Antibiotic Action of Griseofulvin on Dermatophytes

Transcription

1 JOURNAL of BACTERIOLOGY, Mar., 1965 Copyright g 1965 American Society for Microbiology Vol. 89, No. 3 Printed in U.S.A. Antibiotic Action of Griseofulvin on Dermatophytes MOUSTAFA A. EL-NAKEEB, W. L. McLELLAN, JR., AND J.. LAMPEN Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey Received for publication 2 June 1964 ABSTRACT EL-NAKEEB, MOUSTAFA A. (Rutgers, The State University, New Brunswick, N.J.), W. L. McLELLAN, JR., AND J.. LAMPEN. Antibiotic action of griseofulvin on dermatophytes. J. Bacteriol. 89: The concentrations of griseofulvin required to inhibit growth and to produce the characteristic morphological distortions were determined for dermatophytes (highly sensitive), fungal plant pathogens (moderately sensitive), filamentous nonpathogenic fungi (poorly sensitive), and for yeasts and Escherichia coli (insensitive). Addition of griseofulvin to small inocula of the dermatophytes Microsporum gypseum and Trichophyton mentagrophytes produced complete and apparently permanent growth inhibition. If the antibiotic was added to actively growing cultures, the inhibition was only temporary, even with the most sensitive dermatophytes. During growth inhibition, griseofulvin temporarily halted the net synthesis of protein and nucleic acids, and of the amino acid and nucleotide pools. It decreased substantially the incorporation of C14-uridine or C'4-thymidine into nucleic acids of M. gypseum, but not that of C'4-leucine or C14-valine into protein. With a less sensitive culture, T. mentagrophytes x8, the uptake of uridine was inhibited only to a slight extent; the incorporation of leucine was unaffected. A partial protective effect of purine nucleotides against growth inhibition by griseofulvin was observed with one strain of T. mentagrophytes, but not with another strain or with M. gypseum. Brian, Curtis, and Hemming (1946) isolated from culture filtrates of Penicillium janczewskii Zal. a material they called "curling factor," since it produced severe stunting and distortion of the germinating spores of Botrytis allii. Grove and collaborators (1952) proved the identity of the curling factor with griseofulvin-an antifungal antibiotic discovered earlier by Oxford, Raistrick, and Simonart (1939). Studies on the griseofulvin-sensitivity of different organisms showed that dermatophytes were very sensitive, whereas bacteria and yeasts were insensitive (Brian, 1949, 196; Roth, Sallman, and Blank, 1959). There was disagreement concerning the sensitivity of filamentous fungi other than the dermatophytes. The effectiveness of griseofulvin in curing experimental ringworm infections in guinea pigs (Gentles, 1958) emphasized the unusual sensitivity of the dermatophytes, and has led to the use of griseofulvin in the treatment of superficial skin infections caused by these organisms. The chitinous cell walls of fungi were suggested by Brian (1949, 196) to be the site of griseofulvin action. He observed that growth reduction and morphological changes were produced by the antibiotic only in those fungi which possessed chitinous walls. Exceptions to this correlation were noted, however, by Abbot and Grove (1959) and by McNall (196). As part of a general investigation of the action of griseofulvin on fungi, groups of organisms with diverse levels of sensitivity were selected for subsequent biochemical study. Certain general features of the antibiotic action of griseofulvin are presented, especially the effect on the total synthesis of cell constituents and on the incorporation of C14- nucleosides and amino acids into nucleic acids and proteins. MATERIALS AND METHODS Organisms. The strains and their sources are listed in Table 1. Stock cultures on slants were stored at 4 C. Before cultivation in liquid media, subcultures were made on solid media (1.5%, w/v, agar) and incubated at the appropriate temperatures. Media. All filamentous fungi, with the exception of Neurospora crassa, were usually grown on a Neopeptone medium which contained 1 g of Neopeptone (Difco) and 2 g of glucose per liter of tap water. In some experiments, where indicated, the following synthetic medium (AGS) was used (g per liter of distilled water): arginine monohydrochloride, 2.; glucose, 2.; K2HP4, 1.; NaCl, 1.; MgSO4-7H2,.5; citric acid,.4; Fe2(S4)3. 6H2,.1; CuS4-5H2,.1; ZnSO4-7H2;.1; MnSO4-H2,.1. For the cultivation of yeasts and N. crassa, vitamin-enriched Vogel's N medium (Marini, Arnow, and 557

