Separation of fungal sterols by normal-phase high-performance liquid chromatography

Peacock, Gary A.; Goosey, Michael W.

doi:10.1016/s0021-9673(01)96464-7

Cited by 23 publications

(18 citation statements)

References 19 publications

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“…For sediment slurries, low micromolar concentrations may be required (e.g., Fallon et al 1983, Fallon and Boylen 1990, van Duyl and Kop 1990). Note that other investigators have used 10-1-101 mmol/L added isotope-labeled precursors in order to obtain acceptably high activity in sterols or macromolecules in short (minutes to hours) incubations of fungi in culture or fungal enzyme systems (Pierce et al 1979, Arst and Scazzocchio 1980, Polak-Wyss et al 1985, Peacock and Goosey 1989, Henry 1990). One explanation for this is that fungal incorporation of 3H-thymidine into DNA-fraction-contaminating materials was rapidly rising with increased concentration of added thymidine, since fungal rate-saturation level might be expected to lie in the millimoles-per-litre range (Newell 1984; note that fungal mass is usually >98% of total microbial standing crop of standing-dead cordgrass: ).…”

Section: Methodsmentioning

confidence: 99%

“…It may be that millimolar concentrations of added labeled precursors are generally required to maximize assessment of fungal-synthesis rates in natural samples. Note that other investigators have used 10-'-10' mmol!L added isotope-labeled precursors in order to obtain acceptably high activity in sterols or macromolecules in short (minutes to hours) incubations of fungi in culture or fungal enzyme systems (Pierce et al 1979, Arst and Scazzocchio 1980, Polak-Wyss et al 1985, Peacock and Goosey 1989, Henry 1990).…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, there has been only one method published for measurement of total fungal production in naturally decaying litter, and values for instantaneous rate of production were not obtained (Frankland 1975). Isotope-labeling techniques akin to those used to measure bacterial and microalgal productivity have been used in studies of fungal syntheses (e.g., Jack and Anekwe 1972, Taylor and Parks 1978, Pierce et al 1979, Arst and Scazzocchio 1980, Soderhall and Cerenius 1983, Seo et al 1986, Morita et al 1989, Peacock and Goosey 1989, Henry 1990), but in none of these cases was there any attempt to apply radioisotopic techniques to measurement of total fungal productivity.…”

Section: Introducfionmentioning

confidence: 99%

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Toward A Method For Measuring Instantaneous Fungal Growth Rates In Field Samples

Newell¹,

Fallon²

1991

Ecology

174

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JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org.. Ecological Society of America is collaborating with JSTOR to digitize, preserve and extend access to Ecology.Abstract. Methods are available in the literature for the measurement of instantaneous growth rates in field samples of photosynthetic microbes and procaryotic saprotrophic microbes, but there has been no such method for eucaryotic saprotrophic microbes (members of the kingdom Fungi). We have devised a technique for estimating instantaneous growth rates for ergosterol-containing fungi in field material, and preliminarily applied the technique to estimation of fungal productivities and throughputs in two types of standingdead grass (one saltwater, one freshwater), obtaining plausible values (e.g., conversion efficiencies, total fungal production . leaf mass loss, were calculated to be 36-64%). This method is based on measuring rates of radiolabeled acetate incorporation into ergosterol. We examined several potential problem areas for use of radiolabeled precursors in measurement of microbial molecular syntheses, and its extrapolation to microbial productivity. Key findings were: (a) 5 mmol/L added acetate (required for maximization of detection of rates) had positive or neutral impact on 48-h fungal growth, and no shift-up (sudden upward change in rate) or lag in incorporation of acetate into ergosterol occurred between 0.25 and 1.25 h of incubation of naturally decaying leaf (Spartina alterniflora) samples with 5 mmol/L acetate; (b) bacterial assemblages on leaves did not contribute radioactivity to the ergosterol fraction after incubation with radioacetate (as opposed to > 30 Bq per centimetre length of leaf containing fungal mycelium); (c) the average empirical factor for conversion from nanomoles '4C-acetate incorporated to micrograms organic fungal mass produced was 8.2 ,ug/nmol (coefficient of variation, 27%). We tentatively conclude that the acetateto-ergosterol method is tenable for measurement of natural fungal productivity. et al. (1985) when they estimated procaryotic productivity by following the rate of incorporation of '4C-acetate into phospholipid fatty acids. We have tested whether measurement of rate of incorporation of radioacetate into ergosterol can be used in samples of decaying vascular plants (primarily smooth cordgrass, Spartina alterniflora Loisel.; see Newell et al. 1989), as a means of determining productivity of fungal decomposers in field samples. We have examined several areas of potential pitfalls (Staley and Konopka 1985, Karl 1986, Moriarty 1988 including ability of target microbes to take up the precursor molecule, concentrations of precursor required to maximize degree of participation (Bell 1990) of exogenous precursor, effect of presence...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introducfionmentioning

