Antibiotics Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, PO Box 115, Yusung, Taejon 305-333, Korea
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Abstract |
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Introduction |
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In the course of our continuous screening programme to find potent chitin synthase 2 inhibitors from wild and medicinal plant extracts, we found that the extract of leaves of Magnolia obovata inhibited chitin synthase 2 activity. The extract of bark of M. obovata has been used as a stomachic herb in Korea, China and Japan for hundreds of years,15 and the leaves have been eaten as a vegetable in Korea and Japan. A number of Magnolia species have been extensively examined for compounds with pharmacological, antimicrobial and pesticidal activity.16 However, this is the first report that the extract of the leaves of M. obovata could be used as a chitin synthase 2 inhibitor. Here, we report the isolation of obovatol and the synthesis of tetrahydroobovatol, and their inhibitory activities against chitin synthases of S. cerevisiae and C. albicans and whole cells of various pathogenic fungi.
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Materials and methods |
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The strains used in this study were S. cerevisiae YPH499 (ura3-52 lys2-801amber ade20101ochre trp1-63 his-
200 leu2-
1),17 ECY38-38A(pAS6) (MATa chs1-23 chs2::LEU2 cal1/csd2 ura3-52 trp1-1 leu2-2 pAS6)18 and ECY38-38A(pWJC6) (MATa chs1-23 chs2::LEU2 cal1/csd2 ura3-52 trp1-1 leu2-2 pWJC6),18 which were used as sources of chitin synthase 1, 2 and 3 activity, respectively. S. cerevisiae YPH499, the wild type for all three synthases, was grown in YEPD [1% yeast extract (Difco, Detroit, MI, USA), 2% Bacto peptone, 2% glucose]. S. cerevisiae ECY38-38A(pAS6) and ECY38-38A(pWJC6), which can only overexpress chitin synthases 2 and 3, respectively, were grown in YPG [1% yeast extract (Difco), 2% Bacto peptone, 2% galactose] at 30°C. In addition, the chitin synthase-deleted homologous mutant chs2
chs3
(chs2
::hisG/chs2
::hisG chs3
::hisG/ chs3
::hisG) was used for C. albicans chitin synthase 1 (CaCHS1p) activity.14 The strain was grown in Sabouraud dextrose medium at 30°C.
ScCHS1p, -2p and -3p assays
The assays of chitin synthase 2 and 3 prepared from recombinant S. cerevisiae ECY38-38A(pAS6) and ECY38-38A(pWJC6), respectively, were conducted according to the method of Choi & Cabib.19 Cells suspended in 50 mM TrisHCl (pH 7.5) containing 5 mM magnesium acetate were broken by vortex mixing with glass beads. Cell walls were sedimented at 4000g for 5 min and the supernatant fluid was centrifuged at 130 000g for 45 min. The membrane pellet was suspended in 50 mM TrisHCl (pH 7.5) containing 33% glycerol used in the breakage, to a final volume of 1.6 mL/g (wet weight) of cells. Chitin synthase 2 activity was measured by the procedure described previously.19 For the proteolytic activation step, reaction mixtures contained 32 mM TrisHCl (pH 8.0), 1.6 mM cobalt acetate, 1.0 mM UDP-[14C]-N-acetyl-d-glucosamine (400 000 cpm/µmol; NEN, Boston, MA, USA), 2 mL of trypsin at the optimal concentration for activation (2.0 mg/mL), 20 µL of membrane suspensions and 14 µL of sample in a total volume of 46 µL. The mixtures were incubated for 15 min at 30°C. Proteolysis was stopped by adding 2 µL of a soybean trypsin inhibitor solution (4.0 mg/mL) at a concentration twice that of the trypsin solution used, and tubes were placed on ice for 10 min. GlcNAc was added to a final concentration of 32 mM, and incubation at 30°C was carried out for 90 min. For chitin synthase 3 activity,18,19 the assay was carried out as for chitin synthase 2, except that 32 mM TrisHCl (pH 7.5) and 4.3 mM