difco, Detroit, MI, USA) at 4°C. To obtain yeast growth forms, cells were inoculated into Sabourauds dextrose broth, pH 5.6, and incubated for 18 h at 30°C, as a shake culture as described. 15 Late exponential phase cells (18 h) were harvested and washed by centrifugation, and used for the preparation of microsomes. Mycelial forms of C. albicans were prepared by incubating cells in Sabourauds dextrose broth containing 20% (v/v) newborn bovine serum at 37°C for 24 h. 16
Preparation of microsomes of C. albicans
The preparation of microsomes of C. albicans was based on the method of Ator et al. 17 Harvested cells (mycelial growth form: 65- 70% of total cells were hyphae) were suspended in 2.5 vol. of 0.1 M potassium phosphate (pH 7.4, 1 mM ethylenediamine tetra-acetic acid (EDTA)), and centrifuged at 5000g for 5 min. The washed cells were resuspended in a solution of 0.1 M potassium phosphate (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.25 mM phenylmethylsulphonylfluoride (PMSF) at approximately 2 x 10 8 cells/mL and broken by shaking with glass beads (0.45- 0.5 mm) in a B. Braun disintegrator (MSK, B. Braun Biotech. GMBH, Schwarzenberger-WEG, Germany) for a total of 3 min interspersed with periods of cooling. Cell debris was removed by centrifugation at 11,000g for 20 min, and the supernatant was then centrifuged at 105,000g for 1 h. The resulting microsomal pellets were resuspended in a solution of 0.1 M potassium phosphate (pH 7.4), 1 mM EDTA, 20% glycerol, divided into aliquots, and stored at -70>C. Protein concentration was determined by the method of Bradford 18 using bovine serum albumin as standard.
Extraction and purification of medicinal herbs using organic solvents
The plants were purchased from the Korean medicinal herb dispensary (Hanyakyutong Co.) in Seoul and subjected to methanolic extraction as described. 19 Briefly, about 200 g of powders of dried medicinal plants that include Ligustrum lucidum, Anemarrhena asphodeloides, Phellodendron amurense, Coptis rhizoma and Corydalis yanhusuo were extracted with 3 vol. of 80% methanol by refluxing for 3 h. The extracts were filtered, and the filtrates concentrated by evaporation in vacuo to dryness. Total methanolic extracts were subjected to a bioactivity-guided fractionation procedure. Dried filtrates were extracted with 2 vol. of dichloromethane. The dichloromethane- soluble fractions were dried with magnesium sulphate and lyophilized. These organic fractions were further purified by thin layer chromatography (TLC) using n-hexane as solvent. The bands with antifungal activity were extracted with n-hexane and purified with high-performance liquid chromatography (HPLC) employing C-18 octyl column using dichloromethane- ethanol (95:5) and a UV detector. 19 Collected active antifungal fractions were crystallized (e.g. approximately 120 mg of palmatine) from methanol. The purified compounds were identified as berberine and palmatine when they were subjected to mass spectrometry (MS), nuclear magnetic resonance spectrometry (NMR) and infrared (IR) spectral analyses. There was good agreement between the isolated protoberberines and authentic commercial sources (Sigma, St Louis, MO, USA) with respect to MS, IR, and NMR (data not shown).
Agar diffusion susceptibility test
Molten medium (yeast nitrogen base with 1% glucose; YNBG) was allowed to cool to 50°C and 20 mL aliquots were poured into 100 x 15 mm round Petri dishes as described. 20 After the agar had solidified at room temperature, any excess surface moisture or droplets on the agar surface were eliminated by incubating the plates at 35°C for no longer than 30 min. Seven Candida spp. including C. albicans (ATCC10231), C. albicans (ATCC28838), C. albicans (ATCC11651), C. parapsilosis, C. glabrata, C. guilliermondii and C. krusei were used in this study. Inocula of these test Candida spp. were standardized by making water suspensions from the 24 h cultures and adjusting them to match the turbidity of a 10 4 cells/mL standard. Sterile, non-toxic swabs were dipped into the adjusted suspensions and the YNBG agar plates inoculated. The entire dried surface of the agar was streaked three times by rotating the plates 60° each time. Miconazole and dried methanolic extracts of medicinal herbs were dissolved in dimethylsulphoxide (DMSO, final concentration not exceeding 0.3% w/v). Discs (8 mm) (Whatman, Kent, UK) were soaked in these solutions to give final contents of 200 µg of miconazole and 4000 µg of medicinal herbs. The prepared discs, plus blank controls, were applied to the plates, which were placed in an incubator within 15 min, and incubated at 35°C for 48 h. Following incubation, zones of inhibition were measured with a ruler.