2 558 EL-NAKEEB, MCLELLAN, AND LAMPEN J. BACTERIOL. Lampen, 1961) was supplemented with 2% glucose (w/v). Escherichia coli was cultured in M-9 medium (Roberts et al., 1955) to which 1.5% (w/v) glucose was added. Culture conditions. For bacteria and yeast, the cells from a 24-hr slant culture were suspended in 2 ml of medium and added to 1 ml of the same medium in a 25-ml flask. The culture was incubated on a reciprocal shaker (7 rev/min, 8.9-cm stroke) at 3 C for 48 hr. With filamentous fungi, 1-ml volumes of the appropriate medium in 5-ml flasks were inoculated with.1 ml of a suspension of spores or a mycelial homogenate and shaken (7 rev/min, 8.9-cm stroke) at 28 C for 72 hr. Inocula of filamentous fungi. A spore suspension was prepared by adding a few milliliters of sterile medium to a 1- to 2-week-old culture of the fungus. The spores were dislodged gently, and the mycelial fragments were removed by straining through sterile glass wool. The suspension was then diluted to give 16 spores per milliliter. For preparation of the cell homogeniates, 3-dayold shaken cultures of the organism were used. The mycelia were harvested, washed twice, and suspended in saline. The suspension was shaken with glass beads (3 mm in diameter) on a rotary shaker for 3 to 45 min at 28 C. The homogenate was passed through cotton gauze and the myceliumn was collected by cenitrifugation. The sedimerit was washed twice and suspended in saline to give a 2.5% (v/v) suspension. Aseptic conditions were maintained throughout the procedure. Griseofulvin stock solution. Griseofulvin was dissolved in dimethylsulfoxide (Stepan Chemical Co., Northfield, Ill.) at 1 mng/ml, and the solution was stored at 4 C. The required amounts of the antibiotic in microvolumes of the stock solution were added directly to sterile media. Similar volumes of dimethylsulfoxide were incorporated into the control cultures. Estimation of dry weight. The fungi used in this work did not form a homogeneous diffuse growth. Adequate repetitive sampling from a single culture flask was, therefore, almost impossible. The following procedure produced reasonably reproducible results. Individual 1-mil cultures (flasks) were centrifuged. The mycelium was washed with distilled water and blotted with filter papers, dried in an oven at 1 C for 15 to 3 min, and, finally, desiccated under vacuum to constant weight. For bacteria and yeast, 2-ml samples were taken at the stated times from a single flask culture for each griseofulvin concentration. The cells were collected on Millipore discs, washed twice with distilled water (4 C), and dried. All mycelial weights are given on a dry-weight basis. Extraction of nucleic acid and protein. A modification of Schneider's extraction procedure (Schneider, 1957) was developed for use with the dermatophytes. The washed mycelia were extracted three times with 2 ml of boiling water for 5 min, followed by two extractions under each of the following conditions: 1% (w/v) trichloroacetic acid at 4 C for 5 min, 5%/O (w/v) trichloroacetic acid at 9 C for 15 min, and, finally, 1 N NaOH at 9 C for 3 min. This extraction procedure was found to be the best among several tested for fractionating the cell constituents of dermatophytes. Analytical methods. The protein content of the NaOH extracts was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Ribonucleic acid (RNA) and deoxyribonucleic acid (I)NA) in the hot trichloroacetic acid extracts were estimated by the procedures of Mejbaum (Schneider, 1957) and Burton (1956), respectively. Values are expressed as equivalents of yeast RNA or calf thymus DNA (Worthington Biochemical Corp., Freehold, N.J.). The amino acids and nucleotides in the soluble pool (hot water extracts) were measured as leucine and adenylic acid equivalents, respectively, according to the modified ninhydrin (Rosen, 1957) and orcinol (Schneider, 1957) methods. Radioactive tracer experiments. The following C'4-amino acids arid nucleosides were used: L-leucine-1-C'4, L-valine-1 -C'4, thymidine-2-c'4, and uridine-2-c'4 (specific activity, 21., 7.9, 25.7, and 3. mc/mmole, respectively). The labeled nucleosides were obtained from New England Nuclear Corp., Boston, Mass., and the amino acids from Nichem Inc., Bethesda, Md. Microamounts of these C'4-compounds were diluted with 1 mg of the corresponding unlabeled substances in 1. ml of distilled water and sterilized by filtration. The solutions were then added, with or without 1,ug of griseofulvin, to 1-ml cultures (flasks) of Microsporum gypseum (44 hr) or Trichophyton mentagrophytes x8 (72 hr) grown from spore inocula in the AGS medium. At the times specified in Tables 4 and 5, the amino acid and nucleotide pools, nucleic acids, and proteins of individual cultures were extracted, as described above. The radioactivity was measured by adding the different extracts (.1 ml) to the dioxane scintillation mixture (15 ml) of Bray (196) and counting in Tri-Carb liquid scintillation spectrometer series 314 E (Packard Instrument Co., La Grange, Ill.). All counts were corrected for background activity. RESULTS Griseofulvin action on growth and cell structure. The animal pathogens, Il1. gypseum and the Trichophyton strains, were more sensitive to griseofulvin than any of the other microorganisms tested (Table 1). The growth (as determined by dry weight) of A Iternaria tenuis, Cercospora melonis, and Glomerella cingulata (plant invaders) was partially inhibited by moderate levels of griseofulvin. The saprophytic fungi Aspergillus niger, Fusarium nivale, and N. crassa showed only slight inhibition. Griseofulvin produced severe morphological alterations (stunting and curling) in the mycelia of the dermatophytes at all concentrations tested. The other filamentous fungi showed much less distortion, especially at

3 VOL. V9GRISEOFULVIN 89, 1965 ACTION ON DERMATOPHYTES 559 the lower concentrations of the antibiotic (Table 1). Neither the cell mass nor the morphology of Saccharomyces cerevisiae, Candida albicans, or E. coli was affected with levels of griseofulvin as high as 1.tg/ml. Kinetics of the inhibition of dermatophytes by griseofulvin. When griseofulvin was added to Ml. gypseum cultures at the time of inoculation, it inhibited the growth of the fungus to a greater extent and for a longer period than when it was added at 44 hr, by which time considerable cell mass had developed (Fig. 1). There was almost complete inhibition at either time by 5,ug of griseofulvin per milliliter. The effect was only temporary, however, with the 44-hr cultures. The growth rate increased after a lag period, which was dependent on the concentration of griseofulvin. In a similar experiment, permanent inhibition of III. gypseum was observed only when griseofulvin, in concentration higher than 2,ug/ml, was incorporated into the medium at hr together with a small inoculum. Comparable results were obtained with T. mentagrophytes strains K and x8. The cells from cultures which had resumed growth in the presence of the antibiotic did not appear to have developed resistance to its action. Sufficient mycelial suspension (4 mg/1 ml) to produce growth after a 2-day lag was added to medium containing 1,ug of griseofulvin p)er milliliter. After 5 days, the cells were washed and diluted in fresh medium to the original cell concentration. Growth occurred without lag in unsupplemented medium, but was again inhibited for at least 48 hr by 1,ug/ml of the antibiotic. The explanation for this temporary insensitivity may lie in the fact that, under these circumstances, uptake of griseofulvin from the medium had ceased in the original cultures (El-Nakeeb and Lampen, 1965). Ribonucleotides and griseofulvin action. McNall (196) reported that the purine ribonucleotides prevented the inhibition of Al. canis by griseofulvin. A comparable reduction in griseofulvin action could be shown with T. mentagrophytes strain x8 (Table 2). Adenylic and guanylic acids decreased the inhibition of growth from 56 to 19%, when both were present at ltg/ml. 1 Lower concentrations had little effect. The individual nucleotides were much less effective, even at 2 Ag/ml. In contrast to the l)ositive results with strain x8, the reversing effect of purine nucleotides for T. mentagrophytes