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Effects of griseofulvin on dermatophyte metabolism McBride, Barry C. 1965

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EFFECTS OF GRIS£OFULVIN ON DERMATOPHYTE METABOLISM by Barry C. McBride B*Sc, The University of Bri t ish Columbia, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF BACTERIOLOGY AND IMMUNOLOGY We accept this thesis as conforming to the required standards THE UNIVERSITY OF BRITISH COLUMBIA October, 1965 In presenting th i s thes i s in p a r t i a l f u l f i lmen t of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y a v a i l a b l e fo r reference and study. I fur ther agree that per-mission for extensive copying of t h i s thes i s for scho la r l y purposes may be granted by the Head of my Department or by his representatives,, It is understood that copying or p u b l i -ca t ion of t h i s thes i s for f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of The Un ivers i ty of B r i t i s h Columbia 1 Vancouver 8, Canada Date T i i ABSTRACT The si te of action of griseofulvin is not known. The results of this study enlighten some poorly understood aspects of the mechanism of this pro-blem. Griseofulvin did not interfere with the composition of the free amino acid pool or the nucleic acid precursor pool. Oxidation of exogenous gluc-ose was inhibited by griseofulvin, the extent of inhibition being dependent upon the respiratory characteristics of the mycelial preparation, Griseo-14 fulvin altered the pattern of oxidative assimilation of UL- C-glucose, More accumulated in the l i p id and cold TCA soluble fraction and less ^ C was accumulated in the hot TCA, hot NaOH, and insoluble residue frac-tions. A reduced divalent cation concentration induced morphological aber-rations similar to those caused by griseofulvin. Coincident with these studies, investigations were made into some aspects of metabolism of un-inhibited dermatophytes. Glucose stimulated oxygen uptake, when young cel ls grown in media with a low carbohydrate concentration were used for micro-respirometer studies. The level of endogenous respiration was affected by exogenous glucose, when glucose stimulated oxygen uptake by less than 14%. M. gypseum oxidizes 50% of the exogenous glucose and assimilates the re-mainder. A large proportion is assimilated into nitrogenous substances. Cell-free extracts contained glucokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate ac t iv i ty , indicating the presence of the hexose mono-phosphate shunt. i i i TABLE OF CONTENTS Page Abstract i i Table of Contents i i i List of Tables v List of Figures vi Acknowledgements v i i Introduction v i i i REVIEW OF THE LITERATURE Historical Review 1 Dermatophytes 2 Chemical and Physical Properties 3 Structure of Griseofulvin 3 Chemotherapeutic Characteristics 5 Antimycotic Activi ty 6 Acquisition of Drug Resistance 13 Griseofulvin Analogues 13 Metabolism 15 MATERIALS AND METHODS I. Chemicals 19 II . Culture Media 19 III . Chromatography 21 IV. Cell Preparation 22 V. Dry Weight Determinations 23 VI. Extraction and Analysis of Amino Acids 23 VII . Extraction and Analysis of Nucleic Acid Precursors 2k VIII . Oxygen Uptake Studies 25 iv TABLE OF CONTENTS, cont'd Page MATERIALS AND METHODS, cont'd. IX. Oxidative Assimilation Studies 26 14 X. Chromatographic and Electrophoretic Analysis of C 30 XI. Cell-Free Extract Studies 31 RESULTS I. Antibiotic Sensitivity 32 II . Resistance 36 I I I . Reversal of Griseofulvin Inhibition 38 IV. Amino Acids 39 V. Nucleic Acid Precursor Pool 44 VI. Studies on Dermatophyte Respiration 45 VII. Endogenous Resp i rat i on 61 VIII . Oxidative Assimilation 64 IX. Cel1-Free Extract Studies 70 X. Effect of Magnesium on the Morphology of Microsporum gypseum 72 DISCUSSION 78 SUMMARY 87 BIBLIOGRAPHY 90 LIST OF TABLES Table Page 1 Percentage distribution of assimilated griseofulvin 10 2 Protocol for Warburg manometric experiments 27 3 Sensitivity of M. quinckeanum #8 to griseofulvin 34 4 Microscoptic observations of M. quinckeanum #8 grown in griseofulvin 35 5 Sensitivity to griseofulvin 36 6 Effect of nucleic acid precursors 40 7 Protocol for amino acid studies 42 8 Amino acids found in the free amino acid pool of M. quinckeanum #8 43 9 Ultra-violet light absorbing characteristics of the cold TCA soluble pool 46 10 Influence of glucose on endogenous respiration of M. qypseum 62 11 Assimilation of ' \ from UL-'^C-glucose by M. qypseum grown in synthetic medium 65 12 Assimilation of '^C from UL-^C-glucose by M, qypseum grown in yeast extract medium 67 13 Assimilation of ' \ from UL-^C-glucose by M. qypseum in the presence of griseofulvin 69 14 Assimilation of ' \ from UL-'^C-glucose by M. qypseum in the presence of arginine 71 vi LIST OF FIGURES Figure Page 1 The structure of griseofulvin k 2 Effect of culture medium on oxygen uptake kS 3 Effect of culture medium on oxygen uptake 50 k Effect of age on oxygen uptake 52 5 Effect of age on oxygen uptake 53 6 Effect of buffer and pH on oxygen uptake 55 7 Effect of griseofulvin on oxygen uptake 57 =8 Effect of griseofulvin on oxygen uptake 59 9 Oxygen uptake by spores 60 10 Glucose-6-phosphate and 6-phosphogluconate act ivi ty 73 11 Glucokinase act ivi ty 73 12 Glucose-6-phosphate dehydrogenase act ivi ty 7k 13 M. qypseum grown without added divalent cations 76 \k M. qypseum grown without added divalent cations 76 15 M. qypseum grown in the presence of griseofulvin 77 16 M. qypseum grown in the presence of griseofulvin 77 ACKNOWLEDGEMENTS I am deeply indebted to Dr. J . J . Stock for his helpful advice and supervision throughout the course of this study. I would l ike to extend my thanks to Dr. J .J .R. Campbell and Dr. A. Gronlund for their helpful advice and the freedom they allowed me in using their radioactive counting equipment. My thanks also to the Schering Corporation Limited, Montreal, Quebec for kindly donating the sample of purified griseofulvin which was used in these studies. The work in this project was supported by a Medical Research Council Grant, No. MT-757. vi i I INTRODUCTION Griseofulvin is a fungicidal, metabolic by-product of several Pen- i c i l liiium species. Brian ( 1 9 5 4 ) found a compound which caused morpholog-ical aberrations of the germ tubes of Boytris a l 1 i i . and he called this substance the "curling factor". Grove ( 1 9 ^ 7 ) chemically analyzed the "curling factor" and found it to be identical to griseofulvin, a compound or iginal ly isolated by Oxford ( 1 9 3 9 ) . During the next few years the spec-trum of biological act ivi ty was expanded to include most ofhthe fungi con-taining chitinous ce l l walls. Gentles ( 1 9 5 8 ) reported the value of orally administered griseofulvin in the treatment of experimental dermatomycoses of guinea pigs. Other workers (Blank, 1 9 5 9 ; Riehl , 1 9 5 9 ) soon demonstrated the importance of the antibiotic in curing human ringworm. Progress in elucidating the mechanism of action of griseofulvin has been slow. Brian ( 1 9 ^ 9 ) found the antibiotic to have no effect on respira-t ion . Since his studies, there have been many conflicting reports on this issue. Unfortunately these workers used mycelial preparations which were too old and did not possess respiratory systems which stimulated oxygen up-take in the presence of glucose. One purpose of this study was to obtain mycelial preparations with the characteristics necessary to resolve this conf1 ict . Griseofulvin inhibits the uptake of phosphate and nitrogenous com-pounds (Ziegler, 1 9 6 1 ) and has been shown to inhibit the synthesis of nuc-leic acids and protein (El-Nakeeb, 1 9 6 5 ) . These facts correlate and sug-gest an interference with the synthesis of nitrogenous compounds. There-fore, this study w i l l also concern i t se l f with the effects of the antibiotic ix on the patterns of oxidative assimilation of glucose. The effect of gr is -eofulvin on individual enzymes, the free amino acid pool, and the nucleic acid precursor pool was also studied. The suggestion (McNall, I960) that certain nucleotides competitively reverse griseofulvin inhibition also would be tested. It became apparent while outlining a program of investigations into the s i te of action of griseofulvin, that one of the factors which had im-peded earlier studies was the lack of understanding of the normal metabolic behaviour of the dermatophytes. Studying the action of griseofulvin was in essence "putting the cart before the horse". Therefore, an attempt was made to gain an understanding of the patterns of normal metabolic behaviour. Studies in this area include: the effect of glucose on the level of endog-enous respiration, patterns of oxidative assimilation of glucose, and the effects of age and growth medium on oxygen uptake. 1. REVIEW OF THE LITERATURE Historical Review Griseofulvin was f i r s t discovered by Oxford et. al.. (1939) as a metabolic by-product of Penic?Ilium griseofulvum. They investigated the chemistry of griseofulvin but did not extend their studies to an examina-tion of its biological ac t iv i ty . Brian (19^5) found that a compound prod-uced by Penicil1ium janczewsk? caused germ tubes of BoytrIs al118 to be-come distorted, wavy, and stunted. A year later Brian (1946) isolated the compound and named i t the "curling factor 1 1. Following its isolat ion, McGowan (1946) carried out chemical characterization studies. In 1947, the curling factor was shown to be biologically and chemically identical to griseofulvin (Grove, 1947). During the next two years, Brian (1949) demonstrated griseofulvin to be inhibitory to most members of the Family Eumycetes. There were no dermatophytes included in his test organisms. Thus, in 1949 griseofulvin was well characterized chemically and Its broad spectrum of Inhibition determined, but no attempts had been made to determine its value in the treatment of mycotic infections of animals. It remained for Gentles (1958) to demonstrate that orally-administ-ered griseofulvin was effective in eradicating experimental dermatophytic infections of guinea pigs. Similar results were obtained by Lauder and Sullivan (1958) treating Trichophyton verrucosum infections of cat t le . The efficacy of griseofulvin in the treatment of human dermatophytic in -fections was soon established by Williams (1958), Blank (1959), and Riehl 2 (1959) . Dermatophytes Dermatophytes are those fungi capable of Infecting the keratlno-phll Ice tissues of animals. Most of these organisms are from the genera Microsporum, Trichophyton, and Epidermophyton. With only a few exceptions (Stockdale, 1961; Dawson, 1961) no sexual reproductive systems have been found and thus, c lassif icat ion is based on the sporulation characteristics of these organisms and the c l i n i c a l manifestations of disease. Infection is confined to the skin, hair, or nails where the e t i o l -ogic agent remains in the mycelial state. Controversy exists as to whether the fungus-host relationship is parasitic or saprophytic. Kllgman (1955) suggests that the fungus invades only the dead keratinous tissue and is therefore a saprophyte. Wilson (195*0 and Newcomer (195*0 argue that since there is active bodily involvement the mold is invading the l iv ing tissue and is therefore a parasite. The diverse nature of the various dermatomycoses makes it probable that both hypotheses are va l id . Chemical and Physical Properties Griseofulvin is a colourless, odourless, neutral, crystal l ine com-e pound. It has a melting point of 218-219 C, and a molecular weight of 362. It is soluble in N, N-dimethyl-formamide, chloroform, ethyl acetate, benzene, acetone, dioxane, methanol, and dimethyl sulfoxide. It is only s l ight ly soluble in water (30 ug/ml) (Ashton, 1955; Roth, 1959; El-Nakeeb, 1965). The compound has characteristic absorption spectra with maxima at 296 mu and 326 mu in water (Abbot, 1959), 289 mu in butyl acetate (Robinson, I960) 3. and peaks at 325, 291, 263, 235 my in methanol (Grove, 1952). It is fluorescent, emitting light at 450 my when activated at 295 mu (Bedford, 1959). The antibiotic is remarkably stable. Storing for several weeks at 25* C within a pH range of 3.0-8.8, or autoclaving at 121* C for 20 min-utes w i l l not cause any loss of act ivi ty (Brian, 1949). The chemical behaviour of griseofulvin has been intensively studied and those interested are referred to the work of Oxford (1939) and that by a group of chemists at Imperial Chemical Industries (Grove, 1952a; 1952b; 1952c; 1952d; Mulholland, 1952a; 1952b). Of interest is the observation made by the latter group that griseofulvin is hydrolyzed to griseofulvic acid by ethanol ic N HCl and by N H2S0/ f at 100* C. It has a complex behav-iour in aqueous alkaline solution. It gives decarboxy griseofulvic acid, not griseofulvic acid and other neutral products, by treatment in 0.5N NaOH under different conditions. The molecule possesses two features which are rather uncommon in natural products, namely the aromatic chlorine substituent and the spiran system. Structure of Griseofulvin The original structural formula for griseofulvin was proposed by Oxford (1939) and was accepted with slight modifications by Grove (1947). However, on the basis of new spectral and chemical data, Grove (1952a; 1952b; 1952c; 1952d) modified his views and in 1952 he proposed the struct-ural formula that is accepted today (Fig. 1). The detailed nomenclature for this molecule is 7-chloro-4: 6-dimethyl-cumaran-3-one-2-sp i ro-1'-2(21-methoxy-6'-methyl eye 1ohex-21-en-4'one). Figure 1. The Structure of Griseofulvin. The antibiotic molecule is composed of three rings in a spiran system (Fig. 1). An aromatic benzene ring (A) substituted with a chlorine atom and two methoxy groups, a five-membered heterocyclic ring (B) forming a coumarane system with ring (A), and a hydro-aromatic ring (C) attached by an asymmetric carbon to ring (B). Quite recently Brown and Sim (1963) worked out the crystal structure of 5-bromo-griseofulvin by X-ray diffraction. They also calculated the d i f -ferent interatomic distances and covalent angles of the molecule. They found that ring (C) exists In a chair conformation forming about a 120* angle with the plane of ring A and ring B. The latter are planar to each other. 