Broth microdilution susceptibility test
Test compounds dissolved in the stock solution (0.3%, w/v, DMSO), were diluted in Rosewell Park Memorial Institute (RPMI) 1640 medium (pH 7.0) and 100 µL of each agent was applied to 96-well plates. The MIC values of the various agents, including miconazole and amphotericin B, were determined by a broth dilution method using microdilution in 96-well microtitre trays. 21 Each of seven Candida spp. cells was inoculated to approximately 2 x 10 4 cells/mL in 100 µL medium, containing doubling dilutions of the agents. The addition of cell suspension to each well created a final range of antifungal agent concentrations from 100 to 0.05 mg/L. The trays were covered with loose-fitting lids and incubated for 72 h at 35°C. MIC values were determined three times, and only representative data are shown here.
Sterol biosynthesis assay
Washed cells of C. albicans (ATCC10231) were incubated with radiolabelled substrates as described elsewhere. 22,23 Briefly, washed cells were incubated in medium A (yeast nitrogen base (YNB) without amino acid and 1% (w/v) glucose, pH 6.5) for 18 h at 30°C, then the cells were harvested and washed. Washed cells were resuspended in medium B (0.1 mM potassium phosphate buffer and 1% (w/v) glucose). An aliquot consisted of a 980 µL cell suspension (2 x 109 cells/mL) in a 15 mL capped tube at 30°C. Protoberberines or miconazole were dissolved in DMSO (to give a final concentration of 0.12 µM) and cells were preincubated with drug for 10 min. The reaction was initiated by addition of 10 µL of [14C]acetate (1 microcurie (µCi), specific activity 56 mCi/mmol) or L-[methyl-14C]methionine (1 µCi, specific activity 1 mCi/mmol) and the cells were incubated for 3 h. The reaction was stopped by addition of 1 mL 15% (w/v) KOH, 90% ethanol and the samples were saponified at 80°C for 1 h. The non-saponifiable lipids were then extracted with petroleum ether and the samples evaporated to dryness. Extracts were dissolved in 60 µL chloroform and loaded on to TLC plates. The plates were eluted with toluene- diethyl ether (9:1). Bands were visualized under UV light, identified using standards and cut out, and the radioactivity was determined by liquid scintillation counting (Beckman 6500; Beckman, Palo Alto, CA, USA).
Assay of sterol 24-methyl transferase (24-SMT)
Enzyme assays were conducted by a slight modification of the method of Ator et al. 17 The assay mixture contained 0.1 M Tris- HCl (pH 7.5), 0.4 mg microsomal protein, 30 mM KHCO 3, 5 mM MgCl 2, 200 µM desmosterol, and 50 µM [methyl-14C]S-adenosylmethionine (SAM) (0.25 µCi) in a volume of 500 µL, in an assay vial. Aqueous desmosterol solutions were prepared as suspensions with nonionic detergent Triton WR-1339 (tyloxapol; Sigma, St Louis, MO, USA) as described.24 Assays were incubated at 30°C in a shaking water bath for 25 min to determine the initial velocity of the reaction. The reactions were terminated by addition of 500 µL 15% (w/v) KOH, 90% (v/v) ethanol plus [1a,2a(n)-3H]1-cholesterol (0.015 µCi, specific activity 46 Ci/mmol). Extraction of products was carried out as for sterol biosynthesis above. The amount of radioactivity present was determined by double-label scintillation counting.