strain 171 was slight, and the pyrimidine derivatives were without effect. In another experiment, 12 nucleotides and nucleosides (including deoxy derivatives) were incorporated at 1 gag/mnl into cultures of M. gypseum, in liquid and solid media, without altering the inhibitory action of griseofulvin. It should also be noted that essentially identical concentrations of griseofulvin were required to TABLE 1. In vitro growth inhibition of microorganisms by griseofulvin Microorganism Micr osporum gypseum 'richophyton mentagrophytes (k)... T. mentagrophytes x8... T. interdigitalis Cg9o... T. persicolor C Alternaria tenuis C87... Cercospora melonis C61... Glomerella cingulata C Aspergillus niger C Fusarium nivale C Neurospora crassa A Saccharomyces ceo-evisiae LK2G12. Candida albicans Escherichia coli B... Source* S K SC C CC C CC H L V.5 38 Mt 61 M 29 M 65 M 6 M 6 6 Griseofulvin concn (jg/ml) M 2 M 25 M (sa 9 M 1 M 2 M * C = A. A. Campbell, Glaxo Laboratories Ltd., Stoke Poges, England; H = Harlyn Halvorson, University of Wisconsin, Madison; K = E. A. Konopka, Ciba Pharmaceutical Products, Inc., Summit, N.J., L = H. A. Lechevalier, Institute of Microbiology; V = H. J. Vogel, Institute of Microbiology; S = M. Solotorovsky, Rutgers, The State University, New Brunswick, N.J. t Results, expressed as per cent inhibition of growth, are the average of three estimations. Spore inocula were used for all the fungi. The yeasts and E. coli were incubated 48 hr; all other organisms were incubated 72 hr. M = morphological distortion observed

4 56 EL-NAKEEB, MCLELLAN, AND LAMPEN J. BACTERIOL. o 12 J 8 I,( 4 1-j 1 An. E 2 HOURS FIG. 1. Inhibition by griseofulvin of the growth of Microsporum gypseum. Neopeptone medium (3 ml in 125-ml flasks) was inoculated with.1 ml of the mycelial homogenate. Symbols:, no griseofulvin added; A, 1.5,usg/ml; O, 5. iig/ml. Griseofulvin was added (j ) at hr (solid lines) or at 44 hr (broken lines). inhibit the growth of M. gypseum in the synthetic medium (AGS), in Neopeptone medium, and in Vogel's medium supplemented with.5% casein hydrolysate. Nutritional factors do not appear, therefore, to be significant in determining the response of M. gypseum to the antibiotic. Effect of griseofulvin on protein and nucleic acid synthesis. Addition of griseofulvin (1,Ag/ml) to growing cultures of M. gypseum inhibited net protein, RNA, and DNA synthesis for about 6 hr (Table 3). The level of amino acids and nucleo- / A TABLE 2. Effect of nucleotides on the inhibition of Trichophyton mentagrophytes x8 by griseofulvin* Nucleotide Mycelial wtt Per cent of control growth: With Without With Withou t griseo- griseo- griseo- griseofulvin fulvin fulvin fulvin,g/ml A G A, 5 G A G A) 1 G * Neopeptone medium was inoculated with 1% of a 2% (v/v) mycelial homogenate. A and G represent the 5'-ribonucleotides of adenine and guanine which were added to the inoculated medium as sterile solutions. Cultures received either 1,ug of griseofulvin/ml (with griseofulvin) or no antibiotic (without griseofulvin). t Average of two estimations. Amounts are expressed as milligrams per 1 ml. t Growth in culture without added nucleotides or griseofulvin. tides in the water-soluble pool and of protein and nucleic acids then began to rise. This was followed, after 24 hr, by a resumption of growth. The incorporation of C14-uridine and C14- thymidine into nucleic acid (hot trichloroacetic acid extracts) of M. gypseum was inhibited by griseofulvin (Table 4), although the uptake was not eliminated. Incorporation of C'4-leucine and C14-valine into protein (hot NaOH extract) was, however, unaffected. The nature of the C14-labeled material in the hot NaOH extract was examined, since griseofulvin inhibited net protein synthesis but not the TABLE 3. Inhibition by griseofulvin of nucleic acid, nucleotide, amino acid, and protein synthesis in Microsporum gypseum* Mycelial wt Protein SolublNe pood (Nmoles/1 ml) RNADNA ml) (mg/b Ml) (jsg/b Ml) (mg/bo Ml) (mg/b l) oui ol Amls Time Amino acids Nucleotides -Gr + Gr -Gr + Gr -Gr + Gr -Gr -Gr -Gr + Gr - Gr ± Gr hr * M. gypseum was grown from spores in AGS medium for 72 hr before the addition of griseofulvin at zero time. -Gr = no griseofulvin; +Gr = 1lg of griseofulvin per ml.