5 Cherootherapeutic Characteristics The major problem in the treatment of dermatomycosis is to bring the antifungal agent into contact with the fungus. This d i f f icu l ty arises be-cause the keratin present in skin, hair , and nails is highly impervious to topically-applied compounds. This problem had not been resolved when Wilson (1955) defined the properties which an effective antifungal agent should possess. He stated that, "the ideal antifungal drug even for super-f i c i a l mycoses would seem to be one which could be safely administered internally in amounts sufficient to endow the cel ls eventually destined to produce keratin with the power to resist fungi completely, this power per-s is t ing as they became keratinized, and the drug thus exerting i ts effect from within outwards." Gentles (1958a; 1958b; 1959a; 1959b; i960) suggest-ed that this was the mechanism by which griseofulvin exerted its effects when he was able to extract orally-administered griseofulvin from the hair of guinea pigs. Roth (I960) and Lorincz (1958) isolated the antibiotic from the skin of patients treated with the ant ibiot ic . Bedford (i960) found that orally-administered griseofulvin is absorbed into the blood stream via the small intestine. It can then be found distributed through many tissues of the body. It is detected in the highest concentrations in the skin, keratinous tissues (Bedford, 1959; Scott, I960, Gentles, I960), and the lungs and l iver (Bedford, I960). Freedman et. al_. (1962) have shown that in vi t ro griseofulvin w i l l bind to keratin. They feel that the molecule remains bound to the keratin molecule unti l the keratin molecule is enzymatically attacked by the derm-atophyte. The freed antibiotic can then inhibit fungal growth. Recent evidence (Smithe, 1965) showing that dermatophytes can digest keratin makes 6. this hypothesis feasible* The literature dealing with dosage schedules and effectiveness of treatment is voluminous and no attempt will be made to discuss this topic as it is not pertinent to this investigation. Antimycotic Activity Brian (1946) discovered the antifungal properties of griseofulvin. He observed that in the presence of the antibiotic the spores of Boytris  al1i i were able to form germ tubes, but that subsequent development into hyphae was inhibited. In low concentrations of griseofulvin, hyphae be-came wavy. As the concentration was increased, the hyphal tips became spatulate, then the hyphae became thick, and stunted and excessive branch-ing occurred. In very high concentrations, there was a complete inability to develop the hyphal form. These morphological aberrations are induced by concentrations of antibiotic 10 to 100 times less than necessary for inhibition of growth (Brian, 1949; Roth, 1959; Johnson, I 9 6 0 ) . Subsequent studies have shown that all susceptible fungi react similarly (Roth, 1959; Brian, 1949; Bohme, I960). More detailed observations using electron microscopy (Tomomatsu, I960) reveal that the cell wall appears to lose its integrity, splitting into irregular and frayed layers. Cytoplasmic organelles become grossly distorted (Thyagarajan, 1963) and large lipid granules replace much of the cytoplasm. The cytoplasmic membrane when it can be seen is pulled away from the cell wall. The picture is one of total cellular disorganization and indicates involvement of both structural and metabolic systems of the ce l l . Young, actively metabolizing cells are most readily affected (Foley, I960). Banbury (1952) applied griseofulvin to specific areas on the hyphae 7. and found that hyphal distortion occurred only when the application was made near the hyphal t ips . Electron micrographs show that only those nuclei found in the growing tips were abnormal (Thyagarajan, 1963). This resistance with age could be a result of impermeability to the antibiotic as i t is known that there is no translocation of griseofulvin within the hyphae (Banbury, 1952; Aytoun, 1956; I960). The greater resistance of older, less actively metabolizing ce l l s has resulted in confusion as to whether the antibiotic is fungistatic or fungicidal in nature. The major-ity of studies (Brian, 1949; Roth, 1959; Tomomatsu, I960) carried out using mixtures of both old and new hyphae suggest a fungistatic action. However, a study (Foley, I960) in which only young hyphae were used, indicated that griseofulvin is fungicidal. The antifungal spectrum of griseofulvin was intensively studied by Brian (1949)* Of the organisms examined, a l l Basidiomycetes, Ascomycetes, Fungi imperfect!, and Zygomycetes, with the exception of two yeasts, were sensitive to griseofulvin. No Oomycetes were affected. A l l actinomycetes and bacteria were insensitive. Roth (1959) investigated a large number of pathogenic fungi, yeasts, and bacteria. Only the dermatophytic fungi were sensitive. A l l other fungal pathogens as well as the yeast and bacteria were insensitive to concentrations ranging up to 30 "g/ml. The dermatophytes are the most sensitive fungi. Generally they are inhibited by concentrations In the range 0.2 to 10 Mg/ml (Roth, 1959; Brian, I960; Greco, I 9 6 0 ) . Many investigators (Brian, 1949; Banbury, 1952; Aytoun, 1956) could not find any griseofulvin in the ce l l and thus Brian (I960) concluded that griseofulvin exerted its inhibitory act ivi ty by ce l l wall alteration. 8. Boothroyd (1961) and, later, El-Nakeeb (1963; 1965) firmly established that griseofulvin does enter the c e l l . The latter studied the kinetics and requirements for entry and showed that the antibiotic was able to complex with various ce l l components. A discussion of El-Nakeeb's work follows. Except where noted, a l l the work was done with M. gypseum using tritium-labelled griseofulvin. Griseofulvin is taken up by the ce l l by two mechanisms; (a) an i n i t i a l rapid uptake, probably a result of physical ad-sorpt ion, (b) a slow continuous uptake, dependent upon an extracellular energy source, protein synthesis, pH, temperature and the concentration of the ant ibiot ic . Thus, i t appears that griseofulvin enters the ce l l v ia an active transport mechanism. Fractionation (El-Nakeeb, 1964; 1965) of the ce l l reveals that griseofulvin exists free and bound to various ce l l constituents. During the f i r s t twenty-four hours of exposure to the ant ibiot ic , 90% is found free in the h^O-soluble pool, and 10% is bound. At seventy-six hours 50% is free and 50% is bound. The bound antibiotic is divided evenly between nucleic acid and protein fractions. A small amount is present in the ce l l wa l l . This is probably associated with l i p i d as i t can be extracted with acetone. The longer the period of association with the ant ibiot ic , the stronger the complex. Ignoring the small amount found in the residue, the ratio of unbound griseofulvin: griseofulvin bound to nucleic acid: griseo-fulvin bound to protein is 5 0 : 2 5 : 2 5 . When another sensitive organism was 9 . tested the ratio was found to be 7 3 : 6 : 1 7 . El-Nakeeb suggests that binding may be the factor which causes inhibition of growth. This does not seem l i k e l y , because the growth of M. gypseum is inhibited only during the f i r s t twenty-four hours of exposure to griseofulvin and as mentioned ear l ier , 8 0 % of the griseofulvin is bound after th i s . El-Nakeeb ( 1 9 6 5 ) has attempted to correlate the sensi t iv i ty of an organism with i ts ab i l i t y to take up and bind griseofulvin. The completely insensitive organisms, bacteria and yeast, do not take up any antibiotic and i t is concluded that these are resistant because they are impermeable to the ant ibiot ic . Moderately sensitive organisms are capable of taking up large quantities of griseofulvin but they bind only a small percentage. As an attempt at final proof, he compares two sensitive and one resistant dermatophyte (Table 1 ) . This correlation is based on the ab i l i ty to complex with the nucleic acid. A positive correlation cannot be made from these data for a number of reasons. F i r s t , the sample assessed is too small. Second, the amount bound by the nucleic acid of the sensitive T. mentagrophytes is the mean of the other sensitive and resistant strains. Third, the resistant strain was not derived from either of the two sensitive strains. Fourth, as men-tioned before, the majority of the binding by M. gypseum occurs after the organism has become resistant. Before any val id assumptions pertaining to the relationship between sensi t ivi ty and binding ab i l i ty can be made, many more data must be accum-ulated. The importance of binding in the inhibition of growth is s t i l l un-known. On the basis of morphological studies and the inabi l i ty to find Table 1. Percentage distribution of assimilated griseofulvin % of griseofulvin assimilated Organism Bound to Bound to Unbound nucleic acid protein E. c o l i (insens i t ive Neurospora crassa (moderately sens it ive) T. mentagrophytes (part ial ly res istant) T. mentagrophytes (sensitive) M. qypseum (sens i t ive) 86 71 41 50 2.4 9 25 9.5 18 42 25 11. griseofulvin inside the c e l l , Brian (1949; I960) suggested that the ce l l wall is the s i te of action of griseofulvin. He postulated three mechanisms that would account for the ac t iv i ty . (1) Elements conferring r ig id i ty upon the ce l l wall would be al ter-ed or destroyed and as a result internal ce l l pressure would produce swol1 en, abnormal shapes. (2) Intervention with some regulatory mechanism for ce l l wall for-mat ion. (3) Interference with chi t in formation. This postulation was based on his belief that only organisms with chi t in in their ce l l walls were affected by griseofulvin. Subsequent studies by Abbot (1959) and McNall (I960) showed that non-chitinous organisms were also effected, Rhodes (J963) supported Brian's suggestion that griseofulvin was incorporated into the c h i t i n . Ronald (1964) analyzed the monosaccharides of ce l l walls from organisms grown in par t ia l ly- inhibi tory concentrations of griseofulvin and found there was no qualitative or quantitative alteration in the carbo-hydrate composition. There was no griseofulvin found in the ce l l walls . He also demonstrated that protoplasts were able to regenerate a ce l l wall in the presence of griseofulvin. Ronald's studies negate Brian's third suggestion but do not throw any light on the val id i ty of the other poss-i b i l i t i e s . McNall (I960) was the f i r s t to suggest that griseofulvin inhibited the synthesis of macromolecul es.« He found that certain nucleic acid pre-cursors would competitively reverse the growth inhibition induced by griseofulvin. Coupling this with the previously observed phenomena of 12. mitotic arrest in rapidly dividing ce l ls of rats (Paget, 1958), McNall postulated that the antibiotic inhibited a step in RNA polymerization. Unfortunately his results could not be repeated by other workers (E l -Nakeeb, 1963). El-Nakeeb (1965a) made a limited proximate analysis which revealed that ribonucleic acid, deoxyribonucleic acid and protein synthesis are par t ia l ly inhibited for twenty-four hours after being -treated with 14 griseofulvin. During this period, incorporation of C uracil into nucleic 14 acid was inhibited. The incorporation of C-leucine was not affected. Synthesis resumed after twenty-four hours. No conclusions can be drawn from the data for the following reasons: (a) The proximate analysis was not comprehensive. Therefore, i t is not known whether there is Inhibition of a l l synthetic processes or whether inhibition is unique In those systems examined. (b) At seventy-two hours, the griseofulvin-treated mycelia is com-posed of 54% protein and 42% ribonucleic acid. This is an ex-tremely unlikely occurrence and throws doubt on the val id i ty of related data. 14 (c) The incorporation of C-leucine into protein, when protein synthesis is supposedly stopped. The influence of griseofulvin on fungal respiration has been studied by a number of workers. The results are confl ic t ing. Some feel there is an effect (Roth, 1959; Meyer-Rohn, 1962; Ronald, 1964), while others do not (Brian, 1949; Foley, I960; Larsen, i 9 6 0 ) , Ziegler et al. (1961) havefound that griseofulvin suppresses incorporation of phosphate and nitrogen and suggests that this may be a result of uncoupling of oxidative phosphoryla-t ion . 13. A c q u i s i t i o n of Drug Resistance A n t i b i o t i c s exert strong s e l e c t i v e pressure on the genome of sus-c e p t i b l e microorganisms w i t h the r e s u l t that r e s i s t a n t s t r a i n s o f t e n develop. The a b i l i t y of an organism to become r e s i s t a n t t o a chemothera-p e u t i c agent i s an important f a c t o r in e v a l u a t i n g the continued usefulness of that a n t i b i o t i c . There are se v e r a l reports on the development of r e s -i s t a n c e t o g r i s e o f u l v i n . T. rubrum. M. can i s . M. audou i n i . E.. f 1 pccosum^, T. sch o e n l e i n ? . T. tonsurans. and M. quinckeanum #8 (Aytoun, I960; Robinson, I960; Rosenthal, I960; Schwarz, I960) have a l l been shown to develop r e s -i stance in v i t r o upon continued s u b c u l t u r e in i n c r e a s i n g concentrations of g r i s e o f u l v i n . Rosenthal (I960) s t u d i e d the c h a r a c t e r i s t i c s of t h i s acquired r e s i s t a n c e and demonstrated t h a t ; (a) r e s i s t a n c e could not be reversed in  v i t r o f (b) r e s i s t a n c e could be reversed in v i v o by a s i n g l e passage through a guinea p i g , (c) in experimental i n f e c t i o n s , both r e s i s t a n t and non-res-i s t a n t organisms responded s i m i l a r l y t o g r i s e o f u l v i n therapy. In no case could r e s i s t a n c e be increased to a p o i n t which would i n t e r f e r e w i t h therapy. There have been a few reports of the development of r e s i s t a n c e dur-ing prolonged g r i s e o f u l v i n therapy ( I t o , I960; Michael i d e s , 1961). Development of r e s i s t a n c e has been a t t r i b u t e d to enzymatic degrada-t i o n of g r i s e o f u l v i n (Aytoun, I960). Microsporum can i s and Microsporum  gypseum i n a c t i v a t e the a n t i b i o t i c , removing one of i t s f i v e methyl groups. D i f f e r e n t species remove d i f f e r e n t methyl groups. G r i s e o f u l v i n Analogues More than 350 analogues of g r i s e o f u l v i n have been prepared ( A r k l e y , 1963a; 1963b; Walker, 1962; Goodal1, 1963; MacMillan, 1954; Livinggood, I960). The m a j o r i t y of these compounds have been b i o l o g i c a l l y assayed and 14. their act ivi ty compared to griseofulvin (Crowdy, 1959a; 1959b; Abbot, 1959; Boothroyd, I960; Brian, I960; Crosse, 1964). A summary of these studies follows. Replacement of the chlorine by hydrogen or bromine decreases the act ivi ty of the antibiotic (Brian, I960). The minimal amount of griseo-fulvin required to produce the characteristic wavy hyphae of Bovtris al1? i was 0.1 ug. To produce the same effect, 0.75 Hg of dechlorogriseofulvin was required. The diol (7 'chloro-4':6* dihydroxy-4:6 dimethoxy-2*-methyl grisan-3-one) has decreased biological act ivi ty (Abbot, 1959) . The mono demethylated compounds; 4 demethyl, 6 demethyl, and 2' demethyl griseofulvin do not exhibit any in vi t ro biological act ivi ty (Boothroyd, I960). In independent investigations, Crowdy (1959a) and Crosse (1964) studied over 300 analogues, testing both their in vivo and in vi t ro ac t iv i ty . They concluded that: (a) substitution of H, OH, or NH2 for the methoxy group on C2' drast ical ly reduces biological act ivi ty (Crowdy, 1959b) ; (b) in vivo act ivi ty is lost and in vi t ro act ivi ty reduced by saturation of the double bond in ring C (Fig. 1) (Boothroyd, I960); (c) in a homologous series of 2* alkyloxy analogues, the in vi t ro biological act ivi ty increased to maximum on the 2'niipropoxy and 2'n butoxy analogue. However, in vivo act ivi ty was at a maximum in the 2* ethoxy analogues (Crowdy, 1959b) ; (d) the act ivi ty of the analogues is dependent upon the test organ-ism used (Boothroyd, I 960 ) . 