Assay of chitin synthase
The chitin synthase (CS) assays were carried out by the method of Causier et al. 25 The reaction mixture (50 µL) contained 50 µg microsomes that had been preincubated with trypsin and 40 mM Tris- HCl (pH 7.5), 6 mM MnCl 2, 32 mM GlcNAc and 1 mM UDP-N-acetyl-D-glucosamine (GlcNAc) containing 9 nCi UDP-[U- 14C]GlcNAc (271 mCi/mmol). Incubation was carried out at 25>C for 60 min. The reaction was stopped by heating the samples at 100>C for 2 min. The reaction mixture was filtered through glassfibre filter paper (Whatman GF/F glass microfibre filters (Whatman); 2.5 cm diameter, 0.7 µm pore size) that had been soaked in distilled water for 60 min. The reaction tube was washed twice with 50 µL aqueous Triton X-100 (1%, v/v), and the filters were washed with 10 mL distilled water. Radioactivity of the filters was determined by liquid scintillation counting. Nikkomycin Z was used as a positive control for CS inhibition. 26
Chemicals and reagents
Berberine hydrochloride (mol. wt 371.8), palmatine (mol. wt 387.8), miconazole and amphotericin B were obtained from Sigma Chemical Co. Cofactors and other biochemicals were purchased from Sigma, and were of the highest grade available. The following isotopes (specific activity) were purchased from Amersham (Buckinghamshire, UK); [ 14C]acetate (56 mCi/mmol), UDP-[U- 14C]GlcNAc (271 mCi/mmol), [methyl- 14C]SAM (58 mCi/mmol), [ 14C]mevalonate (60 mCi/mmol), [1a,2a(n)- 3H]1-cholesterol (46 Ci/mmol) and L-[methyl- 14C]methionine (1 mCi/mmol). Silica gel plates (Kiesel gel 60F 254), toluene, diethyl ether and chloroform were purchased from Merck Co. (Darmstadt, Germany). YNB, neopeptone, dextrose and Bacto-agar were obtained from Difco Co.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been previously reported that the total MeOH extracts of a dozen medicinal herbs exhibit anti-Candida activity. 27 This study was designed to examine their relative anti-Candida activities, the nature of the inhibitory component of the bioactive extracts and the mode of action of these compounds. Of the 12 MeOH extracts examined, only five showed >30% growth inhibition of C. albicans at 10 mg/assay and were therefore considered active (data not shown). To explore further the anti-Candida potential of the five selected herbal extracts, their inhibitory activity at lower doses (i.e. 4000 µg/assay) was examined using an agar diffusion assay. As summarized in Table I, of the five different extracts, only C. rhizomaand P. amurense were found to exhibit significant anti-Candidaactivity against most of the Candida spp. tested. Following extensive purification of these MeOH extracts, 19 the major active components of two herbal extracts (C. rhizoma and P. amurense) were determined to be berberine and palmatine (Figure 1.) Further biochemical investigation of the anti-Candida activity has focused on these protoberberines with respect to target site, and mechanism of action and relative potency as anti-Candida agents.
|
To examine whether the protoberberines (berberine and palmatine) inhibit the growth of Candida spp. and to determine their relative anti-Candida activity, cells were grown in the presence of each compound and MIC values measured. As summarized in Table II, for C. albicans, berberine gave MIC values in vitro ranging from 125 to 250 mg/L, depending on the isolate tested. MIC values for the other species of Candida were from 4 mg/L (C. krusei) to >500 mg/L (C. parapsilosis). Palmatine was not effective against most Candida species examined, at a dose of >500 mg/L, but caused significant inhibition of C. parapsilosis growth (MIC 15.6 mg/L). Further investigations concentrated on C. albicans (ATCC10231).
|
To determine the target site for the inhibitory activity of the protoberberines, ergosterol biosynthesis was measured in the presence and absence of these compounds. Total cellular sterols in C. albicans were synthesized in the presence of either [ 14C]acetate or [ 14C]mevalonate and berberine. In the first experiment, using [ 14C]mevalonate or [ 14C]acetate as an ergosterol precursor, there was no difference in ergosterol synthesis between the compound-treated or untreated groups (results not shown). In all cases, cells incorporated [ 14C]acetate or mevalonate exclusively into the ergosterol fraction (results not shown). This indicates that the target may not be an enzyme involved in the biosynthesis of the carbon skeleton of ergosterol, from either acetate or mevalonate. In the second experiment, L-[methyl- 14C]methionine was added to C. albicans, which had been cultured previously in a medium devoid of amino acids, in phosphate buffer containing glucose (1%, w/v). Ergosterol biosynthesis was measured by incorporation of L-[methyl- 14C]methionine, and the effects of protoberberines were examined. As shown in Figure 2a, incorporation of L-[methyl- 14C]methionine into the C-24 of ergosterol was strongly inhibited by the presence of berberine or palmatine in a dose-dependent manner; the former compound was the more potent inhibitor. The decrease in the incorporation of L-[methyl- 14C]methionine into ergosterol appears to be a consequence of the inhibition of sterol 24-methyl transferase (24-SMT), which is the only enzyme that catalyses transmethylation from S-adenosylmethionine. The results shown in Figure 2b indicate a dose-dependent inhibition of the rate of L-[methyl- 14C]methionine incorporation into ergosterol by protoberberines. The IC 50 values of the protoberberines in inhibition of ergosterol synthesis from L-[methyl- 14C]methionine were approximately 25 µM (berberine) and 300 µM (palmatine) (Figure 2b.)
