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Effects of griseofulvin on dermatophytes Ronald, William Pattison 1964

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EFFECTS OF GRISEOFULVIN ON DERMATOPHYTES , by W i l l i a m P a t t i s o n Ronald B..Sc», The U n i v e r s i t y o f B r i t i s h Columbia, 1 9 6 1 .  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 t h i s t h e s i s as conforming t o the required standards  THE UNIVERSITY OF BRITISH COLUMBIA April, 1964.  In the  r e q u i r e m e n t s f o r an  British  mission  for reference  for extensive  p u r p o s e s may  be  advanced  of  written  Department  of  by  for  May  3,  the  this thesis  Head o f my  University'of •  shall  I further  and  Columbia,  make, i t f r e e l y  agree for  that  Department  shall  not  Immunology  per-  scholarly or  that;• copying or  f i n a n c i a l gain  Bacteriology  1964  the  permission*  The U n i v e r s i t y of B r i t i s h V a n c o u v e r 8, Canada Date  study*  Library  I t i s understood  this thesis  w i t h o u t my  and  i n p a r t i a l ' f u l f i l m e n t of  degree at  the  c o p y i n g of  granted  representatives.  cation  this thesis  Columbia, I agree.that  available  his  presenting  be  by publi-  allowed  ABSTRACT The s i t e of a c t i o n of g r i s e o f u l v i n , a drug which i s reported t o be f u n g i s t a t i c i n nature, has been under study f o r some time.  As yet though, there i s s t i l l a great deal of  u n c e r t a i n t y as t o which observed e f f e c t s are primary and which are  secondary. I n the study reported here, i n i t i a l experiments were  involved w i t h e f f e c t s of g r i s e o f u l v i n on oxygen uptake, i n correlation with c e l l starvation. glucose o x i d a t i o n a l s o were studied. were noted i n both of these areas. were c a r r i e d out, u t i l i z i n g  The e f f e c t s of the drug on Definite alterations Further investigations  c e l l - f r e e e x t r a c t s and techniques  f o r measurement of dehydrogenases, but these proved unsuccessful*  Amino a c i d metabolism a l s o was surveyed but no evidence  of any a l t e r a t i o n was observed. Attempts t o produce p r o t o p l a s t s from dermatophytes were s u c c e s s f u l , and u t i l i z i n g these s t r u c t u r e s ,  investigations  i n t o the e f f e c t s of g r i s e o f u l v i n on cytoplasmic membrane permeability  and on c e l l w a l l r e s y n t h e s i s , were c a r r i e d out.  In both cases the a l t e r a t i o n s were small and appeared t o be secondary i n nature* I n the f i n a l study, p u r i f i e d c e l l w a l l s of organisms grown i n the presence and absence of g r i s e o f u l v i n , were compared on the basis o f amino a c i d , amino sugar, and sugar content.  No d i f f e r e n c e s were observed i n these preparations.  In  addition,  no  evidence  wlis i n c o r p o r a t e d It the  was  enzymes  into  cell  concluded  is  probably  ence t h a t and  It  was on or  the  pyrimidine  also  found  that  or  biochemical metabolism.  show t h a t  griseofulvin  g r i s e o f u l v i n may p o s s i b l y  synthesis the  concluded  near the  to  walls.  i n v o l v e d i n the  endogenous r e s p i r a t i o n , enzymes.  was  of  mechanisms that  the  cytoplasmic site  may b e  the  substrates  controlling drug's  of  these  site  of  action  and  by  infer-  membrane, i n the  affect  area  of  purine  ACKNOWLEDGEMENTS  I am d e e p l y i n d e b t e d t o D r . J . J . S t o c k o f t h e D e p a r t ment o f B a c t e r i o l o g y a n d Immunology f o r h i s h e l p f u l a d v i c e a n d supervision throughout the course of t h i s study. I w o u l d l i k e t o e x t e n d my t h a n k s t o D r . R . J . B a n d o n i of the Department o f Botany f o r the Neurospora c u l t u r e used i n t h i s s t u d y and f o r h i s h e l p f u l a d v i c e . I a l s o would l i k e to thank D r . G. Strasdine of the N a t i o n a l Research Council f o r s u p p l y i n g , the p u r i f i e d c h i t i n a s e p r e p a r a t i o n , and D r . S. M a r t i n of the N a t i o n a l R e s e a r c h C o u n c i l f o r h i s a d v i c e on the f o r m a t i o n of protoplasts. My t h a n k s g o a l s o t o S c h e r i n g C o r p o r a t i o n L i m i t e d , M o n t r e a l , Quebec f o r k i n d l y d o n a t i n g t h e sample o f p u r i f i e d g r i s e o f u l v i n , w h i c h was u s e d t h r o u g h o u t t h i s s t u d y . The w o r k i n t h i s p r o j e c t w a s s u p p o r t e d B r i t i s h C o l u m b i a S u g a r R e f i n i n g Company L i m i t e d f o r graduate study and r e s e a r c h .  i n p a r t by a Scholarship,  T A B L E OF CONTENTS PAGE INTRODUCTION  1  R E V I E W OF T H E L I T E R A T U R E  3 13  MATERIALS I. II. III. IV. V.  Glassware  13  Reagents  13  Enzymes  15  Media  16  Equipment  IS 20  METHODS I.  Preparation  of Cells  20  II.  Preparation  of Protoplasts  25  III. IV. V. VI.  VII. VIII. IX. X.  Paper  Chromatography  Oxygen Uptake S t u d i e s  33  Dry Weight Determinations  36  Quantitative  Analysis  of Griseofulvin  in  a  Reaction Mixture  37  Dehydrogenase  39  Studies  41  Amino Compound S t u d i e s Quantitative  Assay f o r  Spectrophotometric Griseofulvin  XI.  2$  Visual  and  43  Hexosamines  Studies  on P r o t o p l a s t  on the  of 47  Membranes  Spectrophotometric  S y n t h e s i s by Dermatophyte  Effects  Studies  Protoplasts  on C e l l  Wall 46*  PAGE XII.  Formation of Griseofulvin  Resistant  XIII.  Cell  Wall  Preparation  Purification  XIV.  Cell  Wall  Analysis  and  50  Mutants 5  2 54  EXPERIMENTAL  55  I.  55  II. III. IV. V.  Oxygen Uptake S t u d i e s A m i n o Compound S t u d i e s Chitinase Studies Preparation Studies  7  75  U s i n g Whole C e l l s  S3  of Protoplasts  on the  Effect  of Griseofulvin  on  Cell 94  Membranes VI.  VII.  Studies  2  on the  Effect  Wall  Resynthesis  Cell  Wall  of Griseofulvin  Composition Studies  on  Cell 96* 102  GENERAL DISCUSSION  106*  SUMMARY  114  APPENDIX  117  BIBLIOGRAPHY  119  LIST  OF I L L U S T R A T I O N S PAGE  1.  The  2.  Standard  G r i s e o f u l v i n Curve  38  3.  Standard  Glucosamine-HCI Curve  46  4.  Oxygen Uptake  5.  Effect  6.  Structure  of  of Griseofulvin  by U n s t a r v e d  Increasing  MQ8 P e l l e t s  Ethanol  Oxygen Uptake  b y MQ8 S t a r v e d  Oxygen Uptake  b y MQ6"  (Homogenized)  Effects  Cells of  3  56*  Concentrations for  on  5 Days  Starved  for  Increasing  63 5 Days  Griseofulvin  Concentrations  64  7.  Oxygen Uptake  by Unstarved  MQS. C e l l s  .8.  Oxygen Uptake  b y MQ8 C e l l s  Starved  (Homogenized)  for  65  2& D a y s  . (Homogenized) 9•  Oxygen Uptake  66 b y MQ8 C e l l s  Starved  for  3 Days  (Homogenized) 10.  Oxygen Uptake  67 b y MQ8 C e l l s  Starved  for  4 Days  (Homogenized) 11.  Oxygen. Uptake  63 b y MQ8 C e l l s  Starved  for  5 Days  (Homogenized) 12.  Effect Glucose  13.  14.  69  of G r i s e o f u l v i n on the  and  Oxidation Valves  Diagramatic  Representation  Protoplasts  of  Neurospora  Electron Micrographs a.  T o t a l Endogenous  Protoplast  70 of the  (int)  o f MQ8  e  of  tetrasperma  $5  Protoplasts  showing cytoplasmic  mitochondria  Formation  membrane  (cm)  and. 90  PAGE b.  Protoplast nucleus  c.  (n),  Portion of a limiting  15.  demonstrating  Effect  c y t o p l a s m i c membrane  and n u c l e o l u s protoplast  membrane  of Griseofulvin  (nu).  90  demonstrating  the  distinct  (cm)•  91  on Dermatophyte  Protoplast  Membranes 16.  Effect  of Griseofulvin  (cm),  96 on C e l l  Wall  Resynthesis  101  INTRODUCTION Griseofulvin is a fungistatic drug which is commonlyisolated from the culture media of several Penicillium species. It was f i r s t recognized by i t s characteristic effect of causing the hyphal tips of many fungal species to become stunted and distorted.  Early workers thus referred to this compound as  "curling factor" (17, IS).  In 1947, i t was discovered that  "curling factor" was a previously isolated compound known as griseofulvin (36). Following the discovery of the antifungal properties of griseofulvin, many studies were carried out to determine i t s physiological site of action i n susceptible fungi.  These  attempts met with l i t t l e success and thus experimental work in this f i e l d slowed considerably.  The discovery, in 1956% that  griseofulvin was an effective agent for curing superficial skin infections caused by the dermatophytic fungi (26), caused an immediate revival of interest i n this area.  Studies were again  initiated to determine the site of action of this drug, but up to the present, these efforts have been almost completely unsuccessful.  In fact, much of the literature consists of  conflicting reports and personal opinions rather than definite experimental findings. One area of investigation which shows some promise is that of the oxidative pathways.  Results have shown that uptake  of phosphorus is decreased (12) and that assimilation of nitrogen  i s lowered (71) i n the presence of griseofulvin.  It has been  suggested that an oxidative phosphorylation step i s being inhibited ( 7 1 ) , but conflicting results i n oxygen uptake investigations (15,44,63) have resulted i n a lack of confirmation. It i s the purpose of this study to repeat these oxygen uptake investigations and i n addition to compare the effects of the drug on the respiration of cells which have been starved for various lengths of time.  It appears that a partial deple-  tion of endogenous reserves i n the cells may be responsible for conflicting results. It i s also the purpose of this study to carry out investigations into facets of this problem which have not been previously explored.  For example, individual enzyme systems,  cell-free extracts, membranes, and c e l l walls.  If any effects  of this agent are demonstrated using these materials, more detailed experiments w i l l be undertaken.  3 REVIEW OF THE LITERATURE Historical review.  Griseofulvin is a fungistatic drug  produced by several species of Penicillium.  It was found by  Brian et a l . (17) i n 1945, as a substance produced by Penicillium .janczewskii. which caused abnormal development of young hyphae of Botrytis a l l i i .  A year later these workers re-  ported isolating this compound, which they referred to as "curling factor" (18). properties©  They demonstrated no antibacterial  Just after this, McGowan investigating the chem-  i c a l properties of "curling factor *, proposed the formula 1  C  In 1947, Grove et al* (36) reported the pres-  20 20°9 H  ence of chlorine in the compound and put forward a new formula C^yH-LyO^Cl.  They showed that "curling factor" was i n effect  griseofulvin, a compound isolated from Penicillium griseofulvin in 1939, by Oxford et al* (53).  Oxford had proposed a struc-  ture for griseofulvin and this was supported at f i r s t by Grove and McGowan (36)*  On the basis of infrared and itrltra violet  absorption spectral data however, they were forced to postulate a new structure*  This structure put forward by Grove et a l .  in 1951 (35) is shown i n FIGURE 1.  FIGURE 1.  The Structure of Griseofulvin.  4 The chemistry of griseofulvin has been under study for some time by fellow workers of Grove at the Butterwick Research Laboratories and also by other workers at the Glaxo Laboratories Ltd. The preceding paragraph has primarily been an account of the problems of defining griseofulvin as a chemical entity, but there was also a considerable lag before i t s importance as an antifungal agent was realized.  In 1949, Brian, the original  discoverer of the antifungal effects of griseofulvin, carried out inhibition studies with the drug on a large number of representative fungi (15).  He did not include a representative  dermatophyte i n his test series, thus there was s t i l l no i n d i cation of the medical importance of the drug*  The emphasis was  rather on plant protection and for some years i t was put to this use ( 1 6 ) .  The medical value of the drug was realized in 195£>  when Gentles (26) published his paper on the treatment of ringworm i n guinea pigs by oral administration of griseofulvin* Dermatophytes.  Dermatophytes are usually defined as  those fungi causing superficial diseases of the skin and hair. They are further limited as being i n the genera; Microsporia!, Trichophyton, and Epidermophyton.  It must be remembered though,  that only those members which w i l l actively invade the skin or hair are dermatophytes.  Other non-pathogenic fungi are found  in these genera. The dermatophytes are commonly classed as Fungi Imperfecti but recently, Benedek has demonstrated the formation of cleistothecia, that is perfect fructifications, by two members of Sabouraud's form genus Microsporon by means of symbiosis with  5  Bacillus weidmaniensis Benedek, 193# ( & ) .  Other workers carry-  ing out soil studies, have described the perfect stages of Microsporum gypseum ( 6 5 ) , Microsporum nunum, and Keratinomyces a.jelloi ( 2 2 ) .  The last organism was classed with the dermato-  phytes by Georg et a l . i n 1959 ( 3 4 ) .  This does not necessarily  mean that a l l dermatophytes w i l l be shown to form perfect stages but i t may mean that they have evolved from Ascomycetes. The invasion of tissues by dermatophytes has been termed "superficial".  This means invasion of non-living tissue*  These organisms then, grow mainly i n the dead keratinized areas of the host.  Dermatophytes have been found growing i n the  tissues, causing "deep mycoses".  In these cases they have  been found as a yeast-like form rather than the typical hyphal form.  A similar growth form has been caused using cysteine as  the inducing agent ( 5 7 ) .  Fortunately such deep infections are  very rare and i t i s as yet uncertain how the induction occurs in the body. Medical aspects of griseofulvin.  On the basis of his  i n i t i a l studies, Gentles (26) felt that this drug probably f u l f i l l e d Wilson's definition of an ideal antifungal agent, at least as far as the dermatophytes are concerned*  Wilson (70)  stated that, "the ideal antifungal drug even for superficial mycoses would seem to be one which could be safely administered internally i n amounts sufficient to endow the cells eventually destined to produce keratin with the power to resist fungi completely, this power persisting as they become keratinized,  6 and the drug thus exerting i t s effects from within outward". Through the work of Gentles (26,27,29,30,31,32) and others (54,53), griseofulvin has been shown to do exactly what Wilson's definition requires.  It is incorporated into those cells which  later form the keratin, thus endowing the patient with a tissue which i s inhibitory to the fungus.  Such a mode of action  appears mandatory, as penetration of keratin by topical application, even of griseofulvin, has been found almost impossible. The greatest weight of literature at this time is on the c l i n i c a l experiences with griseofulvin.  As this is not the  subject of this thesis, I w i l l not mention the c l i n i c a l cases and side effects involved i n the use of this drug. Chemical properties of griseofulvin.  Griseofulvin i s a  colorless, odorless, white, crystalline, neutral compound.  It  has a melting point of 218-219°C and is exceedingly thermostable. It readily forms a crystalline mono-oxime, and i t also reacts with phenylhydrazine and with 2,4-dinitrophenylhydrazine although crystalline compounds can not be readily obtained (53).  This  compound has absorption maxima at 296mu and 326u(in water) and at 239mu (in butyl acetate) (59).  (2),  Its characteristic  ultraviolet and infrared absorption spectra were shown by Grove and McGowan (37).  