15. A number of 2' alkylthio derivates have been pnqpared (Livinggood, I960). The 2 methythio and the 2' ethythio analogues are as active as griseofulvin, but the 2' propylthio and the 2' butylthio analogues are less active. Metabo1 ism Although our knowledge of metabolism has increased vastly in recent years, the majority of this information has come from studies on bacteria, yeast, and a few industrially-important molds. Very l i t t l e is known con-cerning the metabolism of dermatophytes. There are two principle reasons for this gap in our knowledge. One is the high rate of endogenous respira-tion which makes interpretation of respiratory experiments d i f f i c u l t . The second is the diversity of methods used in growing and preparing c e l l s , and this makes any correlation of results almost impossible. The f i r s t studies on dermatophyte respiration were carried out by Nickerson and Chadwick (1946). They reported that these organisms have high rates of endogenous respiration and that the rate of respiration is not increased when exogenous substrate is supplied. Similar results have been obtained by a number of workers using a variety of organisms (Brian, 1949; Melton, 1950; Chattaway, 1954). There are even cases of exogenous substrate reducing the rate of respiration (Roth, 1959). The lack of differences between exogenous and endogenous respira-tion precludes manometry as a useful tool to study respiration. To rectify th is , attempts have been made to reduce the endogenous supply by starvation. The results are contradictory. Some workers (Darby, 1950; Webley, 1952; Brown, 1955; Ronald, 1964) report satisfactory results. Whereas others (Bentley, 1953; Hockenhull, 1954) find that starvation reduces both exogen-16. ous and endogenous r e s p i r a t i o n . Because some enzyme systems are more l a b i l e than o t h e r s , extended s t a r v a t i o n periods could r e s u l t in the incom-p l e t e f u n c t i o n i n g of the t o t a l metabolic system. Results obtained in such a case would not be i n d i c a t i v e of r e s p i r a t i o n in the normal s t a t e . In a l l the s t u d i e s r e f e r r e d t o , the organism has been grown i n Sabouraud's b r o t h . This growth medium i s extremely r i c h , c o n t a i n i n g 4% glucose. In an e n v i r -onment where there i s an excess of energy source, organisms w i l l o f t e n produce l a r g e deposits of substances that can be used as endogenous sub-s t r a t e (Giese, 1946). Cochrane (1958) suggests that instead of s t a r v i n g c e l l s , emphasis should be placed on look i n g f o r a medium that w i l l not permit development of endogenous reserves. When Nickerson i n i t i a t e d r e s p i r a t o r y s t u d i e s , he used mycelia scraped from agar. Many workers s t i l l use t h i s technique. This e x p e r i -mental approach s u f f e r s from two severe disadvantages. Media i s c a r r i e d over w i t h the hyphae, p r o v i d i n g an unknown amount of nitrogenous and carbo-hydrate s u b s t r a t e . A l s o , i t i s not p o s s i b l e to make an evenly dispersed c e l l u l a r suspension, thus making v a l i d q u a n t i t a t i v e assays a matter of luck. These problems were p a r t i a l l y overcome when Melton (1950) suggested using mycelia grown i n submerged shake c u l t u r e . Under these c o n d i t i o n s the mycelia grew as round p e l l e t s . However, p i p e t t i n g of c e l l suspensions was d i f f i c u l t and q u a n t i t a t i v e r e s u l t s were d i f f i c u l t t o o b t a i n . Chattaway (1954) was abl e t o ob t a i n oxygen uptake d i f f e r e n c e s by using mycelia which had been broken in a Hughes pr e s s . Presumably, endogen-ous s u b s t r a t e became u n a v a i l a b l e when the i n t e g r i t y of the c e l l was d i s -rupted. The most c o n c l u s i v e advances in the understanding of dermatophyte 17. respiration have come from investigations of individual enzyme systems. Bentley (1953) prepared ce l l free extracts of M.. can i s . J_. mentagro- phvtes. and T. rubrum and found them a l l to contain L-amino acid oxidase. Jensen et. a l . (1957) demonstrated that J_. mentaqrophvtes possessed many of the enzymes responsible for the operation of the Embden-Meyerhoff pathway, hexose monophosphate shunt and tricarboxcylic acid cycle. Similar enzyme systems have been demonstrated in M.. canis. Many investigators have been concerned with the effect of fungicidal agents on respiration. Sodium azide, undecylenic, butyric, and propionic acids drast ically inhibited oxygen uptake. Assuming that a parallel to bacteria can be made, inhibi -tion of oxygen uptake by sodium azide would indicate that a cytochrome oxidase system constitutes the terminal respiratory mechanism in dermato-phytes. 2-4 Dinitrophenol stimulated oxygen uptake, thus the system which couples oxidation to phosphorylation, should be similar to bacteria. Correlating evidence from the three types of experiments, i t can be concluded that in the fungi examined there exists the Embden-Meyerhoff pathway, the hexose monophosphate shunt, a tricarboxcylic acid cycle and a cytochrome oxidase system. The relative importance of the i n i t i a l pathway of glycolysis has not been established. Although these results probably w i l l be applicable to a l l dermatophytes, they must be proven experimentally. Our knowledge of the endogenous respiration of fungi is inadequate, primarily because experimentation in this area has been scarce. The few results available indicate wide species variations (MacMillan, 1956; Blumenthal, 1957; 1963; Midwinter, I960) and a dependency on the previous history of the culture (Winzler, 1940; Blumenthal, 1951; 1963). For ex-ample, the respiratory coefficient of Scopulariopsis brevicaulis (MacMillan, 18. 1956) indicates l ip id to be the endogenous substrate. Similar data for Nocardia coral 1ina (Midwinter, I960) indicate carbohydrate is the sub-strate. In Nocardia coral Una the respiratory quotient varies with the age of the culture, supposedly, because the endogenous substrate changes. Occasionally, and for no known reason, M. quinckeanum #8 wi l l release large amounts of ammonia, possibly because a nitrogenous substrate is being oxid-ized. When analyzing the results of respiratory studies, it is important to know whether the rate of endogenous respiration is affected by the presence of exogenous substrates. The general practice is to subtract the endogenous from the exogenous oxidation values. In many instances this appears to be correct (Blumenthal, 1951; Cochrane, 1951; Stout, 1951; Moses, 1955). However, when an organism has a high rate of endogenous respiration, the poss ib i l i ty exists that a rate limiting metabolic step is saturated (Cochrane, 1958). If this occurs, then it is reasonable to assume that the added substrate wi l l compete with the endogenous substrate and thus lower the rate of endogenous respiration. The remaining alternative, namely, that added substrate wi l l cause an increase in endogenous respiration, has been demonstrated with Nocardia coral! ina (Midwinter, I960). There are no reports in the l i terature on endogenous metabolism of dermatophytes. 19. MATERIALS AND METHODS I. Chemicals A. Griseofulvin The griseofulvin used in this study was a purified sample which was kindly donated by Schering Corporation Limited, Montreal, Quebec. Two griseofulvin solutions were used in this study: 2. 5000 Fg/ml in dimethylsulfoxide (Matheson Coleman and Bell) prepared immediately prior to use, B. D-glucose-UL-1 / fC 14 0.5 mil l icur ies of D-glucose-UL- C having a specific act ivi ty of 150 mil l icur ies per millimole was obtained from International Chemical and Nucl-ear Corporation. A stock solution containing 100 ucuries/ml was prepared by bringing the total volume to 5 ml with d i s t i l l e d water. For oxidative assimilation studies, the stock solution was diluted with glucose and water to give a specific act ivi ty of 3.5 "curies per 3 "moles of glucose. C. A l l nucleotides, nucleosides, purines and pyrimidines were ob-tained from Pabst Laboratories. 11. Culture Media A. Sabouraud's Cerelose Broth 1. 4000 ug/ml dissolved in 95% ethanol and stored at 4* C, Cerelose 40 g Neopeptone (Difco) 10 g Tap water 1000 ml 20. (adjust pH to 5.8-6.0) B. Sabouraud's Modified Broth Glucose — — 10 g Neopeptone -• — 10 g Tap water 1000 ml (adjust pH to 5.8-6.0) C. Yeast Extract Broth Glucose 5.0 g Neopeptone • 7.5 g Yeast extract 5.0 g Dis t i l l ed water 1000 ml (adjust pH to 5.8-6.0) D. Synthetic Medium Glucose 10 g L (+) Arginine monohydrochloride - 10 g K2HPO4 1.0 g NaCl 1.0 g MgS0Zf»7H20 0.5 g MnSO^ — 0.001 g FeC\y6H20 0.01 g Dis t i l l ed water 1000 ml E. Sabouraud's Cerelose Agar Cerelose —— — — 40 g Neopeptone (Difco) 10 g Agar (Difco) 20 g Tap water 1000 ml 21. • Chromatography A l l chromatography was done on Whatman No. 4 paper using the descend-development technique. A. Solvents 1. Acetic acid : n-butanol: water, 2 :1:1, (v/v). 2. Methyl ethyl ketone: n-butanol; water, 2 :2 :1 , (v/v). For every 100 ml of solvent 0.25 ml of eyelohexylamine was placed on the floor of the chromatography tank immediately prior to hanging the chromatogram. 3. Ethyl acetate: pyridine: water, 8 :2 :1 , (v/v). 4. Isobutyric acid: ammonium hydroxide: water, 100:4.2:55.8, (v/v). 5. n-Butanol: water, 86:14 (v/v). B. Developers 1. 0.25% (w/v) ninhydrin in acetone. 2. Si lver nitrate (i) 0.5 ml of a saturated s i lver nitrate solution in 100 ml acetone, add d i s t i l l e d water until precipitate dis-appears ( i i ) 0.5N sodium hydroxide in 80% ethanol ( i i i ) 5% (w/v) Na 2S 203.5H 20 in water Dip through ( i ) , drain, dip through ( i i ) , drain and wash out background in ( i i i ) . 3. Phosphate spray Mix: 1 g ammonium molybdate dissolved in 8 ml water 3 ml 60% perchloric acid 3 ml HCl (concentrated) 2 2 . Make to 100 ml with acetone. Spray mixture l ight ly over chromatogram, dry, and place under ultra-violet light until blue spots appear. 4 . Ultra-violet light Compounds absorbing or fluorescing in ultra-violet light were identified in an ultra-violet cabinet, "Chromato Vue" (Ultra-violet Products, Inc.). IV. Cell Preparation Stock cultures were maintained on Sabouraud's cerelose slants at 4* C. Subcultures were made at s ix week Intervals, The organisms used in -cluded: Microsporum quinckeanum #8 and Microsporum gypseum. or iginal ly ob-tained from Dr. F. Blank. A. Preparation of Inoculum 1. M. quinckeanum #8. A few mis of s te r i l e medium were added to a seven day old slant culture and the spores dislodged. The spore sus-pension was evenly spread over the surface of a Roux flask of Sabouraud's cerelose agar. Following seven days incubation at 25* C, s t e r i l e 3 . 0 mm glass beads and s te r i l e phosphate buffer were added. Spores were dislodged by ro l l ing the glass beads over the mycelial mat. Mycelial fragments were removed by passing the suspension through s te r i l e glass wool. The spore suspension was diluted as necessary to give an optical density of 7 . 4 . ( 6 1 0 mu). 2 . M. gypseum. The same procedure was followed with the excep-tion that spores could be harvested after five days:., incubation. B. Cultivation of organisms Each medium used was dispensed in volumes that were 20% of the total flask capacity. In this way the surface to volume ratio remained constant 23. when different sizes of flasks were used. Flasks were inoculated with a spore suspension, optical density 7.4, at the ratio of 1 ml spores/100 ml of medium. Except where specified, the organisms were incubated in static culture at 25* C. When shake cultures were used, they were grown at room temperature on a Burrel1 "wrist action' 1 shaker at 225 cycles per minute with a 4.0 cm stroke. C. Harvesting mycelia Mycelia were harvested by suction f i l t r a t i on on Whatman No. 42 f i l -ter paper in a Buchner funnel and washed free of medium with a minimum of two l i te rs of 0.066M phosphate buffer, pH 7.0. The washed mycelial mat was peeled off the f i l t e r paper and suspended in the desired amount of buffer. When necessary, aseptic techniques were used. V. Dry Weight Determinations The diffuse, homogenous fungal suspensions developed for these studies permitted adequate repetitive sampling of concentrated mycelial preparations. Two 2 ml aliquots of the ce l l suspension and two of the sus-pending f lu id were pipetted into tared i aluminum weighing dishes. The dishes were dried at 98* C for 24 hours, and then cooled in a dessicator over anhydrous CaCl 2 . The dishes were then weighed as rapidly as possible to prevent absorption of moisture. VI . Extraction and Analysis of Amino Acids Mycelia from 2 ml of a washed ce l l suspension were collected by centrifugation in a 12 ml conical centrifuge tube. The mycelia had a minimum wet weight of 120 mg. Concurrent dry weight determinations were made on another 2 ml aliquot from the same ce l l suspension. The centrifuge 24. tube and its contents were cooled to 4* C. The mycelia were extracted with 3 ml of cold (4* C) 80% ethanol for thirty minutes. This mixture was sti r -red periodically. It was then centrifuged and the clear supernatant re-moved. The supernatant was then dried at room temperature by passing a stream of air over the surface of the liquid. As different weights of mycelia had been extracted, it was necessary to correct for these differ-ences in order to make the results qualitatively comparable. This was done by dissolving the extracts in an amount of distilled water commensurate with the weight differences of the extracted mycelia. Lipids were also extracted by this technique. Removal of lipids was not necessary as they did not influence the chromatographic separation of the amino acids. The samples were analyzed by one dimensional chromatography using n-butanol: acetic acid: water as solvent, and by two dimensional chromato-graphy using n-butanol: acetic acid: water as the first solvent and methyl ethyl ketone: n-butanol: water as the second solvent. VII. Extraction and Analysis of Nucleic Acid Precursors Two gm wet weight of mycelia was placed in a 28 ml Nossal cup toget-her with an equal volume of glass beads and cooled to 4* C. Enough cold (4* C) 0.6N HClO^ was added to make a thick slurry. The mixture was shaken in a Nossal cell disintegrator for 1.5 minutes in 30 second bursts. Be-tween shakings, the Nossal cup was cooled in ice. The contents of the cup were poured into a 50 ml centrifuge cup and extracted with an additional 15 ml of HCIO^  for 20 minutes at 4* C. The mixture was centrifuged and the supernatant collected. The pellet was washed with 5 ml of 0.6N HCIO^ . The pH of the combined supernatants was raised to 6.6 by adding 5N KOH. The precipitated KCIO^ was removed by centrifugation. The supernatant was then 25 stirred for one hour at room temperature with 0.6 ml of a 10% (w/v) aque-ous solution of activated charcoal. The charcoal adsorbs any compounds containing a purine or pyrimidine group. The charcoal was collected by centrifugation and washed with k ml of d i s t i l l e d water. The charcoal pellet was eluted with two 12 ml aliquots of 10% pyridine in 50% ethanol. Further extractions did not release any more compounds absorbing light at 260 mu. Charcoal was removed and washed by centrifugation. The