5 VOL. 89, 1965 GRISEOFULVIN ACTION ON DERMATOPHYTES 561 incorporation of acids. No C'4 (< 1 %) was found as lipid. Acid hydrolysis and paper chromatography showed that, at 3 hr, 75%ly, of the count in the fraction was C'4-leucine; 25% was found in other amino acids. At 15 hr, the radioactive label was randomized, and less than 4% was in amino acids. At 3 hr, 35% of the radioactivity added to the culture was lost as volatile compounds; at 15 hr, 75% of the added radioactivity had been lost. With C'4-valine at 3 and at 6 hr, 8% of the radioactivity appeared as valine, after acid hydrolysis and chromatography. The incorporation of C'4-uridine and C14- leucine was also determined with strain x8 of T. mentagrophytes, which has the unusual property (among the dermatophytes) of accumulating griseofulvin preferentially into its protein, rather than into its nucleic acid fraction (El-Nakeeb, 1963). Even with this organism, griseofulvin did not prevent the incorporation of C14-leucine into protein (Table 5). Although actively-growing cultures of strain x8 were less sensitive to griseofulvin than comparable cultures of M. gypseum, a partial inhibition of uridine uptake was still observed. DISCUSSION The data presented on the antimicrobial spectrum of griseofulvin confirm and extend those TABLE 4. Effect of gr iseofulvin on the incorporation of C'4-labeled uridine, thymidine, leucine, and valine into nucleic acid and protein of Microsporum gypseum* Time hr Uridine Thymidine Leucine Valine -Gr + Gr-Gr + Gr -Gr +Gr -Gr + Gr 5t * For details, see Materials and Methods. Count/min X 1-3 added to 1-mi cultures were 85, 789, 89, and 1,314 for uridine, thymidine, leucine, and valine, respectively. Data for uridine and thymidine represent uptake into the total hot trichioroacetic acid extract. Inhibition of growth by griseofulvin was comparable to that in the experiment of Table 3. -Gr: no griseofulvin; +Gr: 1,ug of griseofulvin per ml. t Expressed as counts per minute X 1-3 per 1-ml culture. TABLE 5. Effect of griseofulvin on the incorporation of C'4-uridine and C14-leucine into nucleic acid and protein of Trichophyton mentagrophytes x8* Time Uridine Leucine - Gr + Gr - Gr + Gr hr.2t * Conditions same as for Table 4. Counts per minute X 1-3 added to 1-mi cultures were 722 and 49 for uridine and leucine, respectively. t Expressed as 1-mi culture. counts per minute X 1-3 per reported by Brian (1949, 196) and by Roth et al. (1959). The degree of inhibition of the various fungi, noted at a particular griseofulvin concentration, depends to a large extent on the method used for evaluating the inhibition. Measurements of the diameter of fungal pellets (Roth et al., 1959) or of radial growth (Brian, 1949) primarily indicate the morphological distortions produced by the antibiotic, and give sensitivities differing from those obtainable by the dry-weight method adopted in this work. The morphological distortions occur at much lower antibiotic concentrations than those required to prevent an increase in cell mass. Growth inhibition is primarily a characteristic of the highly sensitive dermatophytes, and, even among this group, some Trichophyton strains (El-Nakeeb, 1963) show cell distortion without restriction of cell mass. Secondly, the inhibition is greatly affected by the size of the inoculum and the amount of cell material present before the addition of griseofulyin. With dermatophytes, the per cent inhibition produced by griseofulvin was inversely proportional to the log of the number of the spores used to initiate the cultures (El-Nakeeb, 1963). The proposal of Brian (1949, 196) that griseofulvin interferes with chitin biosynthesis is not directly supported by our experimental data, although it is not excluded. It should be noted that A. niger contains large amounts of chitin (Blumenthal and Roseman, 1957), and yet it is relatively insensitive to griseofulvin. Also, cellwall formation by N. crassa protoplasts (prepared according to Bachmann and Bonner, 1959) was not prevented by the antibiotic even at 1,ug/ml, although the resulting cells were distorted (El-Nakeeb, 1963). Neither the glycolysis nor the respiration of