confidence: 99%

See 1 more Smart Citation

Toward A Method For Measuring Instantaneous Fungal Growth Rates In Field Samples

Newell¹,

Fallon²

1991

Ecology

174

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“…Ergosterol was analyzed by HPLC with ultraviolet (UV) detection based on the method proposed by Peacock and Goosey (57). The chromatography was performed with a system consisting of two high-pressure pumps (Jasco 880-PU intelligent HPLC pump I), a manual injector (rheodyne 7125), and a spectrophotometer detector (Jasco 875-UV intelligent UV ⁄ Vis).…”

Section: Sterol Analysismentioning

confidence: 99%

Antifungal Activity of Xanthones: Evaluation of their Effect on Ergosterol Biosynthesis by High-performance Liquid Chromatography

Pinto

Afonso

Duarte

et al. 2011

Chemical Biology &Amp; Drug Design

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The increasing resistance of pathogenic fungi to antifungal compounds and the reduced number of available drugs led to the search for therapeutic alternatives among natural products, including xanthones. The antifungal activity of 27 simple oxygenated xanthones was evaluated by determination of their minimal inhibitory concentration on clinical and type strains of Candida, Cryptococcus, Aspergillus and dermatophytes, and their preponderance on the dermatophytic filamentous fungi was observed. Furthermore, a simple and efficient HPLC method with UV detection to study the effect of the active xanthones on the biosynthesis of ergosterol was developed and validated. Using this methodology, the identification and quantification of fungal sterols in whole cells of Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Trichophyton mentagrophytes were accomplished. In summary, 1,2-dihydroxyxanthone was found to be the most active compound against all strains tested, showing its effect on sterol biosynthesis by reducing the amount of ergosterol detected.

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“…This can be compared with a control profile and profiles given by known SBIs, to establish whether the compound is an inhibitor and, also, to identify at which enzyme sites inhibition may be occurring. 25 The site of inhibition is normally identifiable by observation of increased radiolabel incorporation into an immediate precursor. Secondly, the quantity of labelled ergosterol can be assayed, against the control, at a range of inhibitor concentrations in order to obtain a dose response curve and an ICs0 representing the concentration at which incorporation into ergosterol is reduced to 50% of that of the control.…”

Section: Biochemical Assaysmentioning

confidence: 99%

Guanidinium and amidinium fungicides: A new class of carbocation mimetic ergosterol biosynthesis inhibitors

Arnold¹,

Duriatti²,

Jung³

et al. 1995

Pestic. Sci.

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A novel class of chemical has been designed with the aim of inhibiting the Δ14‐reductase and Δ8‐Δ7‐isomerase enzymes in the ergosterol biosynthesis pathway in fungi. Use was made of knowledge about the mechanisms of both enzymes and the mode of action of known, fungicidal inhibitors of these enzymes. Pioneer examples have been synthesised and have been demonstrated to be potent inhibitors of ergosterol biosynthesis in Ustilago maydis (DC) Corda, acting in the same manner as the commercial fungicide fenpropimorph. They also showed excellent fungicidal activity against Erysiphe graminis DC f. sp. hordei Marchal (powdery mildew of barley) and Puccinia recondita Rob. ex Desm. (wheat leaf rust) in in‐vivo glasshouse tests. Using these compounds as a starting point, systematic structural variation has been carried out. Testing of a wide range of analogues at high volume confirms the potential of this class of compound to control mildew and rust pathogens at levels comparable to those of the standards. Correlation of in‐vivo and enzymatic data is good and the structure‐activity relationship developed for this series of compounds closely parallels that found for the morpholine/piperidine class of fungicides, suggesting a common mode of action.

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Separation of fungal sterols by normal-phase high-performance liquid chromatography

Cited by 23 publications

References 19 publications

Toward A Method For Measuring Instantaneous Fungal Growth Rates In Field Samples

Toward A Method For Measuring Instantaneous Fungal Growth Rates In Field Samples

Antifungal Activity of Xanthones: Evaluation of their Effect on Ergosterol Biosynthesis by High-performance Liquid Chromatography

Guanidinium and amidinium fungicides: A new class of carbocation mimetic ergosterol biosynthesis inhibitors

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