eluate and washings were combined and concentrated by flash evaporation to 2 ml. The purified extracts were stored at -20° C. The extracts were analyzed by paper chromatography and paper electro-phoresis. The electrophoresis apparatus consisted of a water cooled horiz-ontal support, 50 inches long, and was powered by a "PowerPack" Model 3~104 (Buchler Inst. Inc., N . J . ) . Electrophoresis was carried out in 0.1M ammon-ium carbonate (Analar) buffer at 850 volts for 1.5 hours. One dimensional paper chromatography using n-butanol: water assthe solvent was efficient in separating non-phosphorylated compounds. Com-pounds were identified by their ab i l i ty to absorb or fluoresce in u l t ra -violet l ight . Compounds having phosphorylated groups were detected with phosphate spray. VIII . Oxygen Uptake Studies Yeast extract medium inoculated with M. guinckeanum #8 spores as described (Sect. IV A) was harvested after seventy-two hours of incubation. The well-washed mycelium was suspended in 0.066 M phosphate buffer, pH 7.0. The amount of buffer added depended on the concentration of hyphae. Best results were obtained when there was approximately 5 mg dry weight of mycelia/ml. After a few practice attempts, this concentration of hyphae 26. could be closely approximated by visual observation. The mycelial suspen-sion was divided into three equal aliquots; griseofulvin (40 ug/ml) in DMSO was added to one, an equal volume of DMSO was added to another, and water was added to the th i rd . These cultures were held at room temperature for two hours. Two 2 ml aliquots were taken for dry weight determinations. Duplicate Warburg flasks were set up according to the protocol in Table 2. Oxygen uptake was measured at 25° C using the Direct Method outlined in Manometric Techniques (Umbreit, 1959). Manometric data were collected every 15 minutes for the f i r s t two hours and every 30 minutes thereafter. The length of an experiment was determined by the respiratory level of the mycelial preparation. IX. Oxidative Assimilation Studies A. Preparation of ce l l s Thirty hour cultures of M. gypseum were aseptically harvested by f i l -t rat ion. The mycelia were suspended in 200 ml of the f i l tered medium and then divided into two equal aliquots, Griseofulvin (40 ug/ml) was added to one half and an equal volume of s te r i l e water to the other sample. These cultures were incubated five hours at room temperature. They were then harvested and washed by centrifugation. The mycelial suspension which had been incubated with griseofulvin was washed with, and suspended in , 0.066M phosphate buffer, pH 7,0, containing 40 ug/ml of griseofulvin, A Warburg experiment was set up and run as described (Sect, VIII) using D-14 glucose-UL- C, Following the completion of the microrespirometric experi-ment, the Warburg flasks containing ' \ were immediately cooled to 4* C. B, Analysis of the contents of the Warburg flasks 1, The f i l t e r paper from the center well of the Warburg flask Table 2. Protocol for Warburg manometric experiments Endogenous Glucose:; Endogenous Glucose Description of + + + + flask contents Endogenous Glucose Griseofulvin Griseofulvin DMSO DMSO 20% KOH (center wel 1) 0.2 0.2 0.2 0.2 0.2 0.2 Dermatophyte in Phosphate buffer 2.8 2.8 Dermatophyte + Griseofulvin in Phosphate buffer 2.8 2.8 Dermatophyte + DMSO in Phosphate buffer 2.8 2.8 Glucose (15 umoles/ml) (side arm) 0.2 0.2 0.2 Disti1 led water (side arm) 0.2 0.2 0.2 28. was placed in a 5 ml volumetric flask. The center well was then rinsed three times with d i s t i l l e d water and the rinsings added to the volumetric flask. The volume was brought to 5.0 ml with d i s t i l l e d water. This was termed the C02 fraction. 2. 2.6 ml of ce l l suspension were removed from each Warburg flask and placed in a 12 ml conical centrifuge cup. After centrifugation, the supernatant was removed. The mycelial suspension was washed with two 1 ml aliquots of 0.066M phosphate buffer, pH 7.0. These washings were com-bined with the original supernatants and frozen to prevent contamination prior to analysis. This material was termed the supernatant fraction. 3. Fractionation of the mycelia. The ce l ls were fractionated by modification of the procedure used by Roberts (1957). (a) The washed mycelia (step 2) were mixed with 0.3 ml of Superbrite glass beads (grade #110) and ground with a glass s t i r r ing rod for 5 minutes. The mixture was then suspended in 4 ml of cold (4° C) 5% trichloroacetic acid (TCA) at 4° C and extracted for sixty minutes with inter-mittent s t i r r i n g . During every step in the fractionation, the insoluble material was centrifuged down and washed with two 1 ml aliquots of the extracting f l u i d . The washings and the original extracting supernatant were then combined. (b) The pellet from (a) was suspended in 4 ml of 75% ethanol for sixty minutes at 50* C. The supernatant was termed the alcohol soluble fraction. (c) The pellet from (b) was suspended in 4 ml of chloroform: 29. methanol, 2:1 (v/v) for sixty minutes. The supernatant was termed the chloroform-methanol soluble fraction. (d) The pellet from (c) was dried under a current of a i r and then suspended in 2 ml of 5% TCA and heated in a boiling water bath for thir ty minutes. After centrifugation, the supernatant was removed and the procedure repeated with an additional 2 ml of 5% TCA. This was termed the hot TCA soluble fraction. (e) The pellet from (d) was resuspended in k ml of 0.1N NaOH and placed in a boiling water bath for sixty minutes. The supernatant was termed the hot NaOH soluble fraction. (f) The pellet from (e) was suspended in 50% h^SO^ and d is -solved by heating in a boiling water bath. This was termed the insoluble residue. (g) The supernatants from steps (b) and (c) were combined and diluted with 10 ml of water and 10 ml of ether. The ether layer was removed and the aqueous layer extracted twice more with two 10 ml portions of ether. The combined ether extracts were evaporated to dryness and the residue dissolved in k ml of chloroform. This was termed the chloroform soluble fraction. The extracted aqueous solu-tion contained alcohol soluble protein. C. Assay for radioactivity Radioactivity was measured using an automatic planchet sampler (Nuclear Chicago) equipped with a geiger counter having an ultra-thin win-dow. Counts were recorded on a Ratemeter (Nuclear Chicago) Model 181A and 30. and printed by a Printing Timer (Nuclear Chicago) Model C-111B. The assay technique consisted of evenly spreading 0.02 ml of the desired fraction over the surface of a stainless steel planchet and drying this material under an infra-red lamp. This is the " in f in i t e thinness" plating method. It is essential that the l iquid be spread evenly over as large an area as possible. Duplicate samples of each fraction were plated. If the counts of duplicate planchets differed by more than 5%, sampling was repeated. It was essential to reduce the f i l t e r paper in Sample 1 to 14 small fibres to ensure that a l l C02 was released into the basic aqueous solution. This was accomplished by pulverizing with a glass s t i r r ing rod. The fibres were removed by centrifugation and the supernatant plated. One ml of the cel lular residue dissolved in 50% h^SO^ was neutralized with a known volume of ION NaOH prior to plat ing. D. Colon-metric analysis The chemical nature of the isolated fractions was determined c o l o r i -metrically. 1. Protein determinations were made on the hot NaOH, hot TCA, and alcohol soluble fraction using the technique of Lowry (1951). 2. A carbohydrate determination was made on the cel lular residue using the anthrone method (Morris, 1948). 3. Ribonucleic acid In the hot TCA soluble and hot NaOH fractions was determined by the orcinol method (Schneider, 1957) using RNA (N.B.C.) as the standard. 14 X. Chromatographic and Electrophoretic Analysis of C A. The supernatant fraction was analyzed electrophoretically in 31. 0.1M ammonium carbonate buffer (Analar) at 850 volts for 1.5 hours. B. TCA was removed from the cold TCA soluble fraction by ether extraction. The extracted sample was chromatographed in n-butanol: acetic acid: water and methyl ethyl ketone: n-butanol: water and electro-phoresis carried out in 0.1M ammonium carbonate buffer, C, The chromatograms were cut lengthwise into three-quarter inch wide strips and assayed for radioactivity on an Actigraph (Nuclear Chicago) Model IO36, Counts were recorded on a Ratemeter (Nuclear Chicago) Model 1620B. A simultaneous graphic recording was made on a Graphic Recorder (Nuclear Chicago) Model R100, XII. Cell Free Extract Studies A, Cell free extracts of M, quinckeanum #8 spores Spores were harvested from ten Roux flasks and incubated in yeast extract medium for twenty-four hours. The spores were then harvested, washed, and placed in a 12 ml Nossal cup. Two ml of 0.02M trihydroxy amino methane (THAM) buffer, pH 7.2, were added to the spores along with enough Superbrite glass beads to make a thick slurry. The mixture was cooled to 0* C and then shaken in the Nossal for twenty seconds. The con-tents of the Nossal cup were centrifuged at 10,000' x g\jforeten:miihutesnand the supernatant removed. One ml of THAM buffer was added to the pellet and the procedure repeated. The supernatants were combined and stored at -20* C. B, Cell free extracts from M. quinckeanum #8 mycelia The mycelia from three liters of yeast extract medium were harvested, washed, and placed in a chilled mortar together with an equal volume of glass beads. The mixture was ground at h* C for ten minutes. During the 32. grinding, sufficient 0.02M THAM buffer, pH 7.2, was added to form a thick slurry. The paste was centrifuged at 10,000 x g for ten minutes. The supernatant was stored at -20* C. Dehydrogenase activi ty was measured by determining the reduction of triphosphopyridine nucleotide (TPN) in the presence of the specific sub-strate. Reduction was determined by measuring the change in optical dens-ity of the test solution at 3^ 0 my with a Beckman Model B spectrophotometer. 33 RESULTS I. Antibiotic Sensitivity Ai; M. quinckeanum #8 spore suspension was inoculated into yeast ex-tract medium containing griseofulvin. Growth was measured at twenty-four, forty-eight and ninety-six hours. As is shown in Table 3, 1 Hg/ml of griseofulvin slows the rate of growth. As the antibiotic concentration is increased, growth Inhibition increases unti l at a concentration of 4 ug/ml a l l growth is stopped. The cultures were maintained twelve days. During this time there was no evidence of adaptation or development of resistance to griseofulvin. The morphology of M. quinckeanum #8 in griseofulvin was examined by phase contrast microscopy using wet mount preparations. Because of its sensi t ivi ty to low concentrations of the ant ibiot ic , M. quinckeanum #8 was used to study the mode of action of griseofulvin. Microscopic observations are recorded in Table 4. The morphological abnormalities induced by griseofulvin in M. quinckeanum #8 were similar to those reported for other fungi1. For reasons outlined below, it became necessary to change the test organism when this thesis was only one half completed. In an experiment similar to the one described above, three organisms were tested for their su i tab i l i ty as replacements for M. quinckeanum #8 (Table 5). Morphological aberrations were similar to M. quinckeanum. A l l organisms tested were sensitive. M. qypseum was chosen because (a) it is the only organism that w i l l grow well on a synthetic medium, (b) Table 3 . Sensitivity of M. quinckeanum #8 to griseofulvin Growth at 25* C Concentration of griseofulvin 24 hours 48 hours 96 hours ug/ml 0 + 1 +2 +4 1 + 1 +2 + 3 2 ± + 1 + 3 3 ± ± + 1 Table 4. Microscopic observations of M. quinckeanum #8 grown in griseofulvin Concentrat ion of griseofulvin Microscopic morphology of hyphae ug/ml 0 Hyphae were straight and radiated out from their o r ig in . Branching occur-red every 5 to 6 septal units. 1 Hyphae were wavy and occasionally curled back upon themselves, tips were swollen or spatulate. 2 Hyphae were wider in diameter than normal, branching was increased. Rather than growing outwards the maj-ori ty of hyphae were curled backwards tending to form compact pel le ts . 3 Hyphae were very thick and frequently swollen. Branching was frequent, of-ten 3 or k new hyphal elements were formed within one septal unit . h A l l spores germinated, some conidia and up gave rise to as many as 3 or h germ tubes. The germ tubes were short and extremely distorted. 36. Table 5. Sensitivity to griseofulvin Organism Minimum concentration of griseofulvin required to stop growth M, quinckeanum #7 2 ug/ml M. quinckeanum #13 2 Hg/ml M. qypseum 3 Hg/ml i t produces spores in the shortest incubation period, and (c) development of pleomorphism is more readily detected. It has been suggested that the composition of the medium w i l l in -fluence sensi t ivi ty to griseofulvin (Raubitschek, 1961). The ab i l i ty of M. qypseum to grow in a synthetic medium provided a means to satisfactori ly evaluate this hypothesis. Sensitivity tests were performed on Sabouraud's, Sabouraud's modified, yeast extract, and a synthetic medium. Griseofulvin sensi t ivi ty of M. qypseum was the same in a l l media. Thus, antibiotic sensi t ivi ty does not appear to be altered by varying the concentrations of carbohydrates in the medium, the type of carbohydrate or the nitrogen source. 