It is sparingly soluble i n water which  makes i t d i f f i c u l t to handle under physiological conditions. It i s soluble i n ethanol, acetone, acetic acid, butyl acVtate, and N,N-dimethylformamide to name a few of the more common solvents.  7 Antifungal properties of griseofulvin.  A report by  Brian et a l . i n 1946 (17) was the f i r s t recorded observation that griseofulvin was antifungal i n i t s properties.  They noted  that, i n the presence of the drug, hyphae of Botrytis a l l i i were stunted, distorted, and showed increased branching (in low dilutions of griseofulvin) or "waving" (in greater dilutions). They found that higher concentrations of the compound stopped the growth of Botrytis a l l i i conidia, but only after some growth had occurred.  It was also of interest to them that stunting  and distortion of the hyphae occurred at much lower concentrations of griseofulvin than were required to stop conidial germination.  Brian continued his studies by surveying the  effects of this agent on a large group of representative fungi, aetinomycetes, (15)o  bacteria, and even some seeds of higher plants  He found that, " a l l Basidiomycetes, Ascomycetes, Fungi  Imperfecti, and Zygomycetes, with the exception of two yeasts, were sensitive to griseofulvin", and also that, "it has no apparent effect on bacteria, aetinomycetes,  or on certain groups  of fungi (Oomycetes and yeasts); i t inhibits the germination of seeds of higher plants and retards root extension".  His final  conclusion was that griseofulvin acts on only those fungi which have chitinous c e l l walls while fungi with cellulose-containing walls are not affected.  This is a very broad statement and  there appear to be some exceptions (2). Further visual observations have been carried out more recently using electron microscope techniques (10,66).  On close  8  observation of the c e l l wall, i t was seen that the drug caused a loss of integrity.  The wall became much thicker, and splits  appeared u n t i l i t became separated into frayed and irregular layers.  The cytoplasm was seen to become reduced, leaving  remnants of cytoplasmic membrane and large l i p i d storage granules.  Younger, actively metabolizing cells showed more  drastic alteration than older c e l l s . The greater susceptibility of young hyphae to griseofulvin has frequently been mentioned i n the literature (10,15). This was demonstrated more fully by Banbury (7).  He applied  the drug to growing portions of Phyeomyees blakesleanus sporangiophores and noted a curvature.  He noted no such reaction when  the application was made to the non-growing area several m i l l i meters below i t .  Other work has shown that no translocation  of griseofulvin occurs within fungal hyphae and therefore intimate contact between the growing area and the drug i s required ( 2 ) .  It has also been suggested that griseofulvin is  fungistatic to older hyphae, but fungicidal to young growing hyphae ( 2 3 ) .  That i t is fungistatic has been demonstrated  (17,56). Biochemical studies on the action of griseofulvin on fungi are as yet very limited.  Oxygen uptake i n the presence  of the drug has given conflicting results (15,44,63) although i t does seem l i k e l y that a reduction i n respiratory rate occurs (63).  Coupling this latter suggestion with other results, show-  ing a decrease i n phosphate uptake and a decrease in nitrogen  9 assimilation, i t i s f e l t that griseofulvin uncouples a phosphorylation i n a respiratory pathway (12,71).  However, according  to Gentles (33), Rhodes feels that the compound may be incorporated into c e l l wall, giving a modified chitin.  McNall, on  the other hand has proposed interference with nucleic acid synthesis.  He demonstrated partial reversal of the effect of  griseofulvin with purines, pyrimidines, and their nucleotides (47). Resistance by dermatophytes to griseofulvin.  One of the  early questions asked by medical workers i n relation to a new drug, i s ; how easily and therefore how frequently may the susceptible organism become resistant to the drug? course was also the case with griseofulvin.  This of  In May I960, four  separate publications appeared, dealing with the problem of "in vitro" resistance by dermatophytes (4,60,62,64).  The general  conclusions were that a l l dermatophytes tested, acquired some "in vitro" resistance, but that on return to "in vivo" conditions, this resistance was l o s t .  Isolates from patients under griseo-  fulvin therapy were found to have no "in vitro" resistance.  For a  short period the situation looked extremely hopeful but then two cases of "in vivo" resistance were reported (41,50).  One was  experimentally induced, the other occurred naturally. It was suggested that griseofulvin resistance was due to an enzymatic system which degraded the drug (4).  This has been  shown to occur with several species of fungi including Microsporum canis (13).  The mechanism of deactivation was a  demethylation, i n a l l cases tested, but the particular methyl  10 group removed varied with different fungal species. Microsporum canis produced 4-demethylgriseofulvin, from griseofulvin. Griseofulvin analogues.  The structure of griseofulvin i n  relation to i t s activity i s an important property.  However, un-  t i l recently few reports have appeared on the inhibitory action of griseofulvin analogues.  Chemists at the Butterwick Research  Laboratories i n England have reported the preparation and i s o l a tion of many compounds closely related i n structure to griseofulvin, but they have given l i t t l e indication of their inhibitory activities.  In one paper they did briefly mention the anti-  fungal effects of dechlorogriseofulvin and of the bromo analogue of griseofulvin ( 4 6 ) .  In both compounds the chlorine of  griseofulvin has been replaced.  In one instance i t i s by  hydrogen and in the other i t i s by bromine.  They tested the  analogues with Botrytis a l l i i and found that to produce the effect caused by 0.1 ugm./ml. of griseofulvin, they required 0.75 ugm./ml. of the bromo analogue and 6.25 ugm./ml. with dechlorogriseofulvin. A report by Abbot and Grove i n 1959 (1,2) refers to a "diol" (7-ehloro-4 ::6 -dihydroxy-4:6-dimethoxy-2 -methyl grisan!  ,  ,  3-one) which they tested with a strain of Botrytis a l l i i .  They  found the "diol" to be less active than griseofulvin. Finally, a paper by Boothroyd et a l . i n 1961 (13) reports demethylated products of griseofulvin.  These compounds were  found as inactive products of fungal metabolism of griseofulvin. They are 4-demethylgriseofulvin, 6-j.demethylgriseofulvin, and 2' demethylgriseofulvin.  These compounds although very similar i n  11 structure to griseofulvin demonstrated no inhibitory activity. Cell wall constituents of dermatophytes.  The earliest  studies on dermatophytic c e l l walls were carried out by Blank (9) on c e l l wall residues of a l k a l i extractions.  He concluded  that the framework of the walls was composed completely of chitin.  His methods were rather drastic, so that many wall  components were lost.  More recently, mechanical breakage and  analysis of walls by McNall has yielded a different picture (43).  He showed them to contain large quantities of  polysaccharides of glucose and glucosamine, and in addition some protein and l i p i d .  The protein reported by McNall is  possibly a peptide, as a peptide linked to glucosamine has also been reported by Carlson and Knight (19) i n a crude c h i t i nase resistant fraction of a dermatophyte c e l l wall. Fungal protoplasts.  Perhaps the term "protoplast" is  not entirely correct in referring to the fungi, but when fungal hyphae are attacked by mixtures of such enzymes as Streptomyces chitinase,^-l,3-glucanase,  and digestive juice of Helix pomatia.  "protoplast-like" structures are formed©  Of course, this must  be done under conditions of sufficient osmotic strength to prevent osmotic shock or disruption of these fragile structures. Crude digestive juice of Helix pomatia is probably the most commonly used enzyme for the production of fungal "protoplasts". It contains several enzymes which w i l l attack various fungal wall components, so that i t may be employed to produce "protoplasts" of yeasts as well as of filamentous fungi (40),  The  12  techniques normally used with filamentous fungi are modifications (3,43) of those methods described by Bachman and Bonner in their work with Neurospora crassa (5).  As yet, no reports  have been published on the production of "protoplasts" of dermatophytes, although purified c e l l walls of Trichophyton mentagrophytes have been reported to be partially digested by crude snail gut chitinase (19).  A good criterion for the  definition of a "bacterial protoplast" has been put forward by Brenner et a l . (14). fungal protoplasts.  This could also be used for defining  13  MATERIALS I.  Glassware A l l glassware used throughout the course of these e x p e r i -  ments was soaked o r b o i l e d i n a 2% Haemo-sol s o l u t i o n f o r at l e a s t one hour.  I t was then washed and r i n s e d with warm water,  followed by r i n s i n g s w i t h c o l d tap water and f i n a l l y w i t h d i s t i l l e d water. I I . Reagents A. G r i s e o f u l v i n Solutions The g r i s e o f u l v i n used i n t h i s study was a h i g h l y p u r i f i e d sample, which was k i n d l y donated by Sobering Corporation L i m i t e d , Montreal, Quebec.  The f o l l o w i n g  g r i s e o f u l v i n s o l u t i o n s were used i n t h i s study: 1.  2,000 ugm./ml. i n r e d i s t i l l e d b u t y l acetate (b.p.  2.  124° t o 126°C).  2,000 ugm./ml. i n 50% e t h a n o l .  The g r i s e o f u l v i n  was d i s s o l v e d i n a minimum volume of 95% ethanol and then g r a d u a l l y d i l u t e d w i t h d i s t i l l e d water. During the a d d i t i o n of the water, the s o l u t i o n was slowly a g i t a t e d , u s i n g a magnetic s t i r r e r .  If  p r e c i p i t a t i o n occurred while the water was being introduced, a small volume o f 95% ethanol was titrated in.  When the required volume o f water  14 had been added, the solution was brought to i t s final volume with 95% ethanol. 3.  600 ugm./ml• i n redistilled N,N-dimethyl formamide (DMF) (b.p. 151° to 153°C).  B. Chromatographic Reagents 1.  Solvents a. Butanol-acetic acid-water, 2:1:1, (v/v). b. But anol-methyl ethyl ketone-water, 2:2:1, (v/v). c. Ethyl acetate-pyridine-water,  2.  S:2:l,(v/v).  Developers a. 0,5% (w/v) ninhydrin i n n-butanol. b. 0.5 ml. saturated silver nitrate solution in 100 ml. acetone, add d i s t i l l e d water u n t i l precipitate disappears. 0.5 N sodium hydroxide i n  BOfo  ethanol.  5% (w/v) Na2S 0-^.5H20 i n d i s t i l l e d water. 2  3.  Standards a. Amino acids: 15 juM/ml. (0.01 ml„ applied to chromatogram). b. Sugars and amino sugars: 2.0 mgm./ml. (0.01 ml. applied to chromatogram).  G.  Hexosamine Assay Reagents 1.  Glucosamine-HCl stock solution? 50 ugm. glucosamine-HCl /ml. i n d i s t i l l e d water.  Diluted to the  following standards: 5»0 ugm./ml., 10 ngm./ml., 20 ugm./ml., 30 ugm./ml., and 40 ugm./ml. 2.  0.5% (w /v) phenolphthalein i n 95% ethanol.  3.  2% (v/v) acetyl acetone i n 1.0 N sodium carbonate. The acetyl acetone was redistilled and the fraction boiling at 140° to 141°C was used.  4.  Ehrlich's reagent: 2.67% (w/v) p-dimethylaminobenzaldehyde in 95% ethanol and 12 N HC1, 1:1  III.  (v/v)  Enzymes A.  Partially Purified Chitinase A partially-purified sample of chitinase,  isolated  from the intestinal juices of the snail Helix pomatia, was kindly supplied by Dr. G. Strasdine (National Research Council, Ottawa, Canada). B.  Crude Snail Gut Juice "Sue digestif d Helix pomatia" was obtained from 1  L'Industrie Biologique Franchise, 35 a 49, Quai du Moulin de Cage, Gennevilliers (Seine), France.  This  material contains a mixture of enzymes, including chitinase and cellulase.  16 C. Trypsin This proteolytic enzyme was obtained from Nutritional Biochemical Co*  It was used i n the concentration of  0,5 mgm./ml., i n 0.066 M phosphate buffer pH 7.6, D. Ribonuclease This enzyme was obtained from Nutritional Biochemical Co*  It was used i n the concentration of 0,5 mgm./  ml., i n 0 066 M phosphate buffer pH 7.6. o  Media A. Sabouraud's Cerelose Broth Cerelose  40 gm.  Neopeptone (Difco)  i  Tap water  10 gm. 1,000 ml.  (Adjust pH to 5.8 - 6.0) B. Vogel s Medium 1  Sodium citrate  150 gm.  KH P0  4  250 gm.  3  100 gm.  2  NH N0 4  10 gm.  MgS0 .7H 0 . . 4  2  CaCl .2H 0 . .  5.0 gm. (stir)  Trace elements solution  5.0 ml.  2  2  Biotin solution  • • • .  2.5 ml.  Successively dissolve i n 750 ml. d i s t i l l e d water, then bring volume to 1,000 ml.  For Neurospora species supplement w i t h : glucose (20 gm./l.), N.F. casein (5.0 gm./l.), and yeast e x t r a c t (5.0 gm./l.) Trace Elements S o l u t i o n C i t r i c acid  e  . 5.0 gm.  ZnS0^.7H 0  5.0 gm.  2  Fe(NH^) (S0^) '6H 0  1.0 gm.  CuS04.5H 0  0.25 gm.  MnS04.1H20  0.05 gm.  H3BO3  0.05 gm.  2  2  2  2  Na Mn0 .2H 0  0.05 gm.  D i s t i l l e d water  100 ml.  2  j!f  2  Biotin Solution 5.0 mgm. b i o t i n i n 50 ml. d i s t i l l e d water. S y n t h e t i c Medium f o r Amino Acid  Studies  Glucose  14 gm.  (NH2 ) S0^ •  1.2 gm.  FeGl  0.002 gm.  f  2  .  2  -CaCl .2H 0  0.002 gm.  MgS04.7H 0  . 0.002 gm.  2  2  2  Phosphate b u f f e r , 0.066 M,pH 6.8 D i s t i l l e d water  20 ml. 380 ml.  (The phosphate b u f f e r was autoelaved separately).  Equipment A.  Warburg Respirometer This instrument was used i n a l l oxygen uptake studies. The water bath temperature was 30°G.  It was manufactur-  ed by Gilson Medical Electronics, Middleton, Wisconsin. B,  Thunberg Tubes Standard tubes as described (66) were employed.  G.  Spectrophotometers 1.  Spectronic 20, manufactured by Bauseh and Lomb, was used for the hexosamine assay. were used throughout the study.  Two tubes They were  matched to give the same absorbance. 2.  Beckman DU, was employed i n measuring changes i n absorbance i n the protoplast experiments.  It was  also used for determining concentrations of griseofulvin in reaction mixtures, D,  Homogenizer A VirTis  , ,  2 3 homogenizer was utilized for the breakage n  of long hyphal elements into shorter units, E.  Cell Disintegrator Cells were disrupted, to isolate purified c e l l wall material, with a Nossal c e l l disintegrator.  This was  manufactured by McDonald Engineering Company, Cleveland, Ohio.  Ultra-violet Viewer Observation of chromatograms for compounds which absorb or fluoresce in ultra-violet light was carried out with a "ehromato-vue", (Ultra-violet Products, Inc., San Gabriel, California). This viewer contains two separate lamps and f i l t e r giving wavelengths of 366 mu and 253.7 mu.  20  METHODS I.  Preparation of Cells A.  Fresh Whole -Cells Stock cultures of dermatophytes were maintained on Sabouraud's cerelose agar.  The species used were;  Microsporum quinckeanum strain #8 - Dr. F . Blank, MeGill University. Microsporum quinckeanum strain #7 - Dr. F . Blank, MeGill University. Trichophyton asteroides  - Dr. F. Blank, MeGill University.  Trichophyton irientagrophytes strain #666 - Dr. C. W, Emmons, Bethesda, Md. A culture of Neurospora tetrasperma was also used.  It  was obtained from Dr. R. J , Bandoni, University of B.C, Fungi were grown in shake culture in 500 ml. Erlenmeyer flasks containing 50 ml. of liquid medium.  In this case, 0.2  ml. of a sterile 1.0% solution of triton X-100 was added. Static fungal cultures were grown in 500 ml. Erlenmeyer flasks containing 100 ml. of liquid medium. Shake cultures were grown (at room temperature) on a Burrell shaker, at 225 cycles per minute with a l+.Q cm. stroke. Static cultures were grown in a 25°C. incubator.  