magnesium acetate were used. For chitin synthase 1 activity,19 reaction mixtures contained 37 mM TrisHCl (pH 7.5), 0.12% digitonin, 4.8 mM magnesium acetate, 2 µL of trypsin (1.0 mg/mL), 6 µL of membrane suspension and 14 µL of the test sample in a total volume of 41 µL. After 15 min of incubation at 30°C, 2 µL of trypsin inhibitor (2.0 mg/mL) were added and the tubes were placed on ice. GlcNAc (32 mM) and 1.0 mM UDP-[14C]-N-acetyl-d-glucosamine were added as for the chitin synthase 2 and 3 assays, and the mixtures were incubated for 30 min at 30°C. In all cases, the reaction was stopped by addition of 10% trichloroacetic acid and radioactivity of the insoluble chitin formed was counted after filtration through a glass fibre filter (GF/C; Whatman, Maidstone, UK). The concentration of protein was measured by the method of Lowry et al.20 Blank values were measured by addition of 25% aqueous MeOH instead of both enzyme and sample. Percentage inhibition of chitin synthase activity was calculated by subtracting the blank values from both control and test sample values.
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The chitin synthase 1, 2 and 3 activities of the enzyme were confirmed by positive control with polyoxin D and nikkomycin Z (Calbiochem, San Diego, CA, USA). Each isolated and control compound was solubilized in 25% MeOH and distilled water to make a stock solution (1 mg/mL), and an aliquot (14 µL) of the stock was used for each reaction to give a final concentration of 280 mg/L. The inhibitory activities were represented as average values in duplicates obtained from two independent experiments.
CaCHS1p assay
The preparation of microsome and chitin synthase 1 activity of C. albicans was conducted by a method described previously.21 For CaCHS1p activity, the assay was carried out similarly to that for ScCHS2p, except that 32 mM TrisHCl (pH 7.5) and 2 mM cobalt chloride were used. Nikkomycin Z was used as a positive control for CaCHS1p inhibition. Each isolated and control compound was solubilized in 25% dimethyl sulphoxide and distilled water to make a stock solution (1 mg/mL).
Isolation and purification of obovatol from M. obovata
We purified the active component from the extract of M. obovata using the inhibitory activity-guided fractionation method. The leaves of M. obovata were harvested in Taejon, Korea and the isolation procedure of the active compound was the same as that reported previously.22 The dried and milled sample (1 kg) was soaked in chloroformacetone (1:1, v/v, 5 L) at room temperature for 5 days. The extract was concentrated under reduced pressure, and purified by silica gel, C18 column chromatography and preparative thin layer chromatography (TLC). Finally, we purified the active compound by high-performance liquid chromatography (HPLC) (Phenomenex, Ultracarb 10 ODS; 250 x 21.2 mm at 285 nm, flow rate 3.5 mL/min) using a linear gradient rising from 80 to 90% methanol for 50 min. The tR in HPLC of active compound (120 mg) was 35 min. The mass spectroscopic, infra-red spectroscopic and nuclear magnetic resonance data for the purified compound were in good agreement with the spectral data for obovatol (C18H18O3, mol. wt 282 kDa) published previously.22,23
Synthesis of tetrahydroobovatol
The isolated obovatol (50 mg) was dissolved in 50 mL of tetrahydrofuran (THF) and added to palladium (Pd) charcoal (100 mg). The mixture was stirred under a hydrogen atmosphere at room temperature, and allowed to stand overnight. The reaction mixture was then filtered through filter paper (Whatman No. 1). The filtrate was dried, washed twice with MeOH and re-dried in vacuo to dryness to give tetrahydroobovatol as red brown oil (45 mg; C18H22O3, mol. wt 286 kDa).24
The structures of obovatol and tetrahydroobovatol are shown in Figure 1.