11. Resistance During an investigation into the effects of griseofulvin on oxygen uptake, the test organism, M. quinckeanum #8. developed unfamiliar charact-e r i s t i c s . Previous experiments had shown that the maximum oxygen uptake 37 attributable to exogenous substrate was approximately 20% greater than endogenous respiration. In a series of four repeated experiments this difference gradually increased unti l i t became 45% greater than the endog-enous. Coincident with this increase there was a decrease in the sensit-ivi ty of oxygen uptake to griseofulvin. This prompted an investigation into the nature of the organism being used in this investigation. Micro-scopic examination showed the microconidia were longer and thinner than the egg-shaped microconidia from the stock culture s t ra in . The ab i l i ty to form spores had also been reduced. Sensitivity to griseofulvin was studied. These results indicated that M. quinckeanum #8 had developed some resist-ance, as growth was only s l ight ly inhibited by 4 ug/ml griseofulvin. How-ever, increasing the concentration to 20 ug/ml did not increase the extent of inhibi t ion. Beyond this,concentration, growth was completely inhibited. Following these observations, i t was fel t that the organism was a contamin-ant. This hypothesis was made unlikely by the following evidence: (a) Thick walled septate macroconidia could be found in a slant culture. (b) Mycelial homogenates rubbed into scarified skin of a guinea pig produced a pyogenic form of dermatophytic infection, char-acterist ic of M. quinckeanum #8. Reisolation of the organism after passage through the infected guinea pig did not restore its original properties. An increase in metabolic act ivi ty and reduction in sporulation are characteristic of a change to the pleomorphic form (Reiss, 1957; Ito, 1958). There are several degrees of pieomorphism, each defined in part by the nature and quantity of spores produced. In this case, i f a change 38. to pleomorphism was the cause, then it was a very early stage in pleomor-phic development because there were s t i l l many spores produced. The interesting fact emerging from the observed alterations was the development of resistance in a non-selective environment. Up to this time, resistance to griseofulvin had been acquired only when a sensitive fungus was grown in the presence of griseofulvin (Robinson, 1960a; 1960b; Schwarz, I960; Rosenthal, I960), i .e . a selective environment. In this case, there was no selective pressure exerted but resistance developed concurrently with other metabolic. changes in the c e l l . I II . Reversal of Griseofulvin Inhibition McNall (I960) reported that nucleotides (5 Hg/ml) would reverse the inhibitory action of griseofulvin on M. can i s . Unfortunately he did not test the ab i l i ty of these nucleotides to stimulate the growth of control c e l l s . Therefore, a study incorporating more adequate controls was i n i t -iated. Solutions of a variety of nucleotides, nucleosides, purines, and pyrimidine bases were s ter i l ized by f i l t e r ing them through C0.'i3Hmi 11 ipore f i l t e r s . Each s te r i le solution was added to four 500 ml Erlenmeyer flasks containing 100 ml of Sabouraud's medium. Two of these flasks contained griseofulvin (1.1 Hg/ml) and two were without ant ibiot ic . The final con-centration of each nucleic acid precursor was 10 Hg/ml. Each flask was inoculated with 0.1 ml of an M. quinckeanum #8 spore suspension, O.D. 0.37, and incubated for s ix days at 25* C. Following incubation the flasks were cooled to 4* C. The mycelium was then collected on tared.• f i l t e r paper (Whatman #1). Support for the f i l t e r paper was provided by a coarse sintered glass f i l t e r disc. The f i l t e r paper and thoroughly washed mycelia 39. were placed in a taredJ aluminum weighing dish and dried at 96* C for thirty-six hours prior to weighing. Filter paper discs were dried to determine weight loss attributable to drying. As can be seen in Table 6, many of the added compounds stimulated the growth of cells without griseofulvin. If this fact was not recognized, erroneous conclusions could be drawn regarding the ability of these com-pounds to reverse the inhibition of growth caused by griseofulvin. Poss-ibly this happened in the case of McNall (I960). Thymine was able to de-crease the inhibition from 57% to 18%, Unfortunately this experiment could not be repeated because M., quinckeanum #8 became partially resistant to the antibiotic. A similar experiment was carried out using M. gypseum. but none of the compounds were able to reverse inhibition, El-Nakeeb (1963) obtained similar results with his strain of M, gypseum. It would seem that with the possible exception of thymine, none of the compounds tested are effective in reversing inhibition, IV. Amino Acids Ronald (1964) assayed the growth medium of M. quinckeanum #8 and found that griseofulvin did not cause excretion of amino acids. Such an approach would not indicate if formation of certain amino acids had been inhibited and would be dependent on permeability factors which are as yet unknown. For these reasons, it was thought that the extraction of whole cells would provide a more sensitive approach. A ninety-six hour culture of M. quinckeanum #8 grown on yeast ex-tract medium was harvested aseptically on a Buchner funnel and thoroughly washed with sterile 0.066M phosphate buffer (pH 6,6), The mycelium was then distributed evenly into 500 ml Erlenmeyer flasks. The series of Table 6. Effect of nucleic acid precursors on griseofulvin inhibition Weight of mycelia % of growth relative to the culture containing neither griseofulvin nor precursor* % increase in dry weight of griseofulvin cultures Precursor added Control Griseofulvin Control Griseofulvin mg mg Nothing added 37.12 15.84 100.0 42.7 -Aden ine 37.81 16.7 102.0 45.0 0.3 Adenos ine 43.25 21.38 116.5 57.5 -Adenosine 5' monophosphate 37.05 16.58 99.8 44.9 2.2 Cytidine 47.23 24.02 127.2 64.8 -Cytidine 5' monophosphate 40.86 19.69 110.0 53.1 0.6 Guan ine 41.12 19.2 110.8 51.8 -Guanos ine 38.16 17.13 102.7 46.3 0.3 Guanosine 5' monophosphate 39.97 19.77 107.8 53.3 2.4 Uraci1 41.83 19.38 112.9 52.1 -- continued Table 6, continued Precursor added Weight of mycelia % of growth relative to the culture containing neither griseofulvin nor precursor* % increase in dry weight of gr iseofulvin cultures Control Griseofulvin Control Griseofulvin mg mg Uridine 40.49 19.22 109.1 51.8 -Uridine 5' monophosphate 38.70 17.30 104.3 46.6 Thymi ne 41.15 36.05 110.9 97.2 43.5 The growth in the flask containing neither griseofulvin nor added precursor was assumed to be 100%, the percentage of growth in the other flasks was calculated relative to th is . flasks were prepared according to the following protocol (Table 7). Mycelium from the zero time control was immediately harvested and repeatedly washed with 0.066M phosphate buffer, pH 6.6. Buffer was added to make a final volume of 10 ml. Two ml aliquots were withdrawn for dry weight determinations and amino acid extraction. The eight remaining flasks were incubated at room temperature on a Burrell shaker at 225 cycles per minute with a 4.0 cm stroke. One flask of each series was removed at fifteen hours incubation and treated in the same manner as the zero time control. The remaining flasks were removed at twenty-four hours and treated s imilarly. Amino acids were separated by two dimensional paper chromatography and developed with ninhydrin: Solvent 1 (n-butanol: acetic acid: water); Solvent 2 (n-butanol: methyl ethyl ketone: water). Approximate quantita-tive estimates were made by comparing the intensity of ninhydrin positive spots. Fourteen amino acids were identified (Table 8). Those not found, but known to exist in dermatophyte proteins, were histidine, tyrosine, tryptophan, hydroxyproline and threonine. 0_ua1 itatively and quantitatively all preparations were similar at fifteen hours incubation. At twenty-four hours incubation, the amino acids were qualitatively similar in all preparations. The extract from cells grown in the medium without griseofulvin possessed slightly lower amino acid concentrations. Thus, it appears that in an actively growing prepara-tion the demand incurred by protein synthesis tends to reduce the available reservoir of amino acids. The lack of such a decrease in the griseofulvin preparation does not necessarily indicate that the antibiotic acts by in-hibiting protein synthesis. It could merely represent a secondary phenom-Table 7. Protocol for amino acid studies Description of flask Endogenous Glucose Zero + + time Endogenous griseofulvin Glucose griseofulvin control 0.066M Phosphate buffer, pH 6.4 100 ml 100 ml 100 ml 0.066M Phosphate buffer, pH 6.4 + 1% glucose 100 ml 100 ml Griseofulvin 4000 Hg/ml 0.5 ml 0.5 ml 43. Table 8. Amino acids found in the free amino acid pool of M. quinckeanum #8 Alanine Argin ine Asparagine Aspartic acid Cysteine Glutamic acid Glycine Leuc i ne/i so1euc i ne Lysine Meth ion ine Phenylalanine Proline Serine Valine 44. enon associated with generalized growth inhibit ion. The same amino acids were found in a l l extracts so that i t is unlikely that griseofulvin inter-feres with the synthesis of these compounds. No such speculation can be made regarding the amino acids not identified. V. Nucleic Acid Precursor Pool The ab i l i ty of certain nucleic acid precursors to overcome the in-hibitory effects of griseofulvin (McNalI, I960; El-Nakeeb, 1963) prompted investigation into the nature of the precursor pool. Two alterations were anticipated: (a) inhibition of nucleic acid polymerization, which would result in the accumulation of many precursors; or (b) inhibition of a step in the synthesis of a precursor which would result in accumulation of the compound which would be the substrate for the blocked reaction. M. quinckeanum #8 mycelia grown four days in sixteen 1 l i t e r flasks were harvested aseptically and distributed evenly into four 2000 ml Erlen-meyer flasks. Each flask contained 400 ml of modified Sabouraud's medium plus 0.01% Triton X-100. Griseofulvin was added to two flasks to make a final concentration of 10 ug/ml. The cultures were incubated for twenty-four hours on the shaker at room temperature and then harvested and washed. Aliquots were withdrawn for dry weight determinations. Nucleic acid pre-cursors were then extracted. The extracts were analyzed chromatographically for nucleosides, purines and pyrimidines, and electrophoretically for nucleotides. In the course of a ce l l fractionation experiment, the acid-soluble pools were analyzed for their ab i l i ty to absorb light at 260, 280 and 296 mu. These wave-lengths of 1ight were used because certain types of com-pounds have absorption maxima at or near these points. Purines and 45. pyrimidines absorb a t 2 6 0 mu, aromatic amino acids at 280 mu, and griseo-fulvin at 296 mu. The samples were diluted 1:10 and the optical density (0.0.) determined. These specimens were then shaken for sixty seconds with an equal volume of chloroform. The aqueous layer was removed and its 0.0. determined. Both chromatographic and electrophoretic analysis revealed that griseofulvin did not qualitatively alter the characteristics of the nucleic acid precursor pool. Optical density data (Table 9) suggested that quantitative varia-tions are induced by the ant ibiot ic . However, the reduction in O.D. of the griseofulvin-treated'samples following chloroform extraction makes it l ike ly that the differences were due to the presence of griseofulvin. Chloroform easily extracts griseofulvin from aqueous solutions (Table 9 ) . The presence of griseofulvin in the mycelial extract provides further support for the hypothesis (Boothroyd, 1961 ; El-Nakeeb, 1963) that griseofulvin is able to pass across the fungal ce l l wall and into the cyto-plasm. VI . Studies on Dermatophyte Respiration A. .Preliminary investigations Progress in the study of dermatophyte respiration has been frustrated by high levels of endogenous respiration and inadequate manipulation tech-niques. In order to resolve this problem, more suitable ce l l preparations w i l l have to be made. The objective would be to obtain young, easily d is -pensed cel ls which would show increased oxygen uptake in the presence of glucose. To achieve this goal, investigations were made into the influence on respiration of age, growth media, buffer solutions, and the type of growth 46. Table 9. Influence of griseofulvin on the ultra-violet light absorbing characteristics of the cold TCA soluble pool of M. quinckeanum #8 Optical density Before chloroform extraction After chloroform extraction 260 mji 280 mji 296 mp 260 m\i 280 m(J 296 mji Griseofulvin treated cel ls 0.335 0.198 0.120 0.291 0.122 0.64 Control ce l l s 0.275 0.122 0.43 0 .279 0.125 0.48 Griseofulvin in water 0.225 0 . 3 2 0 0.025 0.035 4 7 . obtained. 1. Preparation of a suitable type of cel lular growth The nature of mycelial growth is the linear addition of septated units. This results in a continuum from old to new segments within a single hyphal strand. Electron micrographs (Urabe, 1959) show that the mitochondria and nuclei are more numerous near the hyphal t i p . Thus, respiratory act ivi ty is probably higher in this region. The problem was to obtain a culture in which the majority of the hyphal elements were actively respiring. Both mycelial homogenates and spores were tested as inocula. Spores proved to be superior as they were easier to prepare and there was no residue of old hyphae. The low rate of dermatophyte respiration makes i t necessary to use large quantities of mycelia. A high yie ld of mycelia per ml was obtained when a minimum of 8 x 10^ spores was used to inoculate 100 ml of media. Q 8 x 10° spores in one ml gives an O.D. of 7 . 2 . The inoculated flasks were incubated in static culture at 25* C. The length of incubation was dependent upon the organism used. After forty-eight hours, approximately 75% of M. quinckeanum #8 micro-conidia had germinated. When shaken, the culture appeared turbid, resembl-ing the early " log" phase of a bacterial culture. After seventy-two hours, a l l possible spore germination was complete. The hyphae at this time were an average of sixteen septal elements long. These elements were uniform in appearance and had not formed the vacuoles, characteristic of old hyphae. Concentrated mycelial suspensions obtained from seventy-two hour old cultures could be easily and accurately transferred. us. M. gypseum cultures grow more rapidly than M. quinckeanum #8. and consequently, they reach the same stage of morphological development by th i r ty-s ix hours. 2, influence of culture medium Investigators of dermatophyte respiration have used such concen-trated media that accumulation of endogenous reserves is probably forced upon the organism (Cochrane, 1958). Lower levels of endogenous respira-tion might be attained by growing them in media containing lower concen-trations of nutrients. To examine this poss ib i l i ty , M. quinckeanum #8 was grown in Sabouraud's medium, Sabouraud's modified medium, and yeast extract medium. Mycelia were harvested at seventy-two hours of incubation at 25 C. Five 500 ml Erlenmeyer flasks, each containing 100 ml of medium, provided enough mycelia for four Warburg flasks. Oxygen uptake was measured using glucose as the substrate. Mycelia from both the Sabouraud's media were grey when harvested. The grey color could be removed by centrifugation or f i l t r a t i o n . When cen-trifuged, i t appeared as a layer on top of the white mycelia; when f i l tered i t was trapped in the f i l t e r paper and did not come off with the hyphae. Care was taken to ensure that none of the residue got into the Warburg flasks because i t could have been a potential source of nutrients. There was no observable oxidation of glucose by the mycelia grown on Sabouraud's media (Fig. 2). In the presence of glucose, mycelia from yeast extract and Sabouraud's modified medium increased oxygen uptake by 29% and 24% respectively (Figs. 2 and 3). The endogenous Q02 fluctuated with each experiment making i t imposs-ible to determine i f there was a characteristic value for each medium. 4 9 . 2 3 4 Time (hours) F i g . 2. Effect of culture medium on oxygen uptake by M. quinckeanum # 8 . Mycelia suspended in 0.066M phosphate buffer, pH 7.0, incubated at 25 C. Yeast extract medium, O = endogenous; A = glucose (1 umole/ ml). Sabouraud's medium, © = endogenous; 4 - glucose (1 umole/ml). 