21 A l l cultures were inoculated with either hyphal fragments from liquid growth or with hyphae and conidia from fresh surface growth. Cells were harvested by one of three methods.  In the  f i r s t method they were centrifuged at 2,000 revolutions per minute (rpm) for thirty minutes at room temperature. two methods involved f i l t r a t i o n .  The second  The cells were collected  either on cheese cloth, or on f i l t e r paper supported by a Buchner funnel, under suction*  Depending on the requirements of the  experiment, the cells were washed with 0.066 M phosphate buffer pH 7.0, 0.05 M NaCl, 20% sucrose, or d i s t i l l e d water. B. Starved Cells Using aseptic precautions, whole cells from four flasks were washed by centrifuging at 2,000 rpm for thirty minutes at room temperature, i n sterile centrifuge cups with two volumes of sterile 0.35% NaCl.  This was followed by one washing with  sterile 0.066 M phosphate buffer pH 7.0  The cells were then re-  suspended i n 200 ml. of the phosphate buffer.  This suspension  was divided into four 50 ml. fractions, each of which was placed in a sterile 500 ml. Erlenmeyer flask, to which had been added 0.2 ml. of a sterile 1.0% solution of triton X-100.  The flasks  were placed on the Burrell shaker at room temperature and the cells were starved for two, three, four or five days.  The  starvation medium was replaced after three days by centrifuging down the cells and resuspending them i n fresh phosphate buffer and triton X-100.  22 C. Cell Suspensions for 02 Uptake Studies Whole cells or starved cells were washed by centrifuging at 2,000 rpm for thirty minutes at room temperature, with two volumes of 0,65% NaCl.  The c e l l material from two flasks was resuspended  in 20 ml. of 0.066 M phosphate buffer pH 7.0, to which had been added triton X-100 (0.4 ml. t r i t o n X-100 per 100 ml. buffer) and 20 International Units of p e n i c i l l i n per ml. of buffer.  This  suspension was transferred to a sterile 250 ml. macro VirTis homogenizing flask.  The flask was then placed i n an ice bath and  allowed to cool for ten minutes.  The mycelial pellets were homo-  genized at 23,000 rpm i n a Vir-Tis 23 ,t  three minutes.  M  homogenizer for a total of  This was done by running the homogenizer for  three intervals of one minute with a cooling period of two minutes between each.  The resulting suspension contained short hyphal  elements of approximately four cellular units.  Such a suspension  permitted easier and more accurate measurement of hyphal material being added to Warburg vessels.  It also resulted i n more uniform  oxygen uptake values as variations i n measurement occur with larger hyphal elements which tend to form clumps when being shaken. D. Cell-free Extracts Whole cells or starved cells were centrifuged at 2,000 rpm for forty-five minutes at room temperature.  The wet, packed c e l l  material was partially dried on f i l t e r paper, then weighed.  To  this material was added fine glass beads (Glass Homogenizing Beads, VirTis Company), which had previously been cleaned with sulphuric acid-dichromate solution and then rinsed ten times with 0.066 M  23 phosphate buffer pH 7 . 0 . The glass beads were added i n the ratio of 9.0 gm. to 1.0 gm. of wet, packed c e l l material.  The i  beads and pellets were mixed as thoroughly as possible, then were frozen*  10 gm. of this frozen mixture was placed in a  Wedgewood mortar which had previously been chilled to 0°C.  The  mixture was vigorously ground with a pestle until thawing was observed.  The mortar was immediately returned to the refriger-  ator to refreeze the mixture.  This procedure was continued  until a total of thirty minutes of grinding had been achieved. The mixture was placed i n a 50 ml. centrifuge tube at 4°C to thaw, then 5.0 ml. of 0.066 M phosphate buffer pH 7.0 which had been chilled to 0°C was added.  The buffer was allowed to extract  the c e l l material for several hours, then the tube was centrifuged at 2,500 rpm for thirty minutes at 4 ° C  The liquid phase  was decanted into another 50 ml. centrifuge tube, a second 5.0 ml. aliquot of chilled phosphate buffer was added to the c e l l residue and this was allowed to extract for several more hours. The tube was again centrifuged and the liquid phase pooled with the f i r s t extract.  The pooled sample was centrifuged at 2,500  rpm for sixty minutes at 4°C to remove any residual c e l l material.*  The cell-free supernatant was stored frozen at - 8 ° C .  The c e l l residue was separated from the glass beads by successive sedimentation and decanting with d i s t i l l e d water.  These  washings were centrifuged at 2,500 rpm for thirty minutes at 4°C and the packed residue was stored frozen. E. Acetone Dried Preparations Two flasks of c e l l s , were grown for four days i n shake  culture.  They were harvested by f i l t r a t i o n and the pellets were  washed on the f i l t e r with 500 ml. of 0.6*5% NaCl.  They were then  quickly transferred to a 50 ml. aliquot of acetone at - 6 ° C , and mixed well by s t i r r i n g .  The c e l l material was allowed to settle  and the acetone was decanted off.  This was followed by washing  with three 50 ml. aliquots of acetone at - 8 ° C . After the last de^ canting, the cells were spread on a watch, glass and the acetone allowed to evaporate for several minutes.  The watch glass was  next transferred to a vacuum desiccator and the drying continued under vacuum at 4 ° C . The dried c e l l material was ground with a mortar and pestle and the dry powder stored i n the deep freeze at - 6 ° C :  25 I I . Preparation of Protoplasts A. Preparation of Protoplasts of Neurospora Species Protoplasts of Neurospora tetrasperma were studied i n order to become familiar with their appearance and with the techniques for their formation.  The method of Colvin (20) was used.  It i s a modification of the original method of Bachmann and Bonner ( 5 ) . ing  Colvin s modification is described i n the follow1  section. 100 ml. of Vogel's medium i n a 500 ml. Erlenmeyer flask  was inoculated with a piece of vegetative growth about 0 . 5 cm. i n diameter.  This was incubated i n static culture at 25°C for  forty-eight hours, yielding a large, fuzzy, b a l l - l i k e mass of submerged growth.  This material was transferred to a mixture  of nine parts 22% sucrose to one part "sue digestif d'Helix pomatia" (L'Industrie Biologique Francaise).  The mycelial  pellet was incubated with the enzyme mixture at 35°C for a total of four hours.  A small sample was removed every fifteen  minutes and observed with a phase contrast microscope. Formation of protoplasts started after approximately one hour and continued for the remaining three hours.  To demonstrate the loss of c e l l  wall, d i s t i l l e d water was slowly added under the cover slip to dilute the sucrose and thus cause bursting of the protoplasts due to "osmotic shock". Protoplasts were partially purified by f i l t r a t i o n through glass wool*  Further purification was accomplished by passing  the suspension through a "C" grade Pyrex sintered glass f i l t e r .  Concentration of the protoplasts was achieved by centrifuging at at 12,100XG. for five minutes at 0 ° C .  The pellet was resus-  pended i n a small volume of the supernatant solution.  All  determinations of protoplast numbers were carried out using a Petroff-Hausser counting chamber.  Protoplast diameters were  determined by use of a calibrated ocular micrometer. B. Preparation of Dermatophyte Protoplasts The method of protoplast preparation used previously with Neuospora tetrasperma was found to be less effective with dermatophytes.  After some experimentation, Kinsky s modifif  cation (43) of the technique of Bachmann and Bonner, (5) was used as a basis for forming protoplasts of dermatophytes.  The  following is the modified method which was f i n a l l y employed. Pieces of vegetative growth or hyphal pellets approximately G.5 cm. i n diameter were inoculated into 100 ml. of Sabouraud s cerelose broth, i n a 500 ml* Erlenmeyer flask, and f  the cells were grown for three to four days i n static culture at :2 5°G*  B a l l - l i k e masses of submerged growth similar to  those of Neurospora were formed.  Several of these were  collected by f i l t e r i n g through a perforated aluminum f o i l cup. These were then washed on the aluminum cup with about 50 ml. of 20% sucrose i n 0.066 M phosphate buffer pH 6.8.  The washed  hyphal elements were transferred to a mixture of 20% sucrose and 0.1 M glutathione i n 0.066 M phosphate buffer pH 6 . 8 ( 9 . 0 ml.), sue digestif d'Helix pomatia (1.0 ml.), and p e n i c i l l i n solution containing 2,000 International Units per ml. (0.2 ml.).  This  enzymic mixture had been previously sterilized and partially  27  "purified by passage through a sterile 0.45 u millipore f i l t e r using a syringe with a Swinny f i l t e r adaptor. was incubated at 35°C for fifteen hours.  The final mixture  The resulting proto-  plasts were clarified by f i l t r a t i o n through a C n  rt  grade Pyrex  sintered glass f i l t e r with a prefilter of Whatman No. 1 paper. The protoplasts were concentrated by centrifuging at 7»700XG for ten minutes at 0 ° C .  28 III.  Paper Chromatography A. General Methods Amino acids, amino sugars, sugars, and griseofulvin  hydrolysis products were separated by paper chromatography.  In  a l l cases, descending chromatograms were used, and run at room temperature. The paper used for single dimensional chromatography was prepared as follows. of the paper.  A fold was made here to hang the paper over the  antisiphon bar. first.  A line was drawn 6.0 cm. from one end  Another line was drawn, 3.0 cm. below the  This marked the "origin" or point where the samples  were applied.  The distances between the samples were from 2.0  to 3.0 cm., depending on the numbers and types of samples employed.  The two dimensional chromatograms were marked with  two extra lines.  These were drawn at 6.0 cm. and 9.0 cm. from  one edge of the paper.  A single spot was applied at the point  of intersection of the two 9»0 cm. lines. Samples were applied to the paper using glass capillaries.  The liquid sample was allowed to run onto the  paper u n t i l a spot of about 1.0 cm. i n diameter was obtained. This was dried and then more sample added to the center of the spot.  A l l spotting of samples was done i n a stream of warm a i r  to f a c i l i t a t e drying. Before addition of the paper to the chromatography tank, a volume of the solvent to be employed was placed in the bottom of the tank to allow saturation of the atmosphere.  After about  29 thirty minutes, the paper was placed i n the tank and allowed to equilibrate for at least another thirty minutes.  At the end of  the equilibration period, the solvent was quickly added to the trough and the system was sealed to prevent evaporation.  At the  end of the run, the paper was removed from the tank, the position of the solvent front was marked, and then the solvent was driven from the paper with a stream of warm a i r .  When the paper was  completely dry, the positions of the compounds were determined with the appropriate developing reagents.  Developed spots were  immediately outlined with a pencil line and their colours noted. B. Amino Compounds Amino acids and amino sugars were separated by one dimensional and two dimensional chromatography.  The samples  were placed i n small test tubes and sufficient 1.0 N HC1 was added to insure that a l l amino compounds were i n the hydrochloride salt form.  In the case of HC1 hydrolysates this was not  necessary. Those samples which contained inorganic salts were previously purified by passing them through columns of DOWEX 50 ionized resin.  The columns were i n the H ion state and were +  eluted with 50 ml. of 1.0 N HC1 after a preliminary washing of the sample i n the column with 20 ml. of d i s t i l l e d water* With a l l amino compounds i n the hydrochloride salt form, samples were evaporated to dryness with a stream of warm a i r . This served to remove most of the excess hydrochloric acid.  The  samples were redissolved i n a small volume of d i s t i l l e d water and spotted onto ehromatograms.  When the concentration of amino  compounds i n a sample was uncertain, two or more volumes, containing different amounts of the sample, were spotted onto the chromatogram. Amino acid and amino sugar standards were run parallel to the unknowns.  They were prepared i n a similar manner.  That  i s , placed i n the hydrochloride salt form, evaporated to dryness, and redissolved i n d i s t i l l e d water.  A quantity of 0.15M M of  each amino compound was spotted onto the chromatogram.  The  twenty common amino acids were employed as standards and i n addition, glucosamine and L-galactosamine were used.  The  solvent system used for amino compound separation was: butanolacetic a c i d - water, 2:1:1, (v/v)(6).  In the two dimensional  system the second solvent was: butanol-methyl ethyl ketone water, 2:2:1, (v/v) i n the presence of cyelohexylamine vapours (51)»  In a l l cases Whatman No. 4 chromatography paper was  used.  A l l amino compounds were detected by spraying the  chromatograms with 0.5% ninhydrin in n-butanol and drying i n the oven at 85°to 95°C.  Characteristic colours developed, although  different colours than are normally found were obtained i n the presence of cyelohexylamine. C. Sugars and Amino Sugars Sugars and amino sugars were also separated on Whatman No. 4 chromatography paper.  The sugar standards which were run  parallel to the unknown samples were; glucose, galactose, mannose, ribose, deoxyribose, glucosamine, and galatosamine.  Standards  consisted of 0.01 ml. of a solution of 2.0 mgm. of the sugar per ml. of d i s t i l l e d water.  The solvent system used t o separate these compounds cons i s t e d o f e t h y l acetate-pyridine-water, developing agent used (67)  8:2:1,  (v/v).  The  was s i l v e r n i t r a t e i n acetone,  followed by sodium hydroxide i n ethanol.  The background was  removed w i t h an aqueous s o l u t i o n o f sodium t h i o s u l p h a t e . The spots were brown a t f i r s t , but w i t h d r y i n g t h e i r colour turned to b l a c k . sprayed.  A l l sugar chromatograms were dipped r a t h e r than Care was taken when d i p p i n g through the s i l v e r n i t r a t e  s o l u t i o n t o avoid s t r e a k i n g o f the spots.  Chromatograms u t i l i -  z i n g t h i s solvent system were run f o r twenty-four t o t h i r t y - s i x hours. D. Products o f G r i s e o f u l v i n H y d r o l y s i s A small sample of g r i s e o f u l v i n o r o f m a t e r i a l containing g r i s e o f u l v i n was hydrolyzed w i t h  2.0  ml. o f  2.0  f o r eighteen hours, i n a sealed g l a s s ampoule. from t h i s hydrolysate was c o l l e c t e d and stored. s o l i d residue was hydrolyzed w i t h f o r a f u r t h e r twelve hours.  2.0  N  HC1  at  100°C  The supernate The remaining  ml. o f 6.0 N  HC1  at  100°C  The supernant s o l u t i o n and r e s i d -  u a l p e l l e t were separated and stored* A 1.0 ml. volume o f each of the supernates was evaporated t o dryness with a stream o f warm a i r and then r e d i s s o l v e d i n 0.1 ml. of d i s t i l l e d water.  These samples were then spotted on t o  Whatman No. 4 chromatography paper. The r e s i d u a l p e l l e t and the remaining 1.0 ml. volumes o f the supernates were each extracted with 2.0 m l . of b u t y l acetate. These e x t r a c t s were evaporated t o dryness, r e d i s s o l v e d i n 0.1 ml. of b u t y l acetate, and spotted on t o the chromatogram.  