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MICs were determined by a two-fold serial agar dilution method.24 Obovatol and tetrahydroobovatol were dissolved in 25% MeOH, and polyoxin D and nikkomycin Z were dissolved in distilled water. Human pathogenic fungi were grown on Sabouraud agar medium (Difco), and plant pathogenic fungi were grown on potato dextrose agar medium (Difco). S. cerevisiae ECY38-38A(pAS6) and ECY38-38A(pWJC6) were grown on YPG agar medium. The inoculum sizes of yeasts and spore-forming fungi were 103 cfu/spot and 105 spores/spot, respectively. Antifungal activity was observed after 24 h incubation at 30°C for yeasts and 48 h incubation for fungi at 25°C. The MIC was defined as the lowest concentration of antibiotic that completely inhibited the growth of the organism when compared with a control plate containing no antibiotics.
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Results |
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One of the plant extracts, extract of M. obovata, strongly inhibited ScCHS2p activity (inhibitory activity 75% at a concentration of 280 mg/L) in our assay system and weakly inhibited the growth of Cryptococcus neoformans at 500 mg/L in an agar diffusion method25 (inhibition zone 12 mm). From this result, we selected M. obovata to isolate ScCHS2p inhibitors as antifungal agents and subsequently isolated and purified compounds inhibiting chitin synthase 2.
In vitro inhibitory activities against ScCHS1p, -2p and -3p, and CaCHS1p
Although several chitin synthase inhibitors have been isolated from microbes and higher plants,2633 obovatol and tetrahydroobovatol have not yet been reported as chitin synthase inhibitors. Chitin synthase inhibitory activities were examined by measurement of the formation of chitin with UDP-[14C]-N-acetyl-d-glucosamine. As shown in Figure 2 and Table 1
, obovatol and tetrahydroobovatol strongly inhibited ScCHS2p activity in a dose-dependent manner with IC50s of 38 and 59 µM, respectively. The IC50 of both compounds for chitin synthase 2 represented 3.5 and 2.3 times stronger inhibitory activities, respectively, than that of polyoxin D. To examine whether the observed inhibition was specific for chitin synthase 2, the effects of these compounds on chitin synthases 1 and 3 from S. cerevisiae were also determined. Results showed that these compounds had no effects on chitin synthase 3 activity, whereas they exhibited a weak inhibitory activity for chitin synthase 1 (IC50 > 1 mM). They also inhibited CaCHS1p with an IC50 of 28 and 51 µM, respectively, but these compounds showed weaker inhibitory activities than that of polyoxin D and nikkomycin Z. Based on these data, we consider that obovatol and tetrahydroobovatol are specific inhibitors for chitin synthase 2 from S. cerevisiae. The activities of chitin synthases prepared from the yeast strains and the inhibitory activities of the compound were confirmed with polyoxin D and nikkomycin Z as positive controls, which exhibited chitin synthase 2 and 1 inhibitory activities with an IC50 of 134.5 and 2 µM in our assay systems, respectively (data not shown).31,32
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The mechanism of inhibition of ScCHS2p by obovatol and tetrahydroobovatol was investigated in a kinetic analysis of the inhibition with a LineweaverBurke plot. Double reciprocal plots of the data demonstrated that obovatol and tetrahydroobovatol acted as a mixed competitive inhibitor with respect to UDP-N-acetyl-d-glucosamine substrate (Figure 3). The Ki values of both compounds against UDP-N-acetyl-d-glucosamine were calculated to be 21.8 and 23.1 µM, respectively.