|
|
|
In an attempt to identify other target sites for protoberberines in C. albicans, the effect of these compounds on CS activity in vitro was determined. Palmatine caused a moderate dose-dependent inhibition of CS isolated from yeast or mycelial growth forms (data not shown). The mode of inhibition by palmatine was noncompetitive with a K i of 780 µM. However, berberine had little effect on CS activity (data not shown).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pathways of sterol biosynthesis in fungi are targeted by important antifungal drugs, and many of the enzymes that contribute to fungal cell wall biosynthesis are considered potential targets for novel antimycotics. 30,31 The results of the present study are of considerable significance, as it was demonstrated that a novel class of antifungals, the protoberberines, inhibit key enzymes in the pathways of both ergosterol and chitin biosynthesis. Both palmatine and berberine inhibited microsomal CS from the yeast and mycelial growth forms of C. albicans, although the degree of enzyme inhibition was much less than noted for nikkomycin Z. 32 Palmatine was a non-competitive inhibitor of yeast CS with K i 780 µM (data not shown). These observations indicate that the protoberberines used during the present study are not potent inhibitors of C. albicans chitin synthase. It remains to be seen whether synthetic or semi-synthetic derivatives of these compounds may prove to be more powerful inhibitors of enzyme activity. It will be of interest to establish whether all of the isoforms of CS in C. albicans 33,34 are inhibited by protoberberine antifungals.
When C. albicans yeast cells were incubated in the presence of berberine or palmatine, the protoberberines caused a dose-dependent inhibition of incorporation of radioactivity from L-[methyl- 14C]methionine into ergosterol (Figure 2.) This effect appeared to be a consequence of the inhibition of 24-SMT, the only enzyme that catalyses transmethylation from S-adenosylmethionine. Both berberine and palmatine inhibited 24-SMT activity in microsomes, isolated from the yeast or mycelial growth forms of C. albicans (Figure 3.) Inhibition of 24-SMT activity in vivo will result in the accumulation of ergosta-8,24-dien-3ß-ol and depletion of ergosterol. Both of these effects are likely to contribute to the antifungal properties of the protoberberines. 24-SMT from the mycelial growth form was more susceptible than the yeast enzyme to berberine and palmatine. This may be an important consideration if protoberberines are to be used to treat candidoses in the clinic, as the mycelial form of C. albicans is thought to have an important role during the pathogenesis of infection. 35 The effects of protoberberines on ergosterol biosynthesis in C. albicans appear to involve a specific inhibition of 24-SMT activity, because neither berberine nor palmatine at 1 mM had any effect on other steps of sterol synthesis, when [ 14C]mevalonate or [ 14C]acetate was used in vivo as substrate (data not shown). In a recent study, inhibition of 24-SMT activity was shown to block sterol biosynthesis and cell proliferation in Pneumocystis carinii. 36,37 These results, coupled with the findings of the present study, suggest that 24-SMT may present an important target for antifungals.