The solvent used for this separation was butanol-methyl ethyl ketone-water,  2:2:1, (v/v).  A sample of griseofulvin in  butyl acetate was applied as a control.  Spots were detected  by viewing with an appropriately filtered ultra-violet lamp.  33 IV.  Oxygen Uptake Studie s Oxygen uptake by dermatophytes was followed using a  Warburg manometric apparatus.  The preparation of c e l l material  for these studies has been described i n a previous section. volume of liquid i n the cups i n a l l cases was 2.7 ml.  The  The  general protocol for these studies was as follows:. TABLE I PROTOCOL FOR WARBURG RESPIROMETER CUPS Endogenous  Endogenous & Substrate Griseofulvin  Cell Material in buffer  2.0 ml.  2.0 ml.  2.0 ml.  2.0 ml.  Buffer * (M/l5,pH7.0)  0.28 ml.  0.23 ml.  0.28 ml.  0.23 ml.  Substrate (M/50)  -  -  0.2 ml.  0.2 ml.  Distilled Water  0.2 ml.  0.2 ml.  -  -  -  0.02 ml.  -  Ethanol 50%  0.02 ml.  -  0.02 ml.  -  KOH 20%  0.2 m l o  0.2 ml.  0.2 ml.  0.2 ml.  Total  2.7 ml.  2.7 ml.  2.7 ml.  2.7 ml.  Griseofulvin 2,OOOugm/ml. i n 50% Ethanol  Substrate & Griseofulvin  0.02 ml.  * Buffer is 0.066 M phosphate buffer pH 7.0 containing triton X-100 (0.4 ml. 1.0% triton per 100 ml. buffer) and 20 International Units of p e n i c i l l i n per ml. of buffer. The c e l l material, buffer, griseofulvin, and ethanol were placed i n the Warburg cup. placed i n the side arm.  The substrate or d i s t i l l e d water was The KOH was placed i n the center well  of the cup with a folded piece of f i l t e r paper approximately 2.0 cm. square.  The f i l t e r paper was used to increase the surface  area of the KOH solution. When the contents had been placed i n the cups, these flasks were attached to their matching manometers and the plugs placed i n the side arms.  In both cases the ground glass con-  nections were sealed with "vaspar" (1 part vaseline: 1 part paraffin wax) and secured with small springs.  Vaspar has a  higher melting point than vaseline, so that there i s less chance of i t allowing leakage into or out of the cup. The cups were allowed to shake i n the water bath for ten minutes to allow them and their contents to reach water bath temperature.  A l l tests were carried out at 30°C.  At the end  of this time the seals on the ground glass joints were checked and the manometers were closed.  Endogenous respiration of the  c e l l material was measured for fifteen minutes before the substrate was tipped i n from the side arm.  This was to insure that  a l l flasks were giving similar values and that no leakage was occurring.  A l l readings were determined by returning the fluid  in the closed arm of the manometer to the 15 cm. mark and noting the level of the fluid i n the open arm of the manometer.  The  differences i n values due to alterations i n atmospheric pressure were corrected by comparison to a "thermobarometer".  Since the  temperature i s held constant by the water bath, the only thing which can alter the levels of the fluid i n the manometer i s a change i n atmospheric pressure.  The oxygen uptake values are  i n i t i a l l y recorded i n millimeters (mm.) on the manometer column.  The volumes of the flasks had been measured previously and a "flask constant" determined for each.  When an oxygen uptake  value i n mm. is multiplied by the flask constant the result is the oxygen uptake value in microliters (ul)•  36 V.  Dry Weight Determinations A method of c e l l standardization was required i n order  to have a basis for comparison of oxygen uptake values, Turbidimetric determinations were attempted but were found unsatisfactory with hyphal material.  Wet packed weight was used  but s t i l l gave a low degree of accuracy.  Finally, dry weight  of washed c e l l material was used and this gave good correlation of oxygen uptake values between cultures grown under similar conditions for the same length of time. The c e l l material which was added to the Warburg flasks had been washed and resuspended i n phosphate buffer containing triton X-100 and p e n i c i l l i n .  Two 1 0 ml, volumes of this  suspension were delivered into two dry, tared aluminum cups. Similarly two 1 0 ml. volumes of the phosphate buffer (containing t r i t o n X-100 and penicillin) were also delivered into two aluminum cups.  A l l four cups were placed i n an oven at  100°C  for twenty-four hours. At the end of the drying period, the cups were transferred to a desiccator containing anhydrous CaCl2, where they were allowed to cool.  The dried cells plus cups were then  weighed and the weight of the c e l l material per cup determined. The weighing was done as quickly as possible i n order to minimize any error due to uptake of water by the protein i n the sample.  The weight of the buffer was subtracted from the  combined weight of cells and buffer to give the weight of cells alone.  37 VI.  Quantitative Analysis of Griseofulvin in a Reaction Mixture The method of analysis used i n this study was based on  that given by Robinson and fellow workers i n i960 (59).  A  sample of c e l l material and supernate containing griseofulvin was placed in a test tube and 1.0 ml, of butyl acetate was added. The tube was shaken well and then placed i n the deep freeze for thirty minutes to allow separation of the two phases.  The  butyl acetate was removed from the tube with a Pasteur pipette. A second 1.0 ml. volume of butyl acetate was added and the process was repeated.  The second extract was combined with the  f i r s t and then the total volume was brought to 3.0 ml.  The  absorbance (A) of this solution was measured at 289.5 mu i n a Beckman DU spectrophotometer.  - A control sample containing  cells and supernate but no griseofulvin was treated i n the same way and i t s absorbance subtracted from that of the test sample. A standard curve for the absorbance of griseofulvin i n butyl acetate at 289.5 mu was plotted (FIGURE 2 ) .  The following con-  centrations of griseofulvin were measured against a butyl acetate blank: 2.5 ugm/ml., 5.0 ugm/ml., 10 ugm/ml., and 20 ugm/ ml.  FIGURE 2 Standard  Griseofulvin  Griseofulvin  Concentration  38  Curve  (jigm./ml.)  39  VII.  Dehydrogenase Studies The method of Thunberg was utilized for the determination  of dehydrogenase activity of cells and c e l l free extracts Standard Thunberg tubes were employed.  (63).  The contents of the  tubes are given i n the following protocol. TABLE II PROTOCOL FOR THUNBERG TUBES Endogenous Endogenous & Substrate Substrate & Substrate Griseofulvin Control Griseofulvin Enzyme Source in buffer  1 . 0 ml.  1 . 0 ml.  1 . 0 ml.  -  2 . 0 ml.  2 . 0 ml.  2 . 0 ml.  2 . 0 ml.  2 . 0 ml.  2 . 0 ml.  3 . 0 ml.  2 . 0 ml.  -  -  -  Methy1 . 0 ml. lene Blue  1 . 0 ml.  1 . 0 ml.  1 . 0 ml.  Griseofulvin in 50% ethanol  0 . 0 5 ml.  -  0 . 0 5 ml.  Substrate (M/50)  1 . 0 ml.  -  Phosphate 2 . 0 ml. buffer (M/15, pH 7 . 0 )  Distilled water 2 . 0 ' ml.  1 . 0 ml.  (1/10,000  -  (2,000  Ugm/mL) Ethanol  0 . 0 5 ml.  -  0 . 0 5 ml.  -  0 . 0 5 ml.  Total  6 . 0 5 ml.  6 . 0 5 ml.  6 . 0 5 ml.  6 . 0 5 ml.  6 . 0 5 ml.  50%  The enzyme source was placed in the side arm, the remainder of the contents i n the tube.  The ground glass joint of the  Thunberg tube was sealed with "vaspar" (1 part vaseline: 1 part paraffin wax).  The evacuation outlet was connected to a  vacuum pump and air was removed from the tube. continued for three minutes.  Evacuation was  During this time the tube was  tapped to release dissolved gases.  The effects of "bumping"  of the solutions as gases were released, were kept to a minimum by t i l t i n g the tube.  When evacuation was complete, the  tube was sealed by turning the cap. The sealed tubes were placed i n a water bath at 30°C for ten minutes, to come to temperature.  Each tube was then  tipped, mixing the enzyme with the other contents  0  The exact  time of enzyme addition was noted, and then the time required for reduction of methylene blue was measured.  The end point  was taken as a faint blue solution rather than a colourless one. Tubes were shaken frequently to reduce errors due to precipitation of c e l l material.  41 VIII.  Amino Compound Studies A large amount of c e l l material from Sabouraud's cerelose  broth was washed several times with sterile 0.65% saline using aseptic precautions.  This c e l l material was resuspended i n two  200 ml. volumes of a synthetic medium, one of which contained 25 ugm. of griseofulvin per ml.  Each suspension was then  divided into four equal volumes which were transferred to sterile 500 ml. Erlenmeyer flasks.  One flask of each series  was immediately removed for analysis as a zero time control. A 50 mlo portion of each medium, containing no c e l l material, was also analyzed as a control.  The remaining six flasks were  grown i n shake culture at room temperature.  One flask from  each series was removed for analysis at two days, four days, and six days. The supernatant solutions of the previously mentioned samples were analyzed by the following method.  The contents of  a flask were transferred to a 50 ml. centrifuge tube and centrifuged at 2,000 rpm for thirty minutes at 4°C. The supernate was decanted into a small beaker and an equal volume of 95% ethanol was added. • This mixture was then placed i n a stream of warm a i r and evaporated to a volume of 5*0 ml.  This concentrate was  centrifuged at 2,500 rpm for fifteen minutes at 4°C, to remove denatured protein.  The supernate was then evaporated to dryness  and resuspended i n 1.0 ml. of d i s t i l l e d water. The concentrated sample was passed through a Dowex 50 ionized resin column to remove anions which interfere with paper chromatographic separation of amino compounds.  The column was  6.0 em. long by 0.8" cm. in diameter.  Before addition of the  sample, the resin was placed i n the hydrogen ion form by washing i t with 50 ml, of 1,0 N HC1.  It was then rinsed with freshly  d i s t i l l e d water until no chloride ions could be detected, using a silver nitrate solution as an indicator. After addition of the concentrated amino acid sample to the top of the Dowex 50 H column, anions and replaced H ions +  were washed from the column with 50 ml. of freshly d i s t i l l e d water.  Next, cations were eluted from the column by the  passage of 50 ml. of freshly prepared 1.0 N HC1.  The HC1  eluate was collected and evaporated to dryness i n a stream of warm a i r .  When residual HC1 had been removed by evaporation,  the sample was resuspended i n 1.0 ml. of d i s t i l l e d water. Volumes of 0.05 m l , , 0.1 ml., and 0,2 ml. were spotted onto chromatography paper for separation and identification of amino compounds.  IX.  Quantitative Assay for Hexosamines There are several assays for hexosamines and more speci-  f i c a l l y for glucosamine.  Most of these assays depend on the  acetylation of glucosamine so that N-acetyl glucosamine w i l l also be measured.  The method used i n this study was one  employing Ehrlich's reagent, and i s the technique described i n 1953 by Boas (11). Glucosamine or N-acetyl glucosamine was produced either by acid or enzyme hydrolysis of chitin.  Acid hydrolysis was  carried out i n 2.0 N HC1 for eighteen hours at 100°C.  Higher  concentrations of acid result i n breakdown of glucosamine. Breakdown of N-acetyl glucosamine to glucosamine and acetic acid does occur but the glucosamine i s reacetylated during the assay so that this breakdown does not matter. hydrolysis produces N-acetyl glucosamine.  Enzymic  This enzymic reac-  tion was stopped by using 12% trichloracetic acid (TCA) to precipitate protein. Both samples were diluted with d i s t i l l e d water and then small volumes were applied to Dowex 50 columns.  These columns  had previously been prepared by the addition of 10 ml. of 1.0 N NaOH, followed by 10 ml. of freshly boiled d i s t i l l e d water. Next, 12 m l o of 1.0 N HC1 was passed through each column. Finally, freshly d i s t i l l e d water was passed through the columns until no chloride ions could be detected, using silver nitrate as an indicator. After the amines had been applied to the columns,  undesired anions were removed by the addition of 10 ml* of dist i l l e d water.  Amines were then eluted with 5.0 ml. of 2.0 N HC1.  Usually, 1.0 ml. of this eluate was assayed for glucosamine content.  If the content was low., a larger sample could then be  used* An example of a typical glucosamine assay now w i l l be provided.  1.0 ml. of a Dowex 50 eluate, 1.0 ml. each of three  glucosamine standards (containing 5.0 ugm., 10 ugm., and 20 ugm. per ml.), and 1.0 ml. of d i s t i l l e d water were each placed i n separate graduated test tubes* of phenolphthalein  (0.5%).  To each tube was added a drop  4.0 N NaOH was titrated into each  tube u n t i l the indicator turned red.  The amount of base added  to each tube had been noted, and the difference between each tube and that requiring the most base was calculated.  As the  assay values are influenced by salt concentrations, a l l differences i n volume of a l k a l i added were made up by the addition of that volume of 4.0 N NaCl.  A l l samples were back-  titrated to a colourless end-point, with 0.5 N HC1*  The volumes  i n a l l test tubes were brought to 5.0 ml. with d i s t i l l e d water. 1.0 ml. of acetyl acetone reagent was added to each tube. A l l tubes were stoppered and placed i n a water bath at 89° to 92°C for forty-five minutes. When acetylation was completed, the samples were cooled and 2.5 ml. of 95% ethanol was added to each tube.  Next, 1.0  ml. of Ehrlich's reagent was added to each, mixed well, and then the volume brought to 10 ml. with 95% ethanol.  These mixtures  were allowed to stand for one hour, and then were read at 530 mu  45 o n a B a u s c h a n d Lomb " S p e c t r o n i c The d i s t i l l e d standards the  s a m p l e was  were  used t o  plot  hexosamine  content  of  curve. the  water  test  The  samples were  sample  samine o r N - a c e t y l values*  a  each  unknown was as  were  a blank*  The  (FIGURE 3),  and  determined from  glucosamine  standards.  glucosamine  used as  standard curve  measured  and i n the  spectrophotometer.  20"  The w e i g h t s  calculated  both  HC1,  from  of  this i n  gluco-  these  46  FIGURE 3 Standard Glucosamine-HCI 1.4  Curve  1  IO  20  Glucosamine-HCI (/igm.)  30  40  50  47 X.  Spectrophotometric Studies on the Effects of Griseofulvin on Protoplast Membranes In 1962, Stephen Kinsky reported the use of the spectro-  photometer i n measuring changes i n size of Neurospora protoplasts (43)»  The method used i n this study was essentially the same as  that described by Kinsky. Protoplasts of dermatophytes were prepared by the previously described method©  After centrifuging they were  resuspended in 2.8 ml, of the protoplast preparation medium (containing snail digestive enzyme, buffer, sucrose, and p e n i c i l l i n ) •  glutathione  This medium had been previously clarified by  passage through a 0,45 M millipore f i l t e r . The protoplast suspension was transferred to a cuvette and placed i n a Beckman DU spectrophotometer. measured at 600 mu for several minutes.  Absorbance was  2.8 ml. of the  preparation medium was used as a blank. After these i n i t i a l readings, which were to insure that no alterations i n absorbance were occurring, 0.2 ml. of N,Ndimethyl formamide (DMF), containing 600 ugm. of griseofulvin per ml., was added.  The addition of the griseofulvin was made very  slowly with constant s t i r r i n g , to minimize breakage of protoplasts.  A similar 0.2 ml. of griseofulvin solution was added  to the blank cuvette. taken every two minutes.  Readings of absorbance at 600 mu were A control, containing protoplasts and  DMF but no griseofulvin, was also measured.  