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As shown in Table 2, obovatol and tetrahydroobovatol exhibited low inhibitory activities (MIC 125500 mg/L) against various plant pathogens including Rhizoctonia solani, Colletotrichum lagenarium, Pyricularia oryzae and Botrytis cinerea. In contrast, the compounds showed very potent inhibitory activities (MIC 7.831.25 mg/L) against opportunistic human pathogens, including Coccidioides immitis, C. neoformans and C. albicans. The antifungal activities of obovatol against Candida krusei, Microsporum canis and Trichophyton mentagrophytes were very strong (MIC 31.262.5 mg/L), whereas tetrahydroobovatol exhibited a weak antifungal activity against these species (MIC 125250 mg/L). In addition, obovatol and tetrahydroobovatol showed high inhibitory activities (MIC 15.662.5 mg/L) against various S. cerevisiae.
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Discussion |
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Recently, several types of compound affecting the chitin synthesis of C. albicans have been discovered. For example, protoberberine37 and HWY-28930 inhibit chitin and sterol biosynthesis of C. albicans, and R0-09-314333 inhibits chitin synthase 1 of C. albicans. In C. albicans, obovatol showed a similar inhibitory activity to that of HWY-289, but a weaker activity than that of R0-09-3143 and nikkomycin Z. However, the inhibitory activity of obovatol on ScCHS2p was stronger than that of HWY-289, polyoxin D and nikkomycin Z. In S. cerevisiae, chitin synthases 1 and 3 are more sensitive to nikkomycin derivatives than chitin synthase 2.26 Nikkomycin showed inhibitory activity specifically for chitin synthase 3 in S. cerevisiae.38
Considering the fact that obovatols have biological activity such as ACAT, other than differential inhibitory activity on ScCHS2p and CaCHS1p, this compound or its derivatives may have multiple target sites. Thus, the antifungal activity of obovatol and its derivatives against various pathogenic fungi might be caused in part by potent inhibitory activities for SCCHS2 and CaCHS1, but it could be due to the inhibition of another cellular target alone or together with the inhibition of chitin synthase. However, such possibilities of the antifungal activity of these compounds still remain to be investigated. In S. cerevisiae, obovatol also transiently reduced the number of viable cells of ECY38-38A(pAS6), which can only overexpress chitin synthase 2, although it failed to kill these cells. The failure of obovatol to cause cell death of ECY38-38A(pAS6) is probably due to the low susceptibility of ScCHS2p to obovatol and also to the low solubility of the compound.
In order to obtain some information about structure activity relationships in the hydroxybiphenyl compounds and structural requirements for more potent inhibitors of ScCHS2p, we synthesized tetrahydroobovatol possessing the methyl instead of methylene of obovatol, and its inhibitory activity was examined together with two neolignans, magnolol and honokiol. Tetrahydroobovatol showed a weaker inhibitory activity than obovatol against ScCHS2p (Figure 1 and Table 1
). Interestingly, magnolol and honokiol exhibited no inhibitory activity for ScCHS2p at a concentration of 2 mM (data not shown). The results allow us to consider the possibility that the specific chitin synthase 2 inhibitory activity of neolignans may be dependent on the position of the hydroxyl group. To provide supporting evidence for such a notion, we carried out enzyme kinetics assays of obovatol and tetrahydroobovatol. The results demonstrated that both compounds inhibited chitin synthase 2 activity by mixed competition with UDP-N-acetyl-d-glucosamine, indicating that chitin synthase 2 is one of the cellular targets for the inhibitory activity of obovatol and tetrahydroobovatol.
Based on our present data, further analyses of structureactivity relationships of these compounds would make it possible to design novel synthetic antifungal agents. In this regard, we consider that obovatol and tetrahydroobovatol may be useful lead compounds for development of new antifungal agents through the control of chitin biosynthesis.