6 562 EL-NAKEEB, MCLELLAN, AND LAMPENJJ. BACTERIOL. the sensitive dermatophytes (M11. gypseum and T. mentagrophytes) or of the poorly sensitive N. crassa was affected by griseofulvin (El- Nakeeb, 1963). The respiration of Botrytis allii (Brian, 1949) and Trichophyton rubrum (Roth et al., 1959) is also insensitive to the antibiotic. The concept that nucleic acid biosynthesis, especially the nucleotide polymerization step, might be the target of griseofulvin action was introduced by McNall (196), who observed that the inhibition of lllicrosporum canis by griseofulvin could be prevented in part by ribonucleotides. In support of his postulate, McNall cited the antimitotic colchicine-like effect reported for griseofulvin in rat and bean root cells (Paget and Walpole, 1958, 196). Mitotic interference was not observed, however, in hair roots of human scalp (Burgoon et al., 196). Although it was possible with T. mentagrophytes x8 (but not with related dermatophytes) to demonstrate a partial protective effect of ribonucleotides, the specific suggestion that the polymerization step is a major site of interference is probably not correct. Incorporation of uridine and thymidine into nucleic acid was reduced, but there was no accumulation of soluble nucleotides in inhibited cells (Table 3) nor in culture filtrates (El-Nakeeb, 1963), as might have been anticipated if the main block was at the polymerization stage. It can also be concluded that there is no major breakdown of preformed polynucleotides, such as that produced by mitomycin C (Reich, Shatkin, and Tatum, 1961). Inhibition of the net synthesis of both nucleic acid and protein (Table 3) can be considered to occur rapidly; the doubling time of the organism is about 12 hr. No definite explanation can be provided for the observation that net l)rotein synthesis was halted during a 3 to 6 hr period following addition of griseoful6in, althou,-h incorporation of C'4-amino acids into protein was unaffected. Griseofulvin may have initiated an increased turnover of proteins; however, a detailed exaamination of the kinetics of protein formation and bieakdown will be needed to resolve this apparent discrepancy. No specific site of action of griseofulvin is demonstrated by the findings presented here. Special interest is drawn, however, to the alterations in nucleic acid and protein metabolism, because of the observation (El-Nakeeb and Lampen, 1964, 1965) that large amounts of griseofulvin are taken up by sensitive organisms and are present as complexes with the cellular nucleic acid and protein. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI from the National Institutes of Health, and by a grant from the American Friends of the Middle East, Inc. LITERATURE CITED ABBOT, M. T. J., AND J. F. GROVE Uptake and translocation of organic compounds by fungi. II. Griseofulvin. Exp. Cell Res. 17: BACHMANN, B. J., AND D. M. BONNER Protoplasts from Neurospora crassa. J. Bacteriol. 78: BLUMENTHAL, H. J., AND S. ROSEMAN Quantitative estimation of chitin in fungi. J. Bacteriol. 74: BRAY, G. A A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1: BRIAN, P. W Studies on the biological activity of griseofulvin. Ann. Bot. London 13: BRIAN, P. W Griseofulvin. Trans. Brit. Mycol. Soc. 43:1-13. BRIAN, P. W., P. J. CURTIS, AND H. G. HEMMING A substance causing abnormal development of fungal hyphae produced by Penicillium janczewskii Zal. I. Biological assay, production and isolation of "curling factor." Trans. Brit. Mycol. Soc. 29: BURGOON, C. F., H. J. GRAHAM, R. J. KEIPER, F. URBACH, J. S. BU RGOON, AND E. B. HELWIG Histopathologic evaluation of griseofulvin in Microsporum audouini infections. A. M. A. Arch. Dermatol. 81: BURTON, K A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: ElL-NAKEEB, M. A Antibiotic action and cellular binding of griseofulvin. Ph.D. Thesis, Rutgers, The State University, New Brunswick, N.J. EiL,-NAKEEB, M\1. A., AND J.. LAMPEN Complexing of griseofulvin by nucleic acids of fungi and its relation to griseofulvin sensitivity. Biochem. J. 92:59-6 P. EL-NAKEEB, M. A., AND J.. LAMPEN Uptake of griseofulvin by the sensitive dermatophyte, Microsporum gypseum. J. Bacteriol. 89: GENTLES, J. C Experimental ringworm in guinea pigs: oral treatment with griseofulvin. Nature 182: GROVE, J. F., J. MACMILLAN, T. P. C. MULHOL- LAND, AND M. A. T. IROGERS Griseofulvin. IX'. Structure. J. Chem. Soc., p LOWRY,. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: MIARINI, F., P. ARNOW, AND J.. LAMPEN The effect of monovalent cations on the inhibition of yeast metabolism by nystatin. J. Gen. Microbiol. 24:51-62.

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