43 XI.  Visual and Spectrophotometry Studies on Cell Wall Synthesis by Dermatophyte Protoplasts When fungal protoplasts are resuspended i n the previously  described preparatory medium, from which snail digestive enzyme has been deleted, they soon begin to resynthesize c e l l wall. Two methods were used to investigate this process, visual observations and spectrophotometry measurements. Protoplasts were prepared as previously described.  After  centrifuging, the pellet was resuspended in 0.066 M phosphate buffer pH 6.3, containing sucrose ( 2 0 0 mgm/ml.) and p e n i c i l l i n (40 International Units / m l . ) . centrifuged at  7,700XG  This suspension was again  for ten minutes at 0°C, and the washed  pellet was resuspended i n 7.2 ml. of the washing medium.  2 . 3 mL,  was transferred to each of two cuvettes, and 0.7 ml. was placed i n each of two test tubes.  To one of the cuvettes, 0.2 ml. of  griseofulvin i n N,N-dimethyl formamide (DMF) was slowly added with s t i r r i n g .  The concentration of griseofulvin i n the  solution was 600 ngm. per ml. DMF.  Similarly, 0.05 ml. of  griseofulvin i n DMF was added to one test tube.  Next, 0.2 ml.  of pure DMF was added to the other cuvette and 0.05 ml. was added to the other test tube. The cuvettes were placed i n the Beckman DU spectrophotometer and their absorbances measured at 600 mu over a period of seven hours.  A volume of the solution i n which the protoplasts  were suspended was used as a blank. The samples i n the test tubes were used for the visual  49 observations of sample was a cover s l i p .  utilizing placed  on a c l e a n dry s l i d e and  While observing  a drop of d i s t i l l e d slip.  phase c o n t r a s t microscopy.  water was  due  then covered with  the p r o t o p l a s t s m i c r o s c o p i c a l l y ,  added t o one  P r o t o p l a s t s were t e s t e d i n t h i s way  would rupture  A small drop  t o osmotic shock.  edge o f the to see  cover  i f they  50  XII.  Formation of Griseofulvin Resistant Mutants The following methods were used as reported in a paper  published by Aytoun, et a l . i n I960 (4).  The mutants  eventually employed for further study were obtained using a selection method referred to as a "layer plate method". This procedure consisted of layering a Petri plate with 5.0 m. of Sabouraud s eerelose agar. f  Before solidification,  this agar was seeded with 1.0 ml. of a heavy suspension of dermatophyte spores or hyphal fragments.^  When the agar had  solidified, i t was dried for several hours at 37°Co  A second  thicker layer (10 ml.) of Sabouraud's agar, containing a known concentration of griseofulvin now was added.  The mycelia  which f i r s t appeared on the surface of the upper agar layer were transferred to slopes containing the same concentration of griseofulvin as was present i n the agar layer from which they were isolated.  These isolates were used as a source of ino-  cula for plates containing higher concentrations of griseofulvin. An attempt was made to increase the mutation rate by irradiation with ultra-violet l i g h t .  This was done by taking  the seeded, f i r s t layer of agar and placing i t one foot below a General Electric 15 watt, germicidal, ultra-violet lamp for forty seconds.  The griseofulvin agar layer was added as before.  The results obtained using this additional step were no better than without i t . A second selective method was also employed.  This gave  a quick, easy method for spot checks on the level of drug  resistance of strains, but was not as useful in the original isolation of variants.  It consisted of layering Sabouraud's  agar, containing a known concentration of griseofulvin, i n a Petri plate, and then inoculating i t s center.  Growth away from  the point of the inoculation usually was f a i r l y uniform.  If,  though, one part of the periphery grew faster than the remainder, this edge was assumed to contain resistant hyphal elements and was removed.  The rate of growth away from the point of inocu-  lation also indicated the inhibitory effect of the concentration of griseofulvin present, on the organism tested.  This was done  by comparison with the growth rate on a plate containing no griseofulvin.  52  XIII.  Cell Wall Preparation and Purification The method of c e l l wall preparation used i n this study  was based on the method reported by Carlson and Knight i n 1962 (19), which was a modification of the method of Cummins and Harris (21.).;  Cell material was grown i n Sabouraud's cerelose  broth i n shake culture.  The mycelial pellets were collected on  a Buchner funnel under suction, and were washed three times with 100 ml. volumes of 0.35% NaCl.  Excess moisture was drawn  through the f i l t e r by negative pressure.  Next, wet, packed  c e l l material was transferred to a tared watch glass and weighed. This cellular material was placed in a Nossal disintegrator capsule with glass beads and phosphate buffer.  The  ratio used was 2.0 gm. of c e l l material, to 3.0 gm. of fine glass beads (Glass Homogenizing Beads, VirTis Company), to 6.0ml. 0.066 M phosphate buffer pH 7.0. sule was 13 ml.  The total volume of the cap-  The l i d was placed on the capsule and sealed  with a thin layer of s i l i c a grease.  To insure purity of the  sample, the capsule had been cleaned before use by shaking with a small volume of a warm, detergent solution i n the Nossal apparatus.  This wash was followed by several rinsings with tap  water and f i n a l l y d i s t i l l e d water. When the capsule had been sealed, i t was cooled i n the deep freeze u n t i l the contents reached a temperature of approximately 0°C.  This took a period of about ten minutes.  It was  then placed in the Nossal apparatus and shaken for a total of fifteen minutes.  This was done by a series of one and one-half  53i minute bursts with cooling periods of ten minutes between each. The Nossal apparatus (McDonald Engineering Company, Cleveland, Ohio) reciprocates at 12,000 complete cycles per minute.  The  capsule is constantly cooled during operation by the release of carbon dioxide under pressure, through a nozzle. At the end of the fifteen minutes of shaking, the capsule contents were transferred to a centrifuge cup and sedimented at 8,000 XG for fifteen minutes at O^C.  The supernate was d i s -  carded and the pellet of beads and impure c e l l wall material was transferred to a 100 ml. beaker.  By successive decanting  and resuispension i n 0.05 M NaCl, the c e l l wall material was separated from the heavier glass beads.  The separated  material now was washed five times by centrifuging and resuspending i n 0.05 M NaCl.  After the last centrifuging, the pellet  was resuspended i n 10 ml. of 0.066 M phosphate buffer pH 7.6, containing trypsin (0.5 mgm/ml.) and ribonuclease (0.5 mgm/ml.). This digestion mixture was placed i n a 37°C water bath and allowed to stand, with occasional mixing, for four hours. At the end of the digestion period, the remaining particulate material was centrifuged down at 8,000 XG; for thirty minutes at 0°C.  The pellet was then washed five times with 0.05 M NaCl and  once with d i s t i l l e d water, by centrifuging and resuspending. The remaining pellet was dried over anhydrous  CaCl2,  under negative  pressure i n a vacuum desiccator, at room temperature.  The dried  pellet of purified c e l l wall material then was ground with a small mortar and pestle to allow more accurate weighing of samples*  This powder was stored i n a sealed v i a l i n the  refrigerator, for later analysis.  54  XIV,  Cell Wall Analysis The purified c e l l wall material was qualitatively  analyzed for amino acids, amino sugars, and sugars.  To obtain  these small molecules i t was necessary to f i r s t hydrolyze the large polymers of which they were components.  This was  accomplished by placing 10 mgm. of c e l l wall powder i n a glass ampoule, adding 2.0 ml, of 2.0 N HC1, and sealing the ampoule by heating.  This ampoule was then placed in an oven at 100°C  for eighteen hours, after which time i t was cooled and opened. Next, i t was centrifuged at 2,000 rpm for ten minutes at room temperature and the supernate removed and stored.  The pellet  was resuspended i n 2.0 ml. of 6.0 N HC1, and the ampoule was resealed and heated for a further twelve hours at 100.9c,  This  ampoule was again opened, centrifuged, and the supernate removed and stored*  The pellet was extracted with 2.0 ml, of butyl  acetate and then discarded.  This pellet is believed to have  contained metal or metallic salts resulting from contamination by the Nossal capsule during disintegration. The 2.0 N and 6.0 N HC1 hydrolysates were evaporated to dryness with a stream of warm a i r , thus driving off excess HC1, The dried residues were each resuspended i n 1.0 ml. of dist i l l e d water.  The butyl acetate extract was similarly  evaporated and then resuspended i n 1.0 ml. of butyl acetate. These samples then were analyzed using paper chromatographic techniques.  55  EXPERIMENTAL I.  Oxygen Uptake Studies A o Preliminary Investigations Preliminary experiments into the effects of griseo-  fulvin on dermatophytes were involved with oxygen uptake for several reasons.  The f i r s t was that there were conflicting  results i n this area (15,44,63).  The second reason was that  other workers had shown that nitrogen and phosphorus assimilation by c e l l s , were decreased in the presence of the fungistat (28,29)•  Such results could indicate that oxi-  dative phosphorylation was being inhibited.  The third  reason was that previous work i n this laboratory had indicated that dermatophytes have extremely large quantities of endogenous reserves (67a).  The resulting high rate of  endogenous respiration could be lowered by "starving" the cells in the presence of phosphate buffer, thereby depleting the endogenous reserves and lowering the respiratory rate. Without f i r s t starving this c e l l material, oxidation of substrates could not be demonstrated by oxygen uptake methods. It should be mentioned here that Nickerson and Chadwick (52) report the oxidation of substrates by dermatophytes without involving starvation.  The methods reported by these authors  did not seem suitable in this study.  It was the intention of  this investigator then, to repeat the former experiments and to compare these results with those obtained with starved c e l l material.  The form of growth used throughout these experiments was the fungal pellet which results from shake culture methods.  The  morphology of this form of growth i s in many ways similar to iifycelial mat growth ( 7 2 ) , but there are certain differences. The reasons for choosing growth obtained using the shake culture cultivation method for this study, were the shorter incubation time and the greater ease of handling. A variety of methods of measuring out constant amounts of c e l l material were investigated.  These were, separating  pellets by size as described by Roth, et a l . ( 6 3 ) , weighing wet, packed c e l l material, and f i n a l l y homogenizing for quantitative transfer by pipette. highly unreliable.  The f i r s t two methods were found to be The results obtained from duplicate  Warburg cups were similar only by chance.  Errors were perhaps  due to variations within the hyphae and possibly to variations in the handling techniques.  Homogenized mycelial preparations  did not give the former problems i f the mycelia were broken sufficiently to give hyphal fragments of approximately four c e l l units i n length. bacterial preparations.  This material is more analogous to Unlike bacteria though, the large hy-  phae have the property of sedimenting faster when left standing. To offset this, the mycelial homogenate was constantly agitated when being pipetted into the Warburg vessels. ions were carried out using duplicate cups.  A l l determinatThe experimental  data was plotted by averaging duplicate cup values.  All  «• experiments were repeated once or twice and compared on the basis of the dry weight of c e l l material.  57 Before carrying out any oxygen uptake investigations, i t was necessary to determine the concentration of griseofulvin which would inhibit the growth of the organism under study,  A single organism, Microsporum quinckeanum strain #8  (MQ$), was used for a l l i n i t i a l investigations because i t had the shortest growth and starvation periods (67a),  This  organism was inoculated onto Sabouraud s cerelose agar plates, f  containing known concentrations of griseofulvin.  It was  found that 4 , 0 ugm. of griseofulvin per ml, of medium would completely inhibit the growth of MQ8.  Thus i t was decided  that 4.0 ugm./ml, would be the concentration used i n the Warburg cups, B. Whole Cell Studies The f i r s t experiments were carried out on unstarved, whole pellets as described by Roth, et a l , ( 6 3 ) . are given i n FIGURE 4 .  The results  The decrease i n oxygen uptake i n the  presence of glucose, agrees with the findings of Roth, et a l . , but their reported finding of decreased values i n the presence of griseofulvin was not noted.  Because these authors had  used a higher concentration of griseofulvin (10 ugm./ml. i n the flask), i t was decided to test the effects of increasing concentrations of this drug, on c e l l respiration.  Difficulties  in handling pellets led to the decision to employ washed, homogenized  suspensions.  Three concentrations of griseofulvin (£.0ugm./ml., 16 ugm./ml., and 32 jugm./ml.) were tested on three cellular  FIGURE 4 Oxygen Uptake by U n s t a r v e d MQ 8  Pellets  Endogenous  [Glucose ( 2 . 0 u M )  E n d o g e n o u s + G r i s . (4.0ugm./ml.)  |Glucose+ Gris.  59 preparations (unstarved c e l l material, c e l l material starved for three days, and material starved for five days).  Since the  drug was dissolved i n 50% ethanol, a duplicate series containing ethanol but no griseofulvin, was also tested in each case.  The  final concentrations of ethanol i n each series were: 0.2%, 0,4% and 0.3%.  Both of these test series were compared to the  values from reaction vessels which contained no ethanol or griseofulvin.  The increasing ethanol and griseofulvin concen-  trations caused no differences within each series, but v a r i ations were noted between cultures of different starvation periods*  As an example, representative results for c e l l  material which had been starved for five days are shown in FIGURES 5 and 6. Several conclusions were drawn at this point*  First,  the concentrations of ethanol up to 0.3% were not inhibitory to c e l l respiration.  Second, the presence of griseofulvin did  cause differences in oxygen uptake values of c e l l material which had been starved for various lengths of time, but l i t t l e or no alterations resulted from increasing drug concentrations. Finally, the changes i n oxygen uptake values were different for each starvation time, as shown in FIGURES 7, 9, and 11* At this time i t was felt necessary to determine whether griseofulvin was present i n the same concentrations at the end of a run as i t had been at the start.  This was accomplished  by extracting the contents of each Warburg cup with butyl acetate and measuring the absorbance of the extract at 239.5 mu  (see Methods). was observed.  No alteration in griseofulvin concentration This meant that the drug was not being destroyed  by the cells during the course of the experiment. A study was now undertaken to compare the oxygen uptake values of c e l l s which had been starved for various lengths of time*  The purpose was to determine whether there was any  correlation between starvation time and the effects of griseofulvin on endogenous respiration or glucose oxidation.  