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Acknowledgements |
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Notes |
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References |
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2 . Odds, F. (1996). Resistance of clinically important yeasts to antifungal agents. International Journal of Antimicrobial Agents 6, 1457. [ISI]
3 . Gooday, G. W. (1977). Biosynthesis of the fungal cell wall: Mechanisms and implications. Journal of General Microbiology 99, 111. [ISI][Medline]
4 . Bartnicki-Garcia, S. (1968). Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annual Review of Microbiology 22, 87102. [ISI][Medline]
5 . Shaw, J. A., Mol, P. C., Bowers, B., Silverman, S. J., Valdivieso, M. H., Duran, A. et al. (1991). The function of chitin synthase 2 and 3 in the Saccharomyces cerevisiae cell cycle. Journal of Cell Biology 114, 11123. [Abstract]
6 . Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L. et al. (1986). The Saccharomyces cerevisiae structural gene for chitin synthase is not regulated for chitin synthesis in vivo. Cell 46, 21325. [ISI][Medline]
7
.
Sburlati, A. & Cabib, E. (1986). Chitin synthase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae. Journal of Biological Chemistry 261, 1514752.
8 . Silverman, S. J., Sburlati, A., Slater, M. L. & Cabib, E. (1988). Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences , USA 85, 47359. [Abstract]
9 . 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 , USA 91, 472730. [Abstract]
10 . Bulawa, C. E. (1992). CSD2, CSD3, and CSD4, genes required for chitin synthesis in S. cerevisiae: the CDS2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis. Molecular and Cellular Biology 12, 176476. [Abstract]
11
.
Kollar, R., Petrakova, E., Ashwell, G., Robbins, P. W. & Cabib, E. (1995). Architecture of the yeast cell wall. The linkage between chitin and beta(13)-glucan. Journal of Biological Chemistry 270, 11708.
12 . Cabib, E., Sburlati, A., Bowers, B. & Silverman, S. J. (1989). Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae. Journal of Cell Biology 108, 166572. [Abstract]
13 . Silverman, S. J. (1989). Similar and different domains of chitin synthases 1 and 2 of S. cerevisiae: two isozymes with distinct functions. Yeast 5, 45967. [Medline]
14 . 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, 24169. [Abstract]
15 . Huang, K. C. (1993). Stomachic and wind-dispelling herbs. In The Pharmacology of Chinese Herbs, (Huang, K. C., Ed.), pp. 174. CRC Press, Ann Arbor, MI.
16 . Nitao, J. K., Nair, M. G., Thorogood, D. L., Johnson, K. S. & Scriber, J. M. (1991). Bioactive neolignans from the leaves of Magnolia virginiana. Phytochemistry 30, 21935. [ISI]
17
.
Sikorski, R. S. & Hieter, P. (1989). A system of shuttle vectors and yeast strain designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 1927.
18 . Choi, W. J., Santos, B., Duran, A. & Cabib, E. (1994). Are yeast chitin synthases regulated at the transcriptional or the posttranslational level? Molecular and Cellular Biology 14, 768594. [Abstract]
19 . Choi, W. J. & Cabib, E. (1994). The use of divalent cations and pH for the determination of specific yeast chitin synthetases. Analytical Biochemistry 219, 36872. [ISI][Medline]
20
.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 26575.
21 . Choi, W. J. (1998). The determination of chitin synthases by varying pH and divalent cations in Candida albicans. Journal of Microbiology and Biotechnology 8, 6137.
22 . Kwon, B. M., Kim, M. K., Lee, S. H., Kim, J. A., Lee, I. R., Kim, Y. K. et al. (1997). Acyl-CoA: cholesterol acyltransferase inhibitors from Magnolia obovata. Planta Medica 63, 5501. [ISI][Medline]
23 . Ito, K., Iida, T., Ichino, K., Tsunezuka, M., Hattori, M. & Namba, T. (1982). Obovatol and obovatal, novel biphenyl ether lignans from the leaves of Magnolia obovata. Chemical and Pharmaceutical Bulletin 30, 334753.
24 . Mcginnis, M. R. & Rinaldi, M. G. (1986). Antifungal drugs: Mechanisms of action, drug resistance, susceptibility testing, and assays of activity in biological fluids. In Antibiotics in Laboratory Medicine, 2nd edn, (Victor, L., Ed.), pp. 22381. Williams & Wilkins, Baltimore, MD.