In conclusion, the demonstration that antifungal protoberberines inhibit both 24-SMT and chitin synthase in C. albicans provides a basis for the design of novel antimycotics based on the protoberberine nucleus. These results, and the long history of the successful use of berberine and plant extracts containing this, and related compounds, in Chinese and Korean medicine, provide a basis for the further investigation and development of protoberberines, with potent antifungal activity and low host toxicity.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Broughton, M. C., Bard, M. & Lees, N. D. (1991). Polyene resistance in ergosterol producing strains of Candida albicans. Mycoses 34, 7583.[ISI][Medline]
3 . Georgopapadakou, N. H. & Walsh, T. J. (1994). Human mycoses: drugs and targets for emerging pathogens. Science 264, 3713.[ISI][Medline]
4 . Hitchcock, C. A., Barrett-Bee, K. J. & Russell, N. J. (1987). The lipid composition and permeability to azole of an azole- and polyene-resistant mutant of Candida albicans. Journal of Medical and Veterinary Mycology 25, 2937.[ISI][Medline]
5 . Polak, A. & Hartman, P. G. (1991). Antifungal chemotherapy are we winning? Progress in Drug Research 37, 181269.[Medline]
6 . Huang, K. C. (1993). Antibacterial, antiviral and antifungus herbs. The Pharmacology of Chinese Herbs, pp. 287311. CRC Press, Boca Raton, FL.
7 . Matsuda, H., Tokuoka, K., Wu, J., Shiomoto, H. & Kubo, M. (1997). Inhibitory effects of dehydrocorydaline isolated from Corydalis tuber against type I- IV allergic models. Biological and Pharmaceutical Bulletin 20, 4314.[Medline]
8 . Kubo, M., Matsuda, H., Tokuoka, K., Kobayashi, Y., Ma, S. & Tanaka, T. (1994). Studies of anti-cataract drugs from natural sources. I. Effects of a methanolic extract and the alkaloidal components from Corydalis tuber on in vitro aldose reductase activity. Biological and Pharmaceutical Bulletin 17, 4589.[Medline]
9 . Kubo, M., Matsuda, H., Tokuoka, K., Ma, S. & Shiomoto, H. (1994). Anti-inflammatory activities of methanolic extract and alkaloidal components from Corydalis tuber. Biological and Pharmaceutical Bulletin 17, 2625.[Medline]
10 . Yasukawa, K., Takido, M., Ikekawa, T., Shimada, F., Takeuchi, M. & Nakagawa, S. (1991). Relative inhibitory activity of berberine-type alkaloids against 12-O -tetradecanoylphorbol-13-acetate-induced inflammation in mice. Chemical and Pharmaceutical Bulletin 39, 14625.
11 . Gudima, S. O., Memelova, L. V., Borodulin, V. B., Pokholok, D. K., Mednikov, B. M. Tolkachev, O. N. et al. (1994). Kinetic analysis of interaction of human immunodeficiency virus reverse transcriptase with alkaloids. Molekulianaia Biologiia28 , 130814.
12 . Schmeller, T., Latz-Bruning, B. & Wink, M. (1997). Biochemical activities of berberine, palmatine and sanguinarine mediating chemical defence against microorganisms and herbivores. Phytochemistry 44, 25766.[ISI][Medline]
13 . Vennerstrom, J. L. & Klayman, D. L. (1988). Protoberberine alkaloids as antimalarials. Journal of Medical Chemistry 31, 10847.
14 . Nakamoto, K., Sadamori, S. & Hamada, T. (1990). Effects of crude drugs and berberine hydrochloride on the activities of fungi. Journal of Prosthetic Dentistry 64, 6914.[ISI][Medline]
15 . Ryder, N. S. & Dupont, M. C. (1984). Properties of a particulate squalene epoxidase from Candida albicans. Biochimica et Biophysica Acta 794, 46671.[ISI][Medline]
16 . Davies, A. R. & Marriott, M. S. (1981). Inhibitory effects of imidazole antifungals on the yeast- mycelial transformation in Candida albicans. Microbios Letters 17, 1558.
17
.
Ator, M. A., Schmidt, S. J., Adams, J. L. & Dolle, R. E. (1989) Mechanism and
inhibition of
24-sterol methyltransferase from Candida albicans
and Candida tropicalis. Biochemistry 28 , 963340.[ISI][Medline]
18 . Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Analytical Biochemistry 72, 24854.[ISI][Medline]
19 . Wu, W.-N., Beal, J. L., Leu, R.-P. & Doskotch, R. W. (1977). Alkaloids of Thalictrum. XXI. Isolation and characterization of alkaloids from the roots of Thalictrum podocarpum. Lloydia 40, 38494.[ISI][Medline]
20 . National Committee for Clinical Laboratory Standards. (1993). Performance Standards for Antimicrobial Disk Susceptibility Tests Fifth Edition: Approved Standard M2A5. NCCLS, Villanova, PA.