To  insure that sufficient griseofulvin was present, with the concentrated c e l l homogenate, the concentration was raised to 16 ugm./ml.  Typical results from this study are shown in FIGURES  7 to 11.  These results indicate that griseofulvin had a very  small inhibitory effect on endogenous respiration, decreasing gradually throughout the starvation period.  An effect was  also observed with glucose oxidation values.  Initially,  oxygen uptake due to glucose oxidation was decreased, but with continued starvation, a greater percentage of the glucose was oxidized i n the presence of the drug*  This continued a l l  through the starvation period, until after four days more glucose was oxidized i n the presence of griseofulvin than i n i t s absence*  As an example, the percent alterations of total  glucose and endogenous oxidation values (from FIGURES 7 to 11) are shown graphically i n FIGURE 12. against timer  Three values are plotted  the percent reduction due to griseofulvin, of  the total endogenous oxygen uptake; the percent reduction or increase due to griseofulvin, of the total oxygen uptake resulting from glucose oxidation, and the percent of total  theoretical oxygen uptake due to glucose oxidation (assuming that 134.4 M l . of oxygen would be taken up by the total oxidation of 1.0 uM of glucose)•  The calculations used to  determine these values, are given in the Appendix. These experiments were repeated with Microsporum quinckeanum strain #7, Trichophyton asteroides, and Trichophyton mentagrophytes strain #666.  A similar pattern was observed i n a l l cases,  although the starvation times were longer with these organisms. A question arose at this point in the study, as to whether glucose oxidation was being inhibited at the start of the starvation period or whether glucose was simply not being taken up by the c e l l s .  To answer this question, Warburg cups  were removed from the shaker at the end of the run, and their contents extracted with 1.0-N HC1 for ten minutes at 80°C. Denatured protein was removed by centrifuging for ten minutes at S,000XG.  The supernates were dried, resuspended in dis-  t i l l e d water, and spotted onto paper chromatograms for sugar analysis.  No residual glucose was found, thus indicating that  i t was being taken up and utilized by the c e l l s . The interpretation of these results i s not conclusive. Oxidation values, both for endogenous and for glucose, do indicate an effect of griseofulvin on respiratory pathways. The continuous low inhibitory effect observed with endogenous metabolism suggests that perhaps the site of action of this drug i s i n a normal synthesis pathway.  This might mean that  at least part of this pathway was reversed during endogenous respiration.  Another possibility is that the resynthesis of  62 endogenous substrates i s being blocked.  The latter explanation  seems possible as i t would result i n only a small observable reduction i n comparison to total endogenous respiration.  This  hypothesis i s also supported by the glucose oxidation results* Assuming that as the endogenous reserves are depleted, the requirements of the cells for a source of energy increase, the addition of a substrate such as glucose, in the presence of an inhibitor of synthesis, should result i n increased oxygen uptake values.  Such an increase was observed i n this study.  The  actual site of action of griseofulvin cannot be determined as yet, but from these results i t may be related to the enzymes involved in synthesis, to the cofactors associated with them, or to the energy-transferring systems of the c e l l s . C. Cell-free Extract Studies In an attempt to learn more about the alterations i n oxidation values caused by griseofulvin, cell-free extracts were tested by manometric methods.  Several substrates  (glucose, fructose-1, 6-diphosphate, glueose-l-phosphate, glucose-6-phosphate, and pyruvate) were u t i l i z e d , but no oxygen uptake could be measured using these preparations.  An effort  was made to replace cofactors by adding residual c e l l material which had been heated to 80°C for ten minutes, but this attempt also was unsuccessful.  Because of the lack of results with  manometric methods using cell-free extracts, i t was decided to employ Thunberg techniques to follow dehydrogenase reactions.  FIGURE 5 Effect of Increasing Ethanol Concentrations on Oxygen Uptake by M Q 8 Starved  for  5  Days  6001  i  Time  (hours)  Endogenous Endog. + ethanol(0.2 % ) Endog. + e t h a n o l ( 0 . 4 % ) Endog.+ ethanol ( 0 . 8 % )  G l u c o s e (4.0uM)  _  _  (Slucose 4-ethanol ( 0 . 2 % ) felucose + ethanol(0.4 % ) LGIucos e + ethanol (0.8 °/o)  FIGURE 6 Oxygen Uptake by MQ8 Cecils Starved for 5 Days (Homogenized) Effects of Increasing (SriseofuIvin Concentrations 6001  I  2  3  4  5  6  Time (hours) Endog. 8.0ugm/ml. Gris. Endog.+ I 6 ugm/ml. Gris. and 32 jjgm/ml. +  — -  Glucose + 6,0ugm/ml. Gris. Glucose 1.6ugm/ml. Grls. Glucose + 32 ugm/ml. Grls. (4.0pM) +  .65  FIGURE 7 Oxygen Uptake by Unstarved MQ8 Cells (Homogenized)  Time Endogenous  Endogenous  + Griseof ulvin  (hours) Glucose  Glucose  (2.0>JM.)  -FG r i s e o f u l v i n  66  FIGUR E 8 Oxygen Uptake by MQ8 Cells Starved for 2 1/2 Days (Homogenized)  1  2  3  4  5  6  T i m e (hours) -Endogenous _ Endogenous + Griseofulvin  Glucose (2.0uM) Glucose + Griseofulvin  7  FIGURE 9 Oxygen Uptake by MQ8 Cells Starved for 3 Days (Homogenized)  500-1  T i m e (hours) Endogenous Endogenous + 6riseofu Ivin  Glucose (2.0uM) Glucose + G r i s e o f ulvin  F I G U R E 10 Oxygen Uptake by MQ8 Cells 3501  Starved for 4 Days (Homogenized)  3 0 0 1  2501  a>  2 0 0 H  3  <? " 5 0  I00H  5 0 1  Time (hours) Endogenous Endog enous + G riseofulvin  Glucose (2.0uM) Glucose + Griseofulvin  68  FIGURE  69  II  Oxygen Uptake by MQ8 Cells Starved for 5 Days (Homogenized)  I  2  4  3  5  6  Time (hours) Endogenous Endogenous  + Griseofulvin  Glucose (4.0 uM.) ."  Glucose  + Griseofulvin  FIGURE  70  12  Effect of Griseofulvin on the Total Endogenous and Glucose Oxidation Values  30'  00  M 3 O  c  4) o>  O TJ C UJ  Ho  20^  s:» -•2.  0  M O 0> tO  3 O O TO X O  10  H30  H  o ^ o 3  c o o  0> (0  3 O  . C o  • o> . o •o c til  101  30 <D Q. C  h30  oS  * a  o s  3  O  §& r.  u  _ _  20<  60  3 TJ fl>  o o  -  (0 <0  30'  90 -Endogenous (left  scale)  Glucose (right  scale)  " % of total t h e o r e t i c a l o x i d a t i o n of Glucose (right scale)  D.  Dehydrogenase  The f i r s t whole  cell  protocol  dehydrogenase  was t h a t  given  fructose-1,  glucose-6-phosphate, substrates,  the  It  this  control  c a r r i e d out  on  four  The  substrates  employed.  With  of  substrate  presence  as  the  s u b s t r a t e s were blue  remainder  d i d not  griseofulvin. enzyme  on t h i s it  reaction.  The  reduction  was  was d e c i d e d t o  metabolism for  griseofulvin  for  substrates  as  The p r e s e n c e As r e s u l t s  source,  tested.  two h o u r s .  autoxidation of pyruvate,  become r e d u c e d .  satisfactory,  o f the  to  No a p p r e c i a b l e  o f added  t h e n were used  ten  the  required approximately t h i r t y minutes  was not  these  methylene blue w i t h i n  allowed methylene  the  days.  glucose-l-phosphate,  were  showed no r e d u c t i o n a f t e r  h a d no e f f e c t  cellular  Several  absence  i n the  extracts  reduction, while  endogenous  not  i n the  p r e v i o u s l y mentioned  pyruvate.  that  reduced the  was o b s e r v e d  substrate which  plete  and p y r u v a t e )  while  Cell-free  only  i n TABLE I I I .  for  t i m e was o v e r t w e n t y - f i v e m i n u t e s .  difference  and the  starved  6-diphosphate,  cells  minutes,  reduction  experiments were  m a t e r i a l , w h i c h had been  (glucose,  fifteen  Studies  It the  of  effects.  and  was  the  shown  substrate griseofulvin  in this  investigate  com-  area  other  were  areas  of  72 II.  A m i n o Compound The  effects  Studies  of  griseofulvin  Electron mierographic tions  in cell  wall  concentration  of  sidered  possible  excreted  by the  fulvin. having the  studies  structure  lipid that  nitrogen  and a l s o  in addition,  the  source  The methods  in this  source  (see  of  described  of  The  study  Materials).  it  was  seen  in a l l  when t h e  contents  cultures  containing  increase  i n weight  though,  possible  that  flasks. of  last  hyphae  showed  i n fact,  after  of  a  organism  basal  slight  con-  be griseo-  i n  nature, as  studied  of  amino  was  day  a  first  also  the  was w h e t h e r  any  From d a i l y  day t o  the  analyzed,  medium showed  Those  flasks  after  the  decrease  of  s m a l l amount  were  compounds  Observations  course,  flasks  no g r o w t h  that  The  section.  one  0  of  synthetic  in  was  might  presence  medium e m p l o y e d .  o n l y the of  It  compounds  was  altera-  an increase  cell.  and a n a l y s i s  From t h e  the  show  and d u r i n g e x t r a c t i o n  question  i n the  occurred  fulvin  growth  (10)  widespread.  (MQ$).  i n a previous  first  growth would occur vations  amino  very  c a r b o n a n d ammonium s u l p h a t e  extraction  w e r e made d u r i n g c e l l supernates.  the  o r g a n i s m when grown i n t h e  as  be  indicate  inside  M i c r o s p o r u m q u i n c k e a n u m s t r a i n #6*  have been  to  by B l a n k et a l .  materials  The medium u s e d glucose  appear  a  obser-  of  growth  sixth the  control  continuous  containing  first  day.  in cell  day  griseoIt  was  weight  occurred. A second nates. remaining  After  fact  was  removing  soluble  noted cell  p r o t e i n was  during analysis  m a t e r i a l by  of  the  super-  centrifugation,  precipitated  by the  the  addition  of  ethanol and then heating.  A definite pattern was seen i n the  amount of protein precipitated i n each flask. cultures both gave very l i t t l e soluble protein.  The zero time The griseo-  fulvin culture at two days though, contained approximately three to four times the weight of protein as compared to the control*  At four days and six days, the protein content of  the controls increased steadily.  The griseofulvin cultures  showed no increase i n soluble protein over the amount which had been observed after two days.  The amount of soluble protein  in each of the cultures containing griseofulvin was i n a l l cases higher than that found i n any of the controls. The amino compound extracts for each day were run on one chromatogram.  Three different concentrations of each  sample were applied*  The zero time samples both showed the  presence of about the same quantity of amino compounds i n the highest concentration applied.  An equivalent increase was  observed i n a l l two-day old cultures*  The concentrations of  amino compounds continued to increase in the control flasks for the remaining four days.  The concentrations i n the  cultures containing griseofulvin did not increase after two days. The qualitative assay of the amino compounds showed no differences between the cells grown i n the presence or absence of griseofulvin.  The following spots were recorded and  tentatively identified by their Rf values: lysine, arginine, histidine, aspartic acid, glycine (and/or serine), hydroxyproline, glutamic acid, alanine, proline, methionine, valine,  74 phenylalanine and leucine (and/or isoleucine). cystine and glucosamine were also detected.  Traces of Both media con-  taining no c e l l material were found to be free of ninhydrinreacting compounds.  Thus a l l spots appearing on the  chromatograms originated from the c e l l material, A partial interpretation of these results was attempted* F i r s t , the controls gave results which would be expected of an average culture growing slowly i n a simple medium.  The  amounts of soluble protein and amino acids i n the supernate slowly increased over the whole growth period.  The medium  containing griseofulvin, on the other hand, yielded what seemed to indicate a dead or non-metabolizing culture.  The lack of  growth and of amino acid release after one or two days would support this.  The great increase i n soluble protein which  was observed after two days, i n a l l cultures containing griseofulvin, might indicate auto-degradation and breakage of the cells. The lack of qualitative difference in the amino compounds of the supernate does not conclusively prove that amino acid metabolism i s not affected, as the simpler amino acids have many possible interlocking pathways.  It may indicate though,  that formation of the more complex amino acids i s not inhibited, and that the growth inhibition i s not due to amino acid metabolism being blocked.  75 III.  Chitinase Studies Using Whole Cells The investigation into the effects of chitinase on the  c e l l walls of dermatophytes was initiated for two reasons. F i r s t , the thick c e l l walls possessed by these organisms- made i t very d i f f i c u l t to obtain c e l l free extracts.  It was felt  that the method being used might be destroying many cellular systems, and that by f i r s t weakening the c e l l walls breakage could be caused more easily.  The second reason was to deter-  mine whether the formation of protoplasts might be possible with dermatophytes as i t was with Neurospora species (5). Two 500 ml, Erlenmeyer flasks, each containing 50 ml, of Sabouraud's cerelose medium, were inoculated with a fine suspension of Microsporum quinckeanum strain #8 (MQ8) mycelia. These cultures were grown for six days i n shake culture, at room temperature.  The resulting fine pellets were collected  on Whatman No, 1 f i l t e r paper on a Buchner f i l t e r .  The  pellets were next washed on the f i l t e r , with 500 ml. of 0,85% NaCl,  The washed c e l l material was then resuspended i n 10 ml.  of 0.033 M phosphate buffer pH 6.8.  This suspension then was  treated as described i n TABLE III. The sample used for dry weight determinations was weighed i n comparison to 1.0 ml. of 0.033 M phosphate buffer ph 6.8.  The weight of the buffer was subtracted from the  weight of the c e l l suspension to give the weight of the c e l l material.  The results are given i n TABLE IV.  • The samples of c e l l material were hydrolyzed with acid  i n order to determine the total amount of N-acetyl glucosamine which might be expected from enzymic hydrolysis of the c h i t i n . Several hydrolysis periods were used to determine the optimum conditions for hexosamine measurement. tests may be seen i n TABLE V.  The results of these  77  TABLE III Procedure for Chitinase Studies Using Whole Cell Material MQ8 CELL SUSPENSION (10 ml.) i -j— -j~ j * ; 7 Cell Susp. Cell Susp. Cell Susp. Cell Susp. Cell Susp. Cell Susp. (1.0 ml.) (1.0 ml.) (1.0 ml.) (1.0 ml.) (1.0 ml.) (5.0 ml.) + + -t+ + Dry Weight 3.0 ml. 50 mg.Chiti2.0 ml. 2.0 ml. Determination 3.0 N HC1 3.0 N Hd 2.0 ml. 2.0 NHd nase* i n 15 3.0 NHCL ± ml. of 0.2M Hydrolysis Hydrolysis Hydrolysis Hydrolysis phosphate at 10QOC at 100°C at 100°C citrate at 100OC for 14 hr. for 17 hr. for 20 hr. for 33 hr. buffer pH >5.8 Y Dilute each to 10 ml. with d i s t i l l e d water Centrifuge  I  Hexosamine assay  ^  _  0.5 ml. 1.0 ml. samples for samples microscopic 1.0 ml. observations of 12% TCA I Each sample was diluted to 5.0 ml. with dist. water Dowex 50 columns 2.0 N HC1 eluate (5.0 ml.)  2.0 ml. eluate  3.0 ml.eluate  Hydrolysis at 100°C for 18 hr  Hexosamine assay  Dilute to 10 ml. Centrifuge Hexosamine assay (*The chitinase was a purified preparation isolated from the intestine of the snail Helix pomatia. It was kindly supplied by Dr. G. Strasdine, National Research Council, Ottawa, Canada)•  78 TABLE IV Dry Weight Determination Sample  Cup Weight Cup Sample Weight  Sample Weight  Cell Material  1.3652 gm. 1.3728 gm.  0.0076 gm. 0.0026 gm. 2.6 mgm.  