25 . Hewitt, W. & Vincent, S. (1989). The agar diffusion assay. In Theory and Application of Microbiological Assay, (Hewitt, W. & Vincent, S., Eds), pp. 3879. Academic Press, New York, NY.
26 . Cabib, E. (1991). Differential inhibition of chitin synthase 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycin Z. Antimicrobial Agents and Chemotherapy 35, 1703. [ISI][Medline]
27 . Pfefferle, W., Anke, H. & Bross, M. (1990). Inhibition of solubilized chitin synthase by chlorinated aromatic compounds isolated from mushroom cultures. Agricultural and Biological Chemistry 54, 13814. [ISI]
28 . Kim, S. U., Hwang, E. I., Nam, J. Y., Son, K. H., Bok, S. H., Kim, H. E. et al. (1999). Inhibition of chitin synthase II by catechins from stem bark of Taxus cuspidata. Planta Medica 65, 979. [ISI][Medline]
29 . Jeong, T. S., Hwang, E. I., Lee, H. B., Lee, E. S., Kim, Y. K., Min, B. S. et al. (1999). Chitin synthase II inhibitory activity of ursolic acid, isolated from Crataegus pinnatifida. Planta Medica 65, 2613. [ISI][Medline]
30
.
Park, K. S., Kang, K. C., Kim, K. Y., Jeong, P. Y., Kim, J. H., Adams, D. J. et al. (2001). HWY-289, a novel semi-synthetic protoberberine derivative with multiple target sites in Candida albicans.
Journal of Antimicrobial Chemotherapy 47, 5139.
31 . Hwang, E. I., Yun, B. S., Kim, Y. K., Kwon, B. M., Kim, H. G., Lee, H. B. et al. (2000). Chaetoatrosin A, a novel chitin synthase II inhibitor produced by Chaetomium atrobrunneum F449. Journal of Antibiotics 53, 24855. [ISI][Medline]
32 . Hwang, E. I., Yun, B. S., Kim, Y. K., Kwon, B. M., Kim, H. G., Lee, H. B. et al. (2000). Phellinsin A, a novel chitin synthases inhibitor produced by Phellinus sp. PL3. Journal of Antibiotics 53, 90311. [ISI][Medline]
33
.
Sudoh, M., Yamazaki, T., Masubuchi, K., Taniguchi, M., Shimma, N., Arisawa, M. et al. (2000). Identification of a novel inhibitor specific to the fungal chitin synthase. Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans. Journal of Biological Chemistry 275, 329015.
34 . Lo, Y. C., Teng, C. M., Chen, C. F., Chen, C. C. & Hong, C. Y. (1994). Magnolol and honokiol isolated from Magnolia officinalis protect rat heart mitochondria against lipid peroxidation. Biochemical Pharmacology 47, 54953. [ISI][Medline]
35 . Fukuyama, Y., Otoshi, Y., Miyoshi, K., Hasegawa, N., Kan, Y. & Kodama, M. (1993). Structure of magnolian, a novel trilignan possessing potent 5-lipoxygenase inhibitory activity. Tetrahedron Letters 34, 10514. [ISI]
36 . Chang, B. S., Lee, Y. M., Ku, Y., Bae, K. H. & Chung, C. P. (1998). Antimicrobial activity of magnolol and honokiol against peridontopathic microorganisms. Planta Medica 64, 3679. [ISI][Medline]
37
.
Park, K. S., Kang, K. C., Kim, J. H., Adams, D. J., Johng, T. N. & Paik, Y. K. (1999). Differential inhibitory effects of protoberberines on sterol and chitin biosynthesis in Candida albicans. Journal of Antimicrobial Chemotherapy 43, 66774.
38 . Gaughran, J. P., Lai, M. M., Kirsch, 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. [Abstract]
Received 23 April 2001; returned 19 June 2001; revised 10 August 2001; accepted 30 August 2001