51 There was no correlation between the level of the endogenous QCv, and the ab i l i ty to show increased oxygen uptake in the presence of glucose. The results suggest that growth in a less concentrated medium prod-uced a ce l lu lar preparation possessing catabolic systems which are either not saturated or are capable of being stimulated by exogenous energy sources, 3. Effect of age on oxygen uptake The decision to use seventy-two hour old cel ls was based on the morphological appearance of the hyphae and the ease with which they could be handled. Fortunately, the results had been favorable; however, the poss ib i l i ty existed that better results could be obtained by using younger or older eel Is, Yeast extract medium was inoculated with M. quinckeanum #8 and in-cubated at 25* C. After th i r ty - s ix , seventy-two, ninety-six and 120 hours, enough growth was harvested to carry out a Warburg experiment. Glucose stimulated oxygen uptake 27% in seventy-two hour old cel ls (Fig. 4). The youngest c e l l s , th i r ty-s ix hours, were only stimulated 11%. Ninety-six hour old ce l ls gave similar results (Fig. 5). Oxidation was suppressed by glucose in 120 hour old c e l l s . It is not essential to use precisely seventy-two hour old ce l ls as a variance of four hours, on either side, does not alter the results. The tendency of older cel ls to accumulate endogenous reserves might explain why oxygen uptake is not stimulated when these cel ls are given glucose. The reason why the youngest ce l l s act similarly to old cel ls can-not be explained at present. 52. 500-Time (hours) Fig . 4. Effect of age on oxygen uptake by M. quinckeanum $8. Conditions the same as in Fig . 2. Mycelia harvested at 33 hours after in-oculation, O = endogenous; & = glucose (1 umole/ml). Mycelia harvested at 72 hours after inoculation, O = endogenous; A = glucose (1 umole/ml). 53. Time (hours) Fig . 5. Effect of age on oxygen uptake by M. quinckeanum #8. Conditions the same as Fig . 2. Mycelia harvested at $6 hours after inocula-t ion , O = endogenous; o = glucose (1 umole/ml). Mycelia har-vested at 120 hours after inoculation, A ° endogenous; 0 = glue ose (1 Hroole/ml). 54 4. Influence of buffer on oxygen uptake Previous studies (MacPherson, 1963) in this laboratory have shown that the level of endogenous respiration can be reduced by starvation in phosphate buffer. Possibly this is because an exogenous supply of phosphate is required in the catabolism of endogenous substrates. Seventy-two hour old M. quinckeanum #8 mycelia were harvested from yeast extract medium and washed with 0.85% saline. Equal portions were suspended in 0.066M THAM buffer, pH 7.2, and 0.066M phosphate buffer, pH 6.3, 7.0 and 7.3. THAM buffer lowered the level of endogenous respiration (Fig. 6). Any value this might have had was ofset by the corresponding reduction in oxygen uptake, attributable to glucose. The respiratory levels were not affected by changes in pH in the range of 6.3 to 7.3. This is in keeping with the findings of Nickerson (1946), who found that respiratory act ivi ty was not affected over a pH range of 5.5 to 7.5. It would seem advisable to use phosphate buffer because it supports the highest respiratory ac t iv i ty , thus providing a more comprehensive p ic t -ure of ce l lu lar respiration. B. Influence of griseofulvin on dermatophyte respiration Experiments on the respiratory influence of griseofulvin were i n i t -iated for the following reasons: 1. Previous studies (Brian, 1949; Roth, 1959; Larsen, 1963; Ronald, 1964) had not definitely established positive or negative res-u l t s . 2. New techniques had been developed during this investigation which for the f i r s t time provided a ce l l preparation which res-55. 5 0 0 -1 2 3 4 5 6 Time (hours) F i g . 6. E f f e c t of buf fer and pH on oxygen uptake by M. quinckeanum //8. Mycel ia suspended in 0.066M phosphate bu f f e r , pH 6.3, 7.0, 7.3, O = endogenous; A = glucose (1 umole/ml). Mycel ia suspended in 0.05M THAM b u f f e r , pH 7.2, G = endogenous; 4 = glucose (1 umole/ml). 56. ponded to exogenous substrate without exposing the cel ls to the unknown effects of prolonged starvation. 3. The ce l l suspensions were predominantly young, actively metabol-izing hyphae. As was mentioned ear l ier , i t is at this stage that the fungus is most sensitive to the ant ibiot ic . Other in-vestigators (Brian, 1949; Roth, 1959; Larsen, 1963; Ronald, 1964) had used old hyphae in their experiments. Mycelia were prepared for manometric experiments according to the methods outlined in Materials and Methods. This hyphal preparation was preincubated in griseofulvin for two hours before the substrate was added to insure that the antibiotic had time to enter the ce l l in inhibitory concentrations (El-Nakeeb, 1965a). The amount of griseofulvin required to stop growth is directly related to the amount of ce l lu lar material. Therefore, a high concentration of antibiotic (40 ug/ml) was used in a l l experiments. In every experiment, the solvent used to dissolve griseofulvin (either 95% ethanol or dimethylsulfoxide) was added to a set of control flasks. Neither one of the solvents, in a final concentration of 0.7%, inhibited r.or stimulated oxygen uptake. The experimental results (Fig. 7) indicate that griseofulvin inhib-its oxygen uptake attributable to glucose by 100%. There was not any in-hibition of endogenous respiration as had been observed by Ronald (1964), even when the concentration of griseofulvin was increased to 50, 75 or 100 Hg/ml. The same results were obtained with M. qypseum. When the endogenous-exogenous oxygen uptakes differed by at least 17%, there was 100% inhibit ion. However, when this difference was less 57. L O 2 3 4 Time (hours) F i g . 7. E f f ec t of g r i s e o f u l v i n on oxygen uptake by M. quinckeanum #8. Glucose st imulated oxygen uptake 28%. Condit ions the same as in F i g . 2. O = endogenous; endogenous plus g r i s e o f u l v i n (40 ug/ml); glucose (1 umole/ml) plus g r i s e o f u l v i n (40 ug/ml); A - glucose (1 umole/ml). 58. than 17%, griseofulvin only partially inhibited oxygen uptake (Fig. 8). The degree of inhibition varied but it was generally in the vicinity of 45%. If these mycelia had been prepared from older, more impermeable hyphal elements, the inability of the antibiotic to penetrate these areas might cause such a result. C. Effect of griseofulvin on the respiration of dermatophyte spores Spores harvested from Roux flasks of six day old M. guinckeanum #8 or five day old M. gypseum cultures were suspended in yeast extract medium and incubated for three hours on the shaker at room temperature. The spores were collected by centrifugation and washed four times with 0.066M phosphate buffer, pH 7.0. They were then suspended in phosphate buffer and phosphate buffer containing griseofulvin (40 ug/ml). After incubating another two hours, manometric experiments were begun. Spores from the two organisms behaved very differently in the War-burg flask (Fig, 9). M. gypseum spores, which are predominantly macro-con id ia, had a very high endogenous activity that was not increased by the addition of glucose, M. quinckeanum #8 spores, which are almost 100% microconidia, had very l i t t l e endogenous respiratory activity, but increased oxygen uptake occurred in the presence of glucose. Preincubation in the yeast extract medium enhanced the respiratory activity of M. quinckeanum #8, The macroconidia of M. gypseum were active regardless of whether they had been preincubated or not. The endogenous OA, for macroconidia was 10.8, and for microconidia, 0.2, While there have been many studies on the respiration of mold spores, no work of this nature has been done on dermatophyte spores. The results reported here demonstrate the diverse respiratory characteristics of macro-59. Time (hours) Fig . 8. Effect of griseofulvin on oxygen uptake by M.1 quinckeanum #8. Glucose stimulated oxygen uptake 10%. Conditions the same as in F ig . 2. O = endogenous, endogenous plus griseofulvin (40 Hg/ml); A = glucose (1 pmole/ml); A = glucose (1 umole/ml) plus griseofulvin (40 ug/ml). 60. Tim e (hours) 1 F i g . 9. Oxygen uptake by spores pf M. quinckeanum #8 and M. qypseum. '-Conditions the same as in F i g . 2. To make the resu l t s comparable oxygen uptake is graphed as ul 0 2 per mg dry weight of spores. M. qypseum. O = endogenous, glucose (1 umole/ml). M. quinckeanum  #8, Q = endogenous; A = glucose (1 umole/ml). 61. and microconidia of two dermatophyte species. Griseofulvin has no effect on spore respiration. This is in com-pliance with the fact that the antibiotic is unable to inhibit spore germ-ination. It appears that spores are insensitive, possibly because they are impermeable to griseofulvin. VII . Endogenous Respiration As discussed ear l ier , very l i t t l e is known about endogenous respira-tion of dermatophytes. In the case of these molds, the only known and ac-cepted fact is that the rate of endogenous respiration is extremely high. In order to gain further insight into the problem, experiments were under-taken to test the effects of exogenous glucose on endogenous oxygen uptake. Thirty-six hour cultures of M. gypseum were harvested and used in manometric respirometer tests. The usual protocol was followed except that 14 UL- C-glucose was used as substrate. Table 10 il lustrates the results of an experiment (A) in which the addition of glucose had no effect on the oxygen uptake ab i l i t y of the organ-ism, and an experiment (B) in which the addition of glucose stimulated 0^ uptake. In experiment A, analysis of the released C0 2 indicates that 53% of the added glucose was oxidized. As there was no increase in 0 2 uptake it would appear that endogenous respiration had been suppressed. In experi-ment B, oxygen consumption and C0 2 release correlated, indicating that en-dogenous respiration was not affected by added glucose. It was noted in other experiments that when exogenous-endogenous oxygen uptake differences dropped below 14%, there was a suppression of endogenous respiration. The degree of suppression varied with each prep-Table 10. Influence of glucose on endogenous respiration of M. qypseum 02 consumed Q02 air % of theoretical oxygen uptake % released per mg cel ls 14 % of , H C glucose oxidized to l Z f C0 o % of C0 2 repl aced by l Z t C0 9 "1 cpm Endogenous Glucose 1119 1077 9.6 9.2 less than 0 1.02 x 105 53 19 Endogenous 1104 Glucose 1346 8.04 9.86 60 1.05 x 105 52.6 63. arat ion and could not be standardized even when i t was thought that a l l variables were under r ig id control. The metabolic age of the organism is important in determining whether the ce l l preparation w i l l show increased oxygen uptake in the presence of glucose. in experiment A, the ce l l s were twenty-four hours old and in experi-ment B, th i r ty-s ix hours o ld . Best oxygen uptake values are usually obtain-ed with th i r ty-s ix to forty hour c e l l s . Cells harvested on either side of this incubation time invariably did not register oxygen uptake attributable, manometrically, to glucose. The reason for this is not known, but obviously there is only a certain period during which the ce l l possesses the enzymes to enable i t to increase its respiratory ac t iv i ty . These experiments were not intended as a comprehensive analysis of endogenous respiration, but rather to gain some understanding of how this metabolic system behaves under the experimental conditions imposed by tech-niques used in this investigation. Nevertheless, they do point out two im-portant generalities. F i r s t , in M. gypseum. endogenous respiration is a complex phenomenon. Second, i t is wrong to assume that the amount of sub-strate oxidized can be measured by calculating the difference between con-trol l and substrate oxygen uptake values. More specif ical ly the results show that under the conditions of these experiments, there is no suppres-sion of endogenous respiration when there is a minimum of 14% exogenous oxygen uptake. Between 14 and 1%, it is not possible to predict the extent of the suppressive effect of glucose. When there is l i t t l e or no exogenous oxygen uptake, i t appears that there is extensive suppression of endogenous respiration resulting in the oxidation of approximately 50% of the added 64. glucose. VIII . Oxidative Assimilation El-Nakeeb (1965a) and McNall (I960) have suggested that griseoful-vin inhibits the synthesis of nucleic acid or protein. If their hypoth-esis is correct, the normal assimilatory pattern of the ce l l should be disrupted and reflect these inhibitions. Oxidative assimilation studies of fungi have been confined to proving that assimilation does occur. There have been no reports of a comprehensive fractionation of an assimil-ated substrate. 14 A. Distribution of assimilated C M. qypseum mycelia grown in synthetic medium were prepared for a \k Warburg microrespirometer experiment. UL- C-glucose was used as substrate. At the end of eleven hours, the contents of the Warburg flask were 14 collected and fractionated. The fractions were assayed for C. M. qypseum assimilated 47% of the glucose (Table 11). This is con-siderably less than the 83% and 74% reported respectively for Penicil1ium  chvrsoqenum (Blumenthal, 1957) and Nocardia coral! 1 ina (Midwinter, I960). Of the 47% assimilated, 75% was associated with macromolecular ce l l con-stituents; the remaining 25% was probably metabolic intermediates, as it was soluble in cold TCA. Over 50% of the assimilated glucose was soluble in hot TCA and NaOH. As these fractions contain the majority of the nitrog-enous compounds of the c e l l , there must have been a readily available supply of ammonia. No exogenous ammonia was provided, indicating there had been an endogenous breakdown of a nitrogen containing compound. The large per-centage of ^ C accumulated in nucleic acids was unexpected because these compounds represent only 4% of the ce l l dry weight. The residue is com-1/4 \L Table 11. Assimilation of C frcm UL-C-glucose by M. gypseum grown in synthetic medium Fraction Total counts in fraction x 1C-5 % of total ]kC recovered* 14C assimilated % of total '^C KOH Supernatant Cold TCA soluble Alcohol soluble protein Chloroform soluble Hot TCA soluble NaOH soluble Insoluble residue Total Total counts added % of total counts recovered cpm 13.08 1.96 2.89 0.93 0.9V 2.30 2.72 1.99 26.85 27.91 96 52.5 11.7 3.7 3.6 9.3 11.0 8.1 100 24.8 7.8 7.7 19.5 23.3 16.9 100 * Supernatant not included in calculation of percentages. irk Supernatant included in calculation of percentages. 