Buffer (0.033 M, 1.3728 gm. 1.3778 gm. pH 6.8)  Minus Buffer  0.0050 gm.  Weight in mgm.  -  TABLE V Glucosamine-HCl Content of Hydrolyzed Cell Samples Hydrolysis Absorbance Glicosamine Glucosamine Final 530 mu - HCl (ugm.) -HCl (ugm.) Normality Period in 1.0 ml. c e l l susp.  % Weight Glucosamine -HCl i n c e l l susp.  2N  14 hours  0.055  22.9 ugm.  229 ugm.  8.8%  2N  17 hours  0.058  24.2 ugm.  242 ugm.  9.3%  2N  20 hours  0.050  20.8 ugm.  208 ugm.  8.0%  1.5 N  33 hours  0.057  23.8 ugm.  233 /igm.  9.2%  It was concluded from the similarity of these results to those obtained by Boas (51), that samples for hexosamine assay should be hydrolyzed at 100°C for eighteen hours.  When the  assayed weight of glucosamine-HCl is compared with the dry weight of c e l l material, i t can be seen that glucosamine-HCl would comprise between 9% and 10% of the total c e l l weight. The percentage weight using glucosamine-HCl i s , of course, an a r t i f i c i a l value, but the standards used were glucosamine-HCl. Using these values i t was easy to determine the weights of free glucosamine and free N-acetyl glucosamine on a molar basis.  79 The enzymic hydrolysis of chitin was carried out on a shaker with the flask suspended in a 30°C water bath.  Samples  for hexosamine assay and microscopic examination were taken at: zero time (time when chitinase was added), 15 min., 30 min., 60 min., 90 min., 1 2 0 min., and 150 min.  The hexosamine  samples were a l l taken i n duplicate. The microscopic examination was carried out using an ordinary light microscope.  This is not as satisfactory as a  phase contrast microscope, for observation of c e l l wall alteration.  It was noted though, that after ninety minutes  the tips of hyphae began to swell.  This was taken as an  indication that the enzyme was hydrolyzing at least part of the hyphal wall, although the older portions of the hyphae showed no observable effects. The samples for hexosamine assay were treated with 1 2 % trichloracetic acid (TCA) to stop the reaction and to remove the protein from solution.  The f i r s t step i n the assay was p u r i f i -  cation of the sample by passage through Dowex 5 0 H ionized resin. +  The presence of the amino group on the glucosamine causes i t to bind to the column.  The column was washed and then the glucos-  amine was eluted with 5.0 ml. of 2 . 0 N H C 1 .  3.0 ml. volumes of  the eluate were assayed for hexosamines, but none of the samples showed their presence. This result did not mean that there was no chitinase activity.  It was considered quite possible that the enzyme  was hydrolyzing the chitin into smaller units consisting of several N-acetyl sub-units.  Such units should be soluble,  80 although larger ones might not bind to the Dowex column. To try to test this hypothesis, some of the remaining 2.0 ml. volumes of the 2.0 N HCl eluate were sealed in ampoules and heated at 100°C for eighteen hours. zero time, 90 min., and 150 min.  The samples tested were: The values obtained for  these samples, when assayed for hexosamines, are given i n TABLE VI.  TABLE VI Glucosamine-HCl Content of Hydrolyzed Eluates Sample  Absorbance 530 mu  zero time  0  zero time  0.002  90 min.  0.003  90 min.  0.003  150 min.  0.005  150 min.  0.003  Average  Glucosamine -HCl i n sample (ugm.)  Glucosamine -HCl i n 1.0 ml. of c e l l susp.  % of Glucosamine -HCl by HCl hydrolysis  0.001  0.4 ugm.  4 Ugm.  1.7%  0.003  1.3 ugm.  13 ugm.  5.4%  0.004  1.7 ugm*  17 ugn.  7.3%  The results shown here indicate that a small amount of chitin was hydrolyzed, but that i t was only a small percentage of the amount obtained by acid hydrolysis.  These results are  not entirely conclusive though, as they are at the limit of the sensitivity of the assay. The results do coincide with microscopic observations.  If only the chitin at the hyphal tips i s affected then the percent hydrolysis would be extremely low.  The fact that only  growing tips showed any change, may mean that the chitin i n the c e l l wall i s shielded from the enzyme.  This is supported by  investigations carried out by Prince in I960 (55)•  He found  that the chitinase produced by a streptomycete failed to affect Trichophyton mentagrophytes.  He suggested that some-  thing i n or near the c e l l wall was acting as a shield.  Other  support is found i n the work of Horikoshi and Iida (33,39), who showed that Aspergillus oryzae cells were not altered by chitinase until they had been previously treated with p-1, 3-glucanase. Because of the problems involved in obtaining c e l l wall hydrolysis with purified chitinase preparations, i t was considered f r u i t f u l to investigate the effects of a mixture of enzymes on dermatophyte c e l l walls.  Some preliminary attempts  were made to isolate a bacterial strain which would attack the c e l l walls, but these were unsuccessful.  On further investi-  gation i t was found that the crude intestinal juices of Helix pomatia would cause at least partial weakening of Meurospora crassa c e l l walls ( 5 ) . A- short study was carried out similar to that just described, but with two variations.  F i r s t , crude intestinal  juice of Helix pomatia (L Industrie Biologique Franchise) was 1  utilized.  Secondly, an acetone-dried c e l l preparation was  used rather than whole c e l l s .  The results in this experiment  were very good, a yield of approximately 25% of the expected  82  total yield of glucosamine-HCl was obtained i n two hours.  This  result was obtained from the acid hydrolyzed sample after Dowex 50 separation.  The unhydrolyzed sample gave a yield of ap-  proximately 6% for the same time period.  At this time i t was  decided to make attempts to obtain protoplasts of dermatophytes for studies on the effects of griseofulvin on membrane permeability, and on c e l l wall resynthesis.  IV.  Preparation of Protoplasts A. Preliminary Investigation with Neurospora tetrasperma Before attempting to obtain protoplasts of dermato-  phytes i t was considered more .useful to carry out the procedure of Colvin (20) using Neurospora tetrasperma.  The hyphae of  this species are larger than those of Microsporum quinckeanum strain #8.  Thus protoplasts and structures are more easily  observed.  A l l microscopic examinations were carried out  with a phase contrast.microscope. The protoplast suspension was prepared as described i n a former section.  Samples were removed at fifteen minute  intervals during the incubation period and observed under the microscope.  The general pattern of protoplast extrusion may  be seen i n FIGURE 13.  This is similar to the photographs  shown by Bachmannand Bonner (5). these authors were also observed.  The "blebs" mentioned by These protoplasts were  from 10 to 30 u in diameter. The protoplasts were next purified, concentrated, and resuspended i n a 20% sucrose solution.  Small portions were  removed at thirty minute intervals and challenged with dist i l l e d water.  The results of this study are shown by diagram  i n TABLE VII.  The synthesis of c e l l wall i s seen after thirty  minutes, but the new structures remain fragile u n t i l four hours. The times of course are not absolute.  The observations are  those for about 80% of the protoplast population at the given time.  Upon osmotic shocking, "blebs" as described (5) were  released©  34 The use of Neurospora protoplasts to determine the effects of griseofulvin was now considered.  This was not  possible though, since Neurospora tetrasperma grew in the presence of 3.0 ugm. of griseofulvin per ml. with very slight inhibition.  This was i n the form of slower growth rather than  distortion of the hyphae.  It now became necessary to investi-  gate the possibility of forming protoplasts of ringworm fungi.  FIGURE 13 Diagramatic Representation of the Formation of Protoplasts of Neurospora tetrasperma  residual I cell Two to four  hours  wall  86 TABLE VII Cell Wall Regeneration by Neurospora tetrasperma Protoplasts in 20% Sucrose Time  Protoplast before challenge with d i s t i l l e d water  Protoplast after challenge with d i s t i l l e d water  Comments  Zero Time  In the presence of d i s t i l l e d water, the protoplast swells until the membrane ruptures, and the internal contents are released.  30 min. to 60 min.  A thin wall appears to form but when the c e l l is challenged, the inner protoplast emerges and bursts as before.  after 4 hours  after 12 hours  Q  '0,  r  At this time the challenge with water has no effect. The wall around the c e l l becomes quite r i g i d . After the c e l l wall formation was complete, the cells usually altered i n shape and appeared to grow. This growth did not last for very long, probably because of the lack of a nitrogen source i n the medium.  87 B. Investigations on the Formation of Dermatophyte Protoplasts The formation of protoplasts by the ringworm fungi, through the action of snail digestive enzymes, was considered extremely l i k e l y on the basis of the final results of the hexosamine assays.  This was further supported by work carried  out by Carlson and Knight on purified c e l l wall material of Trichophyton mentagrophytes (19).  They demonstrated the forma-  tion of glucose and N-acetyl glucosamine from the c e l l wall material i n the presence of "crude snail gut chitinase".  The  fact that the acetone-dried cells and purified c e l l wall preparations were attacked did not of course mean that living cells would be vulnerable.  The chitin and glucose polymer  layer could be protected by a glucosamine and peptide layer. Carlson and Knight reported such compounds when they acidhydrolyzed their chitinase resistant, non-dialyzable fraction. The f i r s t attempt to produce dermatophyte protoplasts was with a four day static culture of Microsporum quinckeanum strain #8  (MQ8).  lose broth.  The organism was grown i n Sabouraud s cere1  Colvin*s modification of the method of Bachmann  and Bonner was used.  The observation procedure was the same  as that used for Neurospora studies. The observations using MQ8 almost paralleled those for Neurospora species as described i n FIGURE 1 3 .  The only  variations were that the protoplasts were smaller (from 5 . 0 u to 2 0 u i n diameter) and that they took a shorter time to form. They started to appear after fifteen minutes and continued to  33 form for several hours.  The f i r s t ones formed seemed to come  from the hyphal t i p s , while those which formed later came from older sections of the hyphae. The procedure of resuspending in 20% sucrose and then challenging at thirty minute intervals with d i s t i l l e d water was also followed.  The results were similar to those of  Neurospora species as shown i n TABLE VII.  The "blebs" were  again observed after osmotic rupture. Because of the low yields of protoplasts, variations of Colvin s method were t r i e d . 1  It was f i n a l l y found that a  modification of Kinsky s method (43) gave the greatest numbers. T  Attempts were made to obtain even greater yields by lowering the sucrose concentration from 13% to as low as 10%.  This  provided more protoplasts, but when the sucrose concentration was brought back to 20% sucrose, they did not appear normal. The lowered osmotic strength of the solution causes swelling of the protoplast.  This aids i n i t s extrusion from the hyphae,  but i t also appears to cause rupture and stretching of internal structures.  Therefore, the concentration of sucrose f i n a l l y  used was 13%, It was felt at this time, that further confirmation of the identity of these structures as protoplasts was necessary. Through the kindness of Dr. W. Chase, Department of Pathology, U . B . C , electron micrographs of MQ3 protoplasts were obtained. The following i s a description of the method employed by Dr, Chase. Protoplasts used i n preparation for electron  89 micrographs were separated from the residual hyphal elements in the aforementioned manner, and concentrated by centrifugation. This material was fixed i n veronal-buffered OsG^, 1%, pH 7 . 4 , for twenty minutes.  The pellet was dehydrated in alcohol and  fragments were embedded in Maraglass, according to the treatment of Freeman and Spurlock ( 2 $ ) . Ultrathin sections were cut with glass knives on a Porter-Blum ultramicrotome and mounted on unsupported grids.  Preparations were stained with  lead hydroxide according to the method of Karnovsky ( 4 2 ) . Electron micrographs were taken with a Siemens electron microscope at accelerating voltages of 60 kv. micrographs are shown in FIGURE 14*  The resulting  90 FIGURE 14  E l e c t r o n Micrographs of MQ8 P r o t o p l a s t s  14 a .  P r o t o p l a s t showing cytoplasmic membrane (cm) and mitochondria (mt).  14 b .  P r o t o p l a s t demonstrating cytoplasmic membrane (cm), nucleus ( n ) , and nucleolus (nu).  14 c .  Portion of a protoplast demonstrating d i s t i n c t l i m i t i n g membrane ( c m ) .  the  Brenner, et a l . (14)  have suggested that a " p r o t o p l a s t "  should be defined as, "a s t r u c t u r e i n which the c e l l w a l l i s known to be absent" or "that part of the c e l l which l i e s w i t h i n the c e l l wall*. p l a s t may  They give s e v e r a l c r i t e r i a upon which a proto-  be t e s t e d .  F i r s t , the s t r u c t u r e must be  s e n s i t i v e and observations  osmotically  w i t h an e l e c t r o n microscope must  show no i n d i c a t i o n s of a c e l l w a l l .  These authors a l s o suggest  chemical and immunochemical t e s t s f o r known c e l l w a l l compounds and antigens.  I n a d d i t i o n , they f e e l that i n t e r a c t i o n w i t h  phage would be a good t e s t f o r the presence of c e l l w a l l . F i n a l l y , knowledge of what compounds are released from c e l l w a l l s by the a c t i o n of the enzyme employed, i s mentioned as an i n d i c a t o r of the p o s s i b i l i t y of p r o t o p l a s t  formation.  The p r o t o p l a s t s of dermatophytes which have been described i n t h i s study f u l f i l l s e v e r a l of these standards given by these authors.  The a c t i o n of crude s n a i l gut  chiti-  nase on i s o l a t e d c e l l w a l l s of dermatophytes has been demonstrated to r e l e a s e glucose and glucosamine ( 1 9 ) .  These  compounds do not account f o r a l l of the c e l l w a l l m a t e r i a l , but they do account f o r a l a r g e p o r t i o n of i t . contrast microscopic  observations  Next, phase  i n t h i s study have shown  that the membrane bound contents of one hyphal u n i t are ed, l e a v i n g a c e l l w a l l "skeleton" behind.  extrud-  These membrane  bound spheroids were proven to be o s m o t i c a l l y f r a g i l e . F i n a l l y , the e l e c t r o n micrographs (FIGURE 14)  show that the  s t r u c t u r e s are membrane bound and that no c e l l w a l l i s present. An e x c e l l e n t contrast to these micrographs are those taken by  Blank, et a l . (10)  of normal and g r i s e o f u l v i n t r e a t e d c e l l s .  I n the p i c t u r e s taken by these authors, the t h i c k , r i g i d c e l l w a l l s are very evident.  V.  Studies  on the  The p a p e r  Effect  by Kinsky  excellent  method o f mold  technique  for  used  procedure  this  permeability  of Griseofulvin  i n 1962  (43)  protoplast  of the  show t h e  cell  similar  techniques  were  had any  such effects  effects  used to  and t h e period  o r g a n i s m was MQ8. o f one  graphically absorbancy  hour.  results  decreases are  due  The f i r s t  to  reaction  periods  numbers. although  a  protoplasts  per  ml.  When t h e s e  and t h a t not  i n the  by K i n s k y ,  be  as  seen  the  i t  therefore  He  whether  cuvettes  cells.  