21 . National Committee for Clinical Laboratory Standards. (1992). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Proposed Guideline M27P. NCCLS, Villanova, PA.
22 . Barrett-Bee, K. J., Lane, A. C. & Turner, R. W (1986). The mode of antifungal action of tolnaftate. Journal of Medical and Veterinary Mycology 24, 15560.[ISI][Medline]
23 . Ryder, N. S. (1985). Effect of allylamine antimycotic agents on fungal sterol biosynthesis measured by sterol side-chain methylation. Journal of General Microbiology 131, 1595602.[ISI][Medline]
24
.
Paik, Y.-K., Trzaskos, J. M., Shafiee, A. & Gaylor, J. L. (1984). Microsomal
enzymes of
cholesterol biosynthesis from lanosterol: characterization, solubilization, and partial purification
of NADPH-dependent 8,14-steroid
14-reductase. Journal of Biological Chemistry 259, 1341323.
25 . Causier, B. E., Milling, R. J., Foster, S. G. & Adams, D. J. (1994). Characterization of chitin synthase from Botrytis cinerea. Microbiology 140, 2199205.[Abstract]
26 . Gaughran, J. P., Lai, M. H., Kirch, D. R. & Silverman, S. J. (1994). Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo. Journal of Bacteriology 176, 585760.
27 . Min, B. S., Bang, K. H., Lee, J. S. & Bae, K. H. (1996). Screening of the antifungal activity from natural products against Candida albicans and Penicillium avellaneum. Yakhak Hoeji 40, 58290.
28 . Marriott, M. S. (1975). Isolation and chemical characterization of plasma membranes from the yeast and mycelial forms of Candida albicans. Journal of General Microbiology 86, 11532.[ISI][Medline]
29 . Fridkin, S. K. & Jarvis, W. R. (1996). Epidemiology of nosocomial fungal infections. Clinical Microbiology Reviews 9, 499511.[Abstract]
30 . Hector, R. F. (1993). Compounds active against cell walls of medically important fungi. Clinical Microbiology Reviews 6, 121.[Abstract]
31 . Koller, W. (1992). Antifungal agents with target sites in sterol functions and biosynthesis. In Target Sites of Fungicide Action (Koller, W., Ed.), pp. 119206. CRC Press, Boca Raton, FL.
32 . Cabib, E. (1991). Differential inhibition of chitin synthases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and Nikkomycins. Antimicrobial Agents and Chemotherapy 35 , 1703.[ISI][Medline]
33 . Choi, W.-J., Sburlati, A. & Cabib, E. (1994). Chitin synthase 3 from yeast has zymogenic properties that depend on both the CAL1 and CAL3 genes. Proceedings of the National Academy of Sciences of the USA 91, 472730.[Abstract]
34 . Mio, T., Yabe, T., Sudoh, M., Satoh, Y., Nakajima, T., Arisawa, M. et al. (1996). Role of three chitin synthase genes in the growth of Candida albicans. Journal of Bacteriology 178, 241619.[Abstract]
35 . Lo, H. J., Kohler, J. R., DiDomenico, B., Loebenberg, D., Cacciapuoti, A. & Fink, G. R. (1997). Nonfilamentous C. albicans mutants are avirulent. Cell 90, 93949.[ISI][Medline]
36
.
Urbina, J. A., Vivas, J., Visbal, G. & Contreras, L. M. (1995). Modification of the
sterol
composition of Trypanosoma (Schizotrypanum ) cruzi epimastigotes
by 24(25)-sterol methyl transferase inhibitors and their
combinations with ketoconazole. Molecular and Biochemical Parasitology 73, 199
210.[ISI][Medline]
37
.
Urbina, J. A., Visbal, G., Contreras, L. M., McLaughlin, G. & Docampo, R. (1997).
Inhibitors of 24(25) sterol methyltransferase block sterol synthesis and
cell proliferation in Pneumocystis carinii. Antimicrobial Agents and
Chemotherapy 41, 142832.[Abstract]
Received 15 July 1998; returned 19 October 1998; revised 16 November 1998; accepted 6 January 1999