66. prised predominantly of anthrone positive material and probably represents insoluble ce l l walls . Electrophoresis of the supernatant fraction (Table 11) revealed a single radioactive spot which had the same mobility as glucose. It was 14 assumed that the C in the supernatant was unreacted glucose. Therefore, this value was not included in determinations of the percentage dis t r ibu-t ion . The total radioactivity recovered does not equal 100% for the f o l -lowing reasons: (a) The ^CO, d issolved in the phosphate buffer suspending medium was not measured. (b) Mycelia tends to aggregate and stick to the walls of the War-burg flask, making i t d i f f i cu l t to accurately withdraw an even suspension of organisms for fractionation. B. Influence of media on assimilation of * \ The same procedure used in the preceding experiment was followed, except that the organism was grown in yeast extract medium. As can be seen in Table 12, growth in the more concentrated yeast extract medium only s l ight ly altered the pattern of assimilation. The 30% decrease in assimilation into l i p i d occurred in every experiment. Differ-ences in distribution within the other fractions f e l l within the range of experimental variation. C. Influence of griseofulvin on assimilation of Warburg flasks were prepared according to the procedure outlined in Materials and Methods. Mycelia were collected after eleven hours and then fractionated and assayed for ^ C . The effects of the griseofulvin solvent 67. Table 12, Assimilation of C from UL- C-glucose by M. gypseum grown in yeast extract medium Cold Alcohol Chloro- Hot TCA soluble form TCA NaOH Insoluble C0 2 soluble protein soluble soluble soluble residue %of total m C 54.8 13.9 2,95 2,48 10.10 10,0 6.45 recovered % of total I H C - 30.3 6.40 5.4 22.0 21.8 14.10 assimilated 68 were determined by adding dimethylsulfoxide to one set of flasks. Griseofulvin caused extensive disruption of the normal oxidative assimilation patterns (Table 13)» The most s tr iking effect was the 100% increase of assimilation into the l i p id fraction. Electron micrographs have shown large l i p i d granules in griseofulvin-treated ce l ls but this is the f i r s t biochemical evidence for such an accumulation. The large increase 14 in the percentage of C in the cold TCA extract immediately raised the hope that there had been an accumulation of a metabolic precursor resulting from an inhibited reaction. Chromatographic and electrophoretic analysis 14 of the fraction showed that the increase could be attributed to C glucose. Assimilation into nucleic acids and protein is inhibited to the greatest extent. This is in accordance with the results of proximate analysis re-ported by El-Nakeeb (1965a). In both cases there was approximately 40% inhibi t ion. Incorporation into the ce l l wall was reduced, but a lesser extent than in RNA or protein. Nonetheless, the amount of ^ C 0 2 released indicates that griseofulvin inhibits the oxidation of glucose. As the oxygen uptakes remained the same for preparations with and without griseo-fulvin , i t is assumed that oxidation of endogenous reserves compensated for the reduction in the oxidation of glucose. D. Effect of an exogenous nitrogen source on oxidative assimilation  of ^ C glucose Oxidative assimilatory patterns in resting ce l l suspensions are not necessarily the same in growing c e l l s . Because the latter represents a more normal growth state, i t was decided to fractionate ce l l s supplied with both glucose and a nitrogen source. M. gvpseum grown in synthetic medium was prepared as usual for a Table 13. Assimilation of ' \ from UL-'^C-glucose by M, qypseum in the presence of griseofulvin Fract ion Control ce l l s Total counts in fraction x 105 % of total 14C recovered* % of total ass imi 1 ated Griseofulvin treated ce l l s Total counts % of total in fraction x 105 recovered % of total I1* ass imi 1 ated KOH Supernatant Cold TCA soluble Alcohol soluble protein Chloroform soluble Hot TCA soluble Hot NaOH soluble Insoluble residue Total Total counts added % of total counts recovered** cpm 13.08 1.96; 2.89 0.93 0.91 2.30 •2^72 1.99 26.85 27.91 _96_ 52.5 11.7 3.7 3.6 9.3 11.0 8.1 100 24.8 7.8 7.7 19.5 23.3 16.9 100 cpm 10.99 1.58 6.79 0.95 1.43 1.39 1.71 U53 26.30 27.91 94 44 23.7 3.8 5.8 7.5 6.9 6.2 100 50 6.9 10.7 10.1 12.5 11.1 100 * Supernatant not included in the calculation of percentages, * * Supernatant included in the calculation of percentages. 70. manometric experiment with the exception that 3 H m ° l e s of L - a r g i n i n e were added in a d d i t i o n t o C glucose. M. gypseum cannot u t i l i z e an inorganic source of ammonia f o r growth. The amino a c i d L - a r g i n i n e was chosen because i t i s known to support growth and is a good source of n i t r o g e n . Table 14 shows that the presence of a n i t r o g e n donor reduces the amount of glucose o x i d i z e d from 50% t o 2%. This l a r g e reduction in o x i d a -t i o n may be explained by the f o l l o w i n g reasons: 1. There i s concurrent o x i d a t i o n of the organic n i t r o g e n source and thus competition w i t h glucose f o r o x i d a t i v e pathways, 2, In the presence of an excess of ammonia, there is s t i m u l a t i o n of anabolic systems, the energy requirements being s a t i s f i e d by the o x i d a t i o n of endogenous s u b s t r a t e s . Although there was a l a r g e increase i n the percentage of glucose as-s i m i l a t e d , the p a t t e r n of a s s i m i l a t i o n was q u i t e s i m i l a r t o that of r e s t i n g c e l l s . This i s s u r p r i s i n g , and suggests that there was not a c t u a l l y a r e s t i n g c e l l s t a t e , but rather a c e l l which was merely growing at a slower r a t e . The a d d i t i o n of n i t r o g e n supplemented the endogenous nitrogen supply, 14 enabling the c e l l t o grow more r a p i d l y . The l a r g e amounts of C incorpor-ated i n t o the c e l l w a l l residue i n r e s t i n g c e l l s makes t h i s hypothesis even more tenable. The a d d i t i o n of a nitrogen supply does not a f f e c t the a l t e r a -t i o n s in a s s i m i l a t i o n patterns induced by g r i s e o f u l v i n , but i t does appear t o suppress the extent of the r e s u l t a n t a l t e r a t i o n (Table 14). IX. C e l l - F r e e E x t r a c t Studies C e l l - f r e e e x t r a c t s were prepared from r e c e n t l y germinated M. quinck- eanum #8 m i c r o c o n i d i a . Glucokinase, glucose-6-phosphate dehydrogenase, and Table 14. Assimilation of lHC from UL-C-glucose by M. qypseum in the presence of arginine and griseofulvin Control cel ls Griseofulvin treated eel Is Fraction Total counts in fraction x 105 % of total C recovered* % of, total ass imi 1 ated Total counts in fraction x 105 % of total 14c recovered % of total 14C ass imi1ated cpm cpm KOH 0.31 2 .0 - 0.27 1.7 -Supernatant 1.17 - - 1.27 - -Cold TCA soluble 3.05 19.6 20 .1 4.02 26.2 26.6 Alcohol soluble protein 1.28 8.2 8.4 1.25 8.2 8.3 Chloroform soluble 1.18 7.6 7.8 1.5 9.8 9.9 Hot TCA soluble 3.00 19.3 19.7 2.74 17 .9 18.1 Hot NaOH soluble 3.60 23.2 23 . 7 3.11 20 . 3 20 .6 Insoluble residue 3.12 20.1 20 .6 2.48 16.1 16.4 Total 16.702 100 100 16.637 100 100 Total counts added 17.537 17.537 % of total counts recovered** 95.3 94.8 * Supernatant not included in the calculation of percentages. ** Supernatant included in the calculation of percentages. 72 6-phosphogluconate dehydrogenase act ivi ty was measured by following the reduction of TPN at 340 mu. When the effect of griseofulvin was measured, aliquots of the extract were incubated with the antibiotic (40 ug/ml) at room temperature for one hour prior to performing the assay. The presence of an active glucose-6-phosphate dehydrogenase (Fig. 10) made it possible to demonstrate glucokinase ac t iv i ty . Glucokinase act ivi ty was dependent on magnesium aridATP (Fig. 11). The existence of a hexose monophosphate shunt was indicated when 6-phosphogluconate dehydrog-enase was found (Fig. 10). The Nossal c e l l disintegrator was effective in preparing cell-free extracts from germinating spores; however, when older mycelia were used, hand grinding with a mortar and pestle was necessary. Unlike previous reports (Jensen, 1957; Chattaway, I960), there was a very low level of endogenous TPN reduction. As the earl ier studies had been done with four to s ix day old ce l l s and the present studies with one day old mycelia, the difference might be attributable to aging. This hypothesis was further substantiated when it was found that four day old M. quinckeanum #8 mycelia also had high endogenous act ivi ty (Fig. 12). The addition of griseofulvin to the ce l l free extracts did not affect the act ivi ty of any of the enzymes tested. X. Effect of magnesium on the morphology of Microsporum gypseum A supply of divalent cations is essential for the growth of most organisms. Their best known function is as prosthetic groups in enzyme reactions. It has been speculated (Weibull, 1956) that they are important in maintaining the structural integrity of the ce l l wa l l . In this capacity they are believed to act as linkages binding molecules together. The O.D. at 340mu Fig. 10. 0-40 0-30 -0 2 0 010 73. ^Time(min) Add i t ions : 0.05 ml enzyme (5.45 mg prote in/ml) in 0.02M.THAM buf fer (pH 7 .2), 0.02 ml TPN (0.005M), 0.2 ml substrate (.025M), 0.1 ml MgSOZf.7H20 (0.02M), 0.02M THAM (pH 7.2 to 1.25 ml ) . TPN was added at zero t ime, x = complete system, glucose-6-phosphate substrate with and without g r i s e o f u l v i n ; • = complete system, 6 -phosphogluconate substrate with and without g r i s e o f u l v i n ; o = no substrate with and without g r i s e o f u l v i n . 0 © 5 ' O.D. at 3 4 0 mu F i g . 11, 010 . 0-05 3 4 Time(mio) Add i t i ons : 0.05 ml enzyme extract in 0.02M THAM (pH 7.2), 0.02 ml TPN (0.005M), 0.2 ml glucose (0.025M), 0.1 ml MgSO/^h^O (0.02M), 0.2 ml ATP (0.025M), 0.02M THAM buf fer (pH 7.2) to br ing the volume to 1.25. x = complete system; o = no Mg; • = no ATP; A = no subs t ra te . In each case the curve represents assays done with and without g r i s e o f u l v i n . 74. i • i i i i r 1 2 3 4 5 6 7 T ime Unin) F i g . 12. Add i t i ons : 0.1 ml enzyme extract (6.82 mg prote in/ml) in 0.02M THAM buf fer (pH 7.2), 0.02 ml TPN (0.005M), 0.2 ml glucose-6-phosphate, 0.1 ml MgS0/4.7H20 (0.02M), 0.02M THAM buf fer (pH 7.2) to make a f i n a l volume of 1.25 ml . X a complete system; o = no subs t ra te . 75. f r a y e d , s p l i t c e l l w a l l seen in e l e c t r o n micrographs (Tomomatsu, I960) of g r i s e o f u l v i n - t r e a t e d c e l l s suggested that the l a c k of such a binding group could cause these a b e r r a t i o n s . To t e s t t h i s idea, i t was decided to grow the mold in a medium l a c k i n g d i v a l e n t c a t i o n s . Under such c o n d i t i o n s , the c e l l might be expected to s e l e c t i v e l y u t i l i z e i t s depleted s t o r e of ions f o r metabolic purposes and not f o r maintenance of the c e l l w a l l . A s y n t h e t i c medium l a c k i n g magnesium and manganese was inoculated w i t h M. gypseum spores and incubated at room temperature. Spore germina-t i o n and growth occurred at a normal r a t e . Apparently there were s u f f i c i e n t d i v a l e n t c a t i o n s present, e i t h e r as chemical contaminants or contained by macroconidia, t o support growth. Therefore, these mycelia were used as an inoculum and subcultured i n t o more of the d e f i c i e n t medium. This time growth was slower. Microscopic examination ( F i g s . 13 and 14) revealed that hyphae had become wavy, s w o l l e n , and stunted. Branching was unusually frequent and hyphal t i p s were s p a t u l a t e . These morphological aberrations are the same as those observed in c e l l s grown in g r i s e o f u l v i n . The one c h a r a c t e r i s t i c not observed was the tendency of a n t i b i o t i c - t r e a t e d c e l l s t o c u r l back on themselves. Spatulate and wavy hyphae occur i n low concentrations of g r i s e o -f u l v i n probably because these areas are most s u s c e p t i b l e t o g r i s e o f u l v i n and thus are the f i r s t t o be involved. Another s i m i l a r i t y between the two c e l l p reparations was the ease w i t h which hyphae were d i s i n t e g r a t e d when a very s l i g h t pressure was a p p l i e d , showing a s i m i l a r loss of c e l l w a l l i n t e g r i t y . 76. F i g . 14. M. qypseum grown in a synthet ic medium w i th -out added d iva lent c a t i on s . The mycelium is gross ly d i s t o r t e d . The numerous buddings along the hyphae, probably represent attempts at branching. r Fig . 16. M. gypseum grown in synthetic medium in the presence of griseofulvin (3 Hg/ml). The mycelium is very thick and swollen, branching is frequent. 7 8 . DISCUSSION Sixteen different amino acids were found in the free amino acid pool of M . quinckeanum # 8 . During twenty-four hours of starvation, they remained qualitatively and quantitatively s imilar . There are two possible explanations for th i s . Either the pool remains static because endogenous metabolism does not require amino acids or amino acids are replenished as they are used. The pool could be replenished either from the break-down of protein or from the synthesis of new amino acids from endogenous substrates. The energy requirements would be f u l f i l l e d by the high rate of endogenous respiration. The reduction in concentration of the pool in the presence of gluc-ose suggests that glucose is stimulating biosynthetic mechanisms. This concept is consistent with the results from oxidative assimilation studies which show that a large percentage of glucose is assimilated into nitrog-enous compounds. The amino acid pool would provide a convenient source of ammonia for these syntheses. The status of endogenous respiration during substrate oxidation is best studied using isotopic techniques. The least troublesome method is 1 4 to measure the C 0 2 released during oxidation of a labelled substrate. Blumenthal ( 1 9 5 7 ; 1 9 6 3 ) investigated the problem of endogenous res-erves in P. chrysogenum and N. crassa. The picture that emerges from his studies is that the response of endogenous metabolism to the concurrent metabolism of an exogenous substrate is a complex phenomena influenced by 79. age, environmental conditions, and substrate. For example, neither gluc-ose nor acetate suppresses the endogenous respiration of P_. chrvsogenum grown in glucose medium. However, when i t is grown in acetate medium, acetate but not glucose does suppress endogenous respiration. The studies reported here on M_. qypseum. while not as comprehensive as Blumenthal's (1963), do point out the complexity of endogenous metabol-ism. In young ce l l s in which oxygen uptake was not stimulated by glucose, there was an 18% suppression of endogenous oxidation. In older c e l l s , in which oxygen uptake was stimulated by glucose, there was no suppression of endogenous oxidation. It is interesting that regardless of whether or not glucose increases oxygen uptake, there is always approximately 50% of the glucose oxidized. Cochrane (1958), discussing mold respiration in general, suggested that endogenous respiration would be suppressed i f exogenous sub-strate competed for a reaction-saturated by endogenous substrate. If the reaction in question occurred before any oxidative reactions, the result would be the simultaneous oxidation of both exogenous and endogenous sub-strates without an increase in oxygen uptake. If this hypothesis is used to explain the results obtained in the present investigations, the follow-ing conclusions can be made: (a) when glucose does not stimulate oxygen uptake, there is competi-tion between the exogenous and endogenous substrate at the s i te of the saturated reaction; (b) when oxygen uptake is increased, 14% or more, endogenous sub-strate is not saturating any oxidative systems required by gluc-ose, and thus there is no competition; (c) when oxygen uptake differences drop below 14% there is partial 80. saturation of a shared system resulting in a varying degree of competition. A decrease in the suppression of endogenous respiration would also occur i f the glycolytic pathways had altered to favour the oxidation of glucose. Cell-free extracts prepared from activated microconidia and four day old mycelia provided additional information on the nature of endogenous metabolism. Activated spores which showed low endogenous act ivi ty in mano-metric experiments possessed cel l-free extracts with low endogenous dehydrog-enase ac t iv i ty . It is not known whether they lack endogenous reserves or the enzymes to u t i l i z e them. There was a high level of endogenous dehydrog-enase act ivi ty in cel l-free extracts from four day old mycelia. This ob-servation correlates with the results of manometric experiments which show that these cel ls had a high level of endogenous respiration. Cell-free extracts prepared from mycelia might provide a more simplified system in which to study the nature of endogenous reserves. Any knowledge of dermatophyte respiration has come from studies of individual enzymes (Bentley, 1953; Chattaway, 1954; I960) and by inferred evidence from studies on respiratory inhibitors (Nickerson, 1946; Melton, 1951; Chattaway, 1956). Investigators have relied on these techniques because they have not been able to produce ce l l preparations satisfactory for manometric studies. Acceptable techniques have now been established for the organisms M. quinckeanum #8 and M. gypseum. The metabolic age appears to be an important factor in determining whether an organism's respiratory act ivi ty can be stimulated by added sub-strate. Metabolically, this age has only been defined by its increased 81. r e s p i r a t o r y a b i l i t y . M o r p h o l o g i c a l l y , i t can be defined as mycelia of approximately 16 s e p t a l u n i t s o r i g i n a t i n g from e i t h e r a microconidium or macroconidium. The incubation p e r i o d required t o reach t h i s developmental stage is dependent upon the organism. f\0 quinckeanum #8 requires seventy-two hours, whereas M. qypseum requires only t h i r t y - s i x hours. This can be a t t r i b u t e d to e i t h e r d i f f e r e n c e s in germination time or d i f f e r e n c e s in growth r a t e . Although t h i r t y - s i x hours i s optimum f o r M. qypseum. i t does not always give c o n s i s t e n t l y good r e s u l t s . This i s a t t r i b u t e d to the rates at which i n d i v i d u a l macroconidial u n i t s germinate. In a t h i r t y ^ s i x hour c u l t u r e , t h ere are ungerminated spores as w e l l as spores which have germinated i n t o long hyphal s t r a n d s . As macroconidia have a high l e v e l of endogenous r e s p i r a t i o n , the number remaining ungerminated would i n f l u e n c e the r e s p i r a t o r y c h a r a c t e r i s t i c s of the c e l l p r e p a r a t i o n . Swanson (1965) made a comprehensive proximate a n a l y s i s of M. quinck- eanum #8 and found t h a t , w i t h the exception of c h i t i n , the percentage com-p o s i t i o n of c e l l u l a r c o n s t i t u e n t s remained constant in the d i f f e r e n t ages of c e l l s used f o r r e s p i r a t o r y s t u d i e s . C h i t i n content increased at t h i r t y -s i x hours, but then decreased to the previous l e v e l at forty-seven hours. There is no i n d i c a t i o n of the d e p l e t i o n or accumulation of a c e l l u l a r com-ponent which could serve as an endogenous s u b s t r a t e . However, these r e s u l t s are q u a n t i t a t i v e ; they do not i n d i c a t e whether endogenous su b s t r a t e s were being formed by q u a l i t a t i v e changes o c c u r r i n g w i t h i n the chemical c o n s t i t -uents. It i s a l s o p o s s i b l e that during aging, there are changes in the r e l a t i v e q u a n t i t a t i v e importance of a l t e r n a t e g l y c o l y t i c pathways which could account f o r changes i n o x i d a t i v e c a p a b i l i t i e s . Growth i n l e s s concentrated media produced, mycel i a w i t h an enhanced 82. ab i l i ty to consume oxygen in the presence of glucose. This might be at-tributable to a decrease in the accumulation of endogenous reserves. These studies have established the c r i t e r i a for obtaining mycelial preparations satisfactory for respiratory studies. The fact that the c r i t e r i a established for M. quinckeanum #8 also apply to M. gypseum raises the hope that they can be generalized to include many or a l l dermatophytes. In any case, these new techniques should fac i l i t a t e the study of dermato-phyte metabolism. The concentration of free amino acids was decreased when resting ce l l suspensions were incubated in the presence of glucose. When griseo-fulvin was present this decrease did not occur, indicating that the ant i -biotic might be interfering with the assimilation of amino acids. Oxida-t ive assimilation studies provided direct evidence that griseofulvin does inhibit the synthesis of nitrogenous compounds. Therefore, the inabi l i ty to u t i l i z e endogenous reserves of amino acids can probably be attributed to an inhibition of protein and nucleic acid synthesis. Depriving M. gypseum of divalent cations results in the development of morphological abnormalities similar to those induced by griseofulvin. It is tempting to extend this parallel and suggest that griseofulvin reduces the available supply of divalent cations, possibly through the formation of chelates or metal complexes. However, this assumption cannot be made solely on the basis of morphological evidence. Many antibiotics and fungistatic agents are metal chelators. These compounds generally induce a generalized cel lular response such as that observed with griseofulvin. Thus, there is precedent for the chelating act ivi ty of antifungal agents. Although these results are not conclusive, they do point out a previously unexplored area 83. of investigation which should be developed further. Griseofulvin inhibits the oxidation of glucose in young, sensitive mycelia of M. quinckeanum #8 and M. qypseum. The extent of inhibition is related to the degree which glucose stimulates oxygen uptake. When oxygen uptake is stimulated by more than 14%, there is 100% griseofulvin inhib i -t ion. When the percentage increase drops below 14%, there is only partial inhibit ion. If increased respiratory ab i l i ty is linked to changes in the relative importance of different glycolytic pathways, the complex nature of respiratory inhibition could be attributed to griseofulvin acting on a specific glycolytic pathway. As this pathway was u t i l ized to a greater extent, there would be a corresponding increase in the degree of inhibi t ion. An interesting correlation exists between the partial inhibitory effect of griseofulvin and the decreased repression of endogenous respiration when glucose stimulates oxygen uptake by less than 14%. These developments could also be linked to altered glycolytic pathways. Griseofulvin does not inhibit endogenous respiration, possibly because oxidation of endogen-ous reserves occurs via another pathway. It does not seem l ike ly that griseofulvin uncouples oxidative phos-phorylation, as Ziegler (1961) and Roth (1959) suggested. Compounds in-ducing this type of metabolic disruption increase the rate of respiration. For example, 2,4-dinitrophenol stimulates oxygen uptake in Microsporum  audouini by 29%. Griseofulvin causes the opposite response. How griseo-fulvin causes respiratory inhibition is unknown, and at this point is beyond speculation. There are grounds for speculation about the area in the oxidative pathways that is being inhibited. Since both endogenous and glucose respiration have some common pathways, certain of these can be 84. ruled out as being affected. These include the tricarboxcylic acid cycle and the terminal respiratory chain. Nothing is known about the nature of dermatophyte endogenous reserves, thus, precluding further definition of the affected system. It appears, therefore, that griseofulvin inhibits a step in the respiratory pathway which occurs prior to the TCA cycle. It would be dangerous to conclude that a l l oxidative reactions occurring before the TCA cycle are blocked unti l more is known about the influence of exogenous substrate on the level of endogenous respiration. The inabi l i ty of griseofulvin to inhibit the respiration and germina-tion of spores suggests that spores are resistant to the ant ibiot ic . It would be interesting to repeat El-Nakeeb's (1965c) experiments and determ-ine i f insensitivity could be correlated with the inabi l i ty of spores to take up and bind griseofulvin. 14 In the presence of griseofulvin, C-glucose accumulated in the cold TCA soluble pool. This could have been caused by one of the following reasons. (a) There was a general slowdown in the metabolism of the c e l l . (b) There was inhibition of the i n i t i a l reaction in the u t i l i za t ion of glucose. The only glycolytic pathways that have been found in dermatophytes are the Embden-Meyerhof' and the hexose mono-, phosphate shunt. In M. gypseum, the results presented indicate only the latter. Cell-free extract studies show that griseo-fulvin does not affect glucokinase ac t iv i ty , thus making it doubtful that inhibition of the i n i t i a l reaction is a valid hypothes i s . (c) There was feedback inhibition resulting from the impeding of a 85. reaction at some point along the glycolytic pathway. On the basis of the present knowledge on the action of griseo-fulvin, i t is not possible to discriminate between hypothesis (a) or (c). Oxidative assimilation experiments revealed that griseofulvin par t ia l ly inhibited the oxidation of glucose and the assimilation of '^C into nucleic acid protein and ce l l wa l l . The interdependence of these metabolic systems precludes these results from indicating the primary s i te of action of the ant ibiot ic . The decrease in assimilation of C into nucleic acid and protein correlates with observations of Zeigler (1961) who found phosphate and nitrogen uptake to be reduced, and with El-Nakeeb (1965a), who found synthesis of these components to be par t ia l ly inhibited. El-Nakeeb (1964) has suggested that binding of griseofulvin by nucleic acid causes inhibi t ion. This does not seem l ike ly because binding occurs with whole ribonucleic acid and therefore should not interfere with its synthesis. 14 Assimilations of C into l i p i d is increased in the presence of griseofulvin. There are other nutritional and metabolic disruptions which induce increased l i p id synthesis. Included among these are, inhibition of respiration (Woodbine, 1951), suboptimal levels of nitrogen (Raveux, 1948) and suboptimal levels of phosphate (Maas-Forster, 1955). Ziegler (1961) has shown that griseofulvin reduces the uptake of phosphate and nitrogen-ous compounds. Current investigations have shown that the antibiotic in-hibits respiration and reduces the ce l l s ab i l i ty to u t i l i z e nitrogenous compounds. The result is that griseofulvin induces a situation similar to that caused by the nutritional or respiratory deficiencies known to stimul-ate the accumulation of l i p ids . Thus, it appears that the increased assim-86. i lat ion of C into l ipids ?s a secondary phenomenon resulting from other metabolic disturbances. The complete inhibition of glucose oxidation is the most conclusive indication of the s i te of griseofulvin action. The associated inhibition of assimilation of C could be explained by the fact that oxidative and synthetic systems share some pathways. For example, the hexose monophos-phate shunt oxidizes glucose and at the same time supplies ribose~5-phos-phate for nucleic acid synthesis. Interference with a reaction within such a shared metabolic system could result in inhibition of both oxidative and synthetic mechanisms. This example could be modified and apply equally well to the formation of amino acids. Correlating this hypothesis with the hypothesis used to explain the inabi l i ty of the antibiotic to inhibit endog-enous respiration, i t would appear that griseofulvin is inhibiting a reac-tion occurring early in one of the glycolytic pathways. Another poss ib i l i ty which cannot be ruled out is that griseofulvin is non-spec i f i c a l l y inhibiting a number of cel lular functions resulting in a generalized slowing down of metabolism. This could occur by non-spedific binding to a substance common to many reactions. i 87. SUMMARY Investigations were made into the effects of griseofulvin on some aspects of the metabolism of M. quinckeanum #8 and M. gypseum. In many instances i t was necessary to f i r s t ascertain how these metabolic systems functioned in normal, uninhibited c e l l s . The following findings may be 1isted. 1. Easily manipulated mycelial suspensions are obtained when the growth from a concentrated inoculum is harvested during the early stages of mycelial development. 2. Oxygen uptake in M. guinckeanum #8 and M. gypseum is stimulated by glucose provided that (a) the organism has been grown in a medium with a low carbohydrate concentration, and (b) the organism has grown to a length of approximately 16 septal units. 3. THAM buffer (pH 7.2) depressed the level of both endogenous and exogenous respiration. 4. Endogenous respiration of M. gypseum was suppressed by an exog-enous supply of glucose when oxygen uptake had been increased by less than 14%. The less the increase, the greater the suppression. There was no suppression when oxygen uptake was increased above 14%. Macroconidia of M. gypseum had a high level of endogenous respiration, but the situation was reversed in M. guinckeanum #8 microconidia. 5. The presence of the hexose monophosphate shunt was indicated when glucokinase, glucose-6-phosphate and 6-phospho-gluconate act ivi ty was 88. found in cel l-free extracts from both spores, and four day old mycelia of M. quinckeanum #8. Griseofulvin had no effect on these systems. The mycelia had high endogenous dehydrogenase activi ty whereas the spores had very l i t t l e . 6. Under the conditions imposed by techniques used in these inves-ts tigations, M. gypseum oxidized 50% of UL- C-glucose and assimilated the remainder. Of the assimilated glucose, 50% was associated with nitrogenous compounds, 17% with ce l l wa l l , 8% with l i p i d , and the remainder with the metabolite pool. In the presence of arginine, the pattern of assimilation 14 remained the same but 98% of the C-glucose was assimilated. 7. In a medium lacking added divalent cations, M, quinckeanum #8 develops morphological abnormalities similar to those induced by griseo-fu lv in . 8. The growth of M. gypseum and M. quinckeanum #8 was inhibited by 3 ug/ml and 4 ug/ml of griseofulvin respectively. The morphological abnor-malities induced by lower'concentrat ions of griseofulvin were similar to those reported for other fungi. 9. M. quinckeanum #8 developed partial resistance to griseofulvin in a non-selective environment. 10. Griseofulvin caused neither qualitative nor quantitative changes in the amino acid pool or the nucleic acid precursor pool of M. quinckeanum £8. 11. A variety of nucleotides, nucleosides, and purine and pyrimid-ine free bases were unable to reverse the inhibitory effect of griseoful-vin on M. gypseum. 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