described,  studies  Kinsky,  are  concentrations  shown in  while  and p r e c i p i t a t i o n . i n F I G U R E 15a, size  o f the  occurred.  did  not  protoplasts. end o f  the  protoplast the  same  The n u m b e r s  a p p r o x i m a t e l y 3.3  were  compared w i t h  was r e a l i z e d t h a t  t h e y were  small  i n protoplast  alterations  a  increase  remained almost  were  x  of 10^  those  extremely low size  could  measured. A second  centrations  series  o f e x p e r i m e n t s was c a r r i e d u s i n g  o f a p p r o x i m a t e l y 3.2  the  griseofulvin  already  alteration in  may h a v e  on  investigation,  b e g i n n i n g and the  size  a  size.  of protoplasts,  i n the  protoplast  increase  to  lysis,  showed no g r e a t  slight  the  used  swelling,  alterations  Similarly  of these  According  at  also  o f dermatophyte  were as  shrinkage  observations  but  an  O b s e r v a t i o n s w e r e made o v e r  experiments,  show a n y d e f i n i t e Microscopic  from  only  c e r t a i n drugs  In this  membranes  The r e s u l t s  i n F I G U R E 15.  of  determine  The p r o t o c o l a n d t e c h n i q u e s  not  Membranes  i n protoplast  membrane.  on the  gave  preparation  measuring a l t e r a t i o n s to  on C e l l  x 10°  protoplasts  per  conml.  The r e s u l t s that  both  test  absorbancy partially  are  sucrose  due  to  due  to  experiment.  run.  slight  as  the  counts  slight In  to  The  slow  slope  is  increase  test  and  lysis  i n average  or  the  amount  of  griseofulvin.  An- i n c r e a s e d  tation  i n the  test  also  The  sample  is  as  the  by  slow  demonstrated  the  end o f  the  resulted  were  in  resuspended.  was t h a t  the  de-  than  that  absorbancy  indicate  examinations size  amount  did  i n the  swelling  had  of'  i n d i c a t e d by the  that inditest  been  precipivalues  stirring.  interpretation  conclusive.  Griseofulvin  permeability  of the  of these  observations  appears to  have  c y t o p l a s m i c membrane.  i n  precipitation.  protoplast  by t h e  after  caused  readings  These  also  continuous  protoplasts  after  small  is  size,  i n absorbancy  caused  recorded  It  s a m p l e was g r e a t e r  i n numbers.  words a  is  brown c o l o u r e d  at  series  observed  descreasing  slope  T h i s was  cuvettes  swelling,  before  increase  other  of  A s was p r e v i o u s l y s t a t e d ,  t h e r e was no d e c r e a s e  sample.  of the  i n the  steep  first  i n protoplast  observation i n this  c o n t r o l sample.  a  of the  precipitated  of absorbancy  is  slopes  of the  protoplasts.  contents  It  DMF w a s a d d e d .  increase  remainder  d e c r e a s e s may b e d u e  cate  factor  reduced.  An immediate  cuvettes  Microscopic  is  give  The i n i t i a l  dilution  of larger  The m a j o r  the  a  of the  s t i r r i n g the  crease  the  samples  r e s u l t i n g when t h e  precipitation  both  i n FIGURE 1 5 b .  control  concentration  character  by  and  during the  background, partially  described  some  is  not  effect  From the  very on small  the  FIGURE  15  Effect of Griseofulvin on Dermatophyte Protoplast Membranes 4 0 ' ro O  E  30 « 20  O O  to  10  0  10  20 Time  i i 3 0 40 (min.)  15a. Low Protoplast  i 50  —i 60  Concentrations  to o E O O  <0  140-  0  i 10  i 20 Time  15b. H i g h  i 30  40  50  (min.)  Protoplast  Concentrations  N,N-dimethylformamide (DMF) Griseofulvin in D M F  60  97 effect of such a high concentration of drug i t seems l i k e l y that the membrane is not the main object of the drug's attack. It i s much more probable that the primary target is simply located on or near the membrane.  This is supported by the effect  observed i n the oxygen uptake values with glucose as the substrate.  The enzymes involved i n the oxidative assimilation  of glucose are probably associated with the c e l l membrane. Thus small alterations i n the membrane might simply be an indication of disruption of these enzymes attached to i t .  A better pos-  sible locus for the site of action might be the cellular energy mechanisms.  These are involved in maintaining osmotic equilib-  rium of the c e l l and are also involved i n the glycolytic and assimilative pathways.  If this was the case, the swelling of  the protoplasts i n the presence of 18% sucrose would be very slow, so that only a small difference would be observed during the course of this experiment.  Supporting evidence for this  hypothesis i s found i n the report by McNall on the partial reversal of the action of griseofulvin, by the addition of purines and pyrimidines (47), which are central figures i n cullular energy systems.  98  VI.  Studies  on the E f f e c t  on C e l l  Wall  of  Griseofulvin  Resynthesis  Electron micrographic observations presence of  of  cell walls.  plasts  When i t  fulvin  a  s t u d y was  would i n h i b i t  t r a c i n g the  absorbancy  at  observations  metry  data  microscopic It period,  were  given  the  samples.  a rapid  This  slows  rate  level.  At  The t o t a l  The  It inhibition  rates  increase  the of  test  is  i n the  samples  i n absorbancy  a  or thinner c e l l  of  Spectrophoto-  concluded that initial  while VIII. lag one  period during  absorbancies  of  both  and continues  at  a  lags  just  begin behind  i n both samples is  of  Micro-  slightly longer  short  less  c o n t r o l , which could indicate  is  hours.  which time they  sample  synthesis  crude  show a n i n i t i a l  showing a  s i x hours at  a l l times  than i n the dense  until  i n both  of  MQ8 b y m e a s u r e m e n t seven  both samples  increase  proto-  carried  l i s t e d i n TABLE  Following this  is  moderate  control.,  that  layering  Experiments were  g r a p h i c a l l y i n F I G U R E 16,  seen  control.  absence  simultaneously.  g r i s e o f u l v i n sample  which there  dermatophyte  the  griseo-  a period of  are  grown i n  determine whether  process.  made  observations  c a n be  than the  i n i t i a t e d to  mu, o v e r  600  is  found that  c e l l w a l l i n the  this  cells  show a t h i c k e n i n g a n d  c e l l w a l l formation of  scopic  less  was  would resynthesize  chitinase,  out,  (10,66)  griseofulvin  of  i n the the  are  test  more  to the the  same.  sample,  formation of  a  wall. g r i s e o f u l v i n causes a  c e l l wall resynthesis,  but  slight that  after  the  f i r s t thin wall has been formed the drug then may cease to exert any effect.  The lower, total absorbance increase obser-  ved, may indicate the formation of an irregular c e l l wall, as reported by Blank, et a l . ( 1 0 ) . again s t i l l uncertain.  The site of this action is  Combined with the results of the c e l l  membrane studies i t is probable that the site of action of the drug is on or near the membrane.  The resulting minor dis-  ruption could cause the slight inhibition of c e l l wall resynthesis observed here.  100 TABLE VIII Microscopic Observations of Cell Wall Resynthesis by Protoplasts Time  Control Sample Containing DMF*  Test Sample Containing Griseofulvin i n DMF*  30min.  Protoplasts appeared normal. Ruptured when challenged with d i s t i l l e d water.  As in control.  1 hr.  Protoplasts s t i l l appeared normal and ruptured when challenged•  As i n control.  2 hr.  Between 20% and 30% of protoplasts showed a very thin c e l l wall. When the sucrose was replaced with d i s t i l l e d waiter the inner protoplast usually burst out of this thin wall and ruptured. Most s t i l l burst upon challenging.  Less than 10% of protoplasts exhibited a thin c e l l wall. Most s t i l l ruptured immediately upon challenging.  3 hr.  Between 60% and 70% showed the presence of a thin c e l l wall.- About 10% showed a thicker c e l l wall which would not rupture even when a l l sucrose was replaced. The remainder burst upon challenging.  Between 40% and 50% showed thin c e l l walls. The remainder burst upon challenging.  4 hr.  A l l protoplasts exhibited thick c e l l walls. No bursts were observed upon challenging.  Approximately 80% showed thick c e l l walls. Most of the remainder had thin walls although the occasional free protoplast was observed.  5 hr.  As at 4 hours.  A l l protoplasts exhibited thick walls. No bursts were observed upon challenging.  6 hr.  As at 4 hours.  As at 5 hours.  7 hr.  As at 4 hours.  As at 5 hours.  * N,N-dimethylformamide  F I G U R E 16 Effect of Griseofulvin on Cell Wall Resynthesis  14 0 1 ro O  130' E  I 20  O  o  no • 100  1  T"  i  2  3 3 Time  4  (hours)  N,N-dimethylformamide ( D M F ) Griseofulvin  i n DMF  5  6  102 VII  Cell Wall Composition Studies The results obtained i n the resynthesis study indicate  that a c e l l wall is formed i n the presence of griseofulvin. They do not indicate though, whether this wall has the same composition as a normal c e l l .  On the basis of the electron  micrographs taken by Blank et a l . (10), i t would appear quite possible that the composition was not the same.  An investi-  gation was therefore undertaken to qualitatively analyze the c e l l walls of MQ$ cells, which had been grown i n the presence and absence of griseofulvin.  To increase the scope of the  experiment, i t was decided that a mutant of MQ8, which had developed resistance to the drug under "in vitro" conditions, should be isolated and analyzed at the same time. A. Isolation of a Griseofulvin Resistant Mutant As a f i r s t step i n the isolation of a mutant, the stock MQ8 culture was again tested for griseofulvin sensitivity.  It  was inoculated onto a series of plates containing griseofulvin in the following concentrations: 1.0 ugm./ml., 2.0 ugm./ml., 3.0 ugm./ml., 4.0 ugm./ml., 8.0 ugm./ml., and 16 ugm./ml.. The results are shown in TABLE IX. three and six days©  Observations were made at  103 TABLE I X Sensitivity of MQ8 to Griseofulvin Griseofulvin Concentration (ugm./ml.)  Hyphal "curling" 3 days  6 days  Growth 3 days  6 days  0  0  0  ^3  +4  0  0  0  -3  +4  1.0  0  +  +2  •+3  1,0  0  0  +3  +4  2.0  0  +1  +1  . +2  2.0  +  +2  +1  +2  3.0  +2  +4  +  +1  3.0  -+2  +4  -  4.0  -  +1 0  0  . 0  0  0  0  0  0  4.0  +4*  8.0  +4*  8.0  +4*  16.0  +4*  mm  0  0  16.0  + 4*  m  0  0  -  * Inocula showed extreme distortion.  These results agree generally with those found previously for MQ8.  This strain which was sensitive to 4.0 ugm. of  griseofulvin (G.) per ml. was now subjected to a "layer plate" containing 2.0 ;igm. G./ml.  The f i r s t hyphae to appear on the  surface were transferred to two "layer plates" containing 4.0 jugm.G./ml. six days.  Growth appeared on the surface of the plate after This was immediately transferred to two "layer  plates" containing 8.0 jugm.G./ml.  Growth appeared on the  surface of one plate after ten days and on the other after twelve days.  The growth from the ten day plate was trans-  ferred to two "layer plates" containing 16 ugm.G./ml. growth appeared on the surface by fourteen days.  No  This step  was repeated, but with no success even after twenty days.  The  MQ8 mutant, resistant to 8.0 p.gm. G./ml. (referred to as MQ8R), was maintained on Sabouraud's cerelose agar containing this concentration of drug. B. Analysis of Cell Walls The mutant strain (MQ8R) and the parent (MQ8)! were separately inoculated into Sabouraud's cerelose broth containing griseofulvin.  The media contained: no griseofulvin (G.),  8.0 ugm. G . / m l . , and 16 p.gm. G./ml.  This original inoculation  pattern was not satisfactory as the parent (MQ8) would not grow in the presence of 8.0 ugm. G./ml. and 16 jigm. G./ml.  These  two flasks of media then were replaced with a medium containing 3.0 ^igra. G./ml.  The growth periods for these cultures varied  greatly as may be seen i n TABLE SX.  105 TABLE X  Growth Periods of Cultures Used i n Cell Wall Studies Culture  MQ8  Griseofulvin Concentration  0  Growth period  4 days  MQ8R 3.0 ugm./ml. 14 days  0  8.0 ugm./mL l6ugmvfeL  3 days  21 days*  14 days  7  * Even after this length of time there was less than i n the other cultures.  The c e l l walls of these cultures were purified and analyzed according to the procedure given in Methods, Chromatographic analysis of the crude c e l l wall hydrolysates was also carried out during the purification process, prior to the trypsin and ribonuclease treatment.  The results of this preliminary  analysis indicated the presence of the sugars, glucose and ribose, and possible mannose and galactose.  The amino sugar,  glucosamine, was also found in high concentrations.  The amino  acid chromatogram contained at least 16 spots and appeared to be a protein hydrolysate.  These results were the same for a l l of  the c e l l wall preparations. The analysis of the purified c e l l wall preparations showed the presence of the sugars);; glucose, mannose, and galactose and the amino sugar, glucosamine, not observed in any of these preparations.  Galactosamine was An unidentified  compound which ran approximately half-way between glucosamine and galactose was observed i n a l l cases.  No differences i n  sugar or amino sugar content were noted among the various c e l l  wall preparations, either qualitatively or i n the relative quantitative observations.  The densest spot appeared to be  glucose, followed by glucosamine, mannose, galactose, and f i n a l l y , the unidentified spot. The amino acid analysis of the purified walls did not indicate any qualitative differences among the five c e l l wall hydrolysates.  The following eleven amino acids were tenta-  tively identified i n a l l samples: lysine, histidine, arginine, aspartic acid, glycine, glutamic acid, alanine, proline, valine, phenylalanine, and leucine (and/or isoleucine). Traces of an amino compound which was possibly cystine were also observed. In the analysis for griseofulvin products, the griseofulvin 2 . 0 N and 6 . 0 N HC1 hydrolysates and the precipitate extract, were run as standards.  A pure griseofulvin sample  was also run as a standard, and had an Rf of 0 . 8 7 .  A single,  unidentified, light-blue fluorescent spot was observed i n trace amounts i n the 2 . 0 N HC1 hydrolysate and i n high concentrationsr. i n the 6 . 0 N NCI hydrolysate.  It was not observed  i n the butyl acetate extract of the precipitate. pound had an Rf of approximately 0 . 7 3 .  This com-  No griseofulvin was  observed i n any of the hydrolysates. Cell wall preparations were chromatographed and observed under ultra-violet l i g h t .  No fluorescing or absorbing  spots were observed i n the hydrolysates.  A spot having a  yellow fluorescence was observed i n a l l extracts of the precipitates.  This spot may be disregarded at this time, as  107 i t is obviously not a griseofulvin hydrolysis product. These results indicate that although cells grown i n the presence of griseofulvin show a structural alteration microscopically (10), they do not alter appreciably i n the chemical content of the assayed compounds.  Qualitative differences i n  amino acid, amino sugar, and sugar content could not be demonstrated.  Similarly, no differences i n the relative quantita-  tive values of these compounds could be observed.  Finally,  the absence of griseofulvin products i n the hydrolysates is a good indication that griseofulvin i s not incorporated to form a modified chitin, as suggested by Rhodes (33)»  108  GENERAL DISCUSSION The i n i t i a l review of the literature, with respect to the antifungal properties of griseofulvin, indicated a variety of unexplained facts about i t s mode of action.  First,  it  causes stunting and distortion of growing hyphal tips (18),  A  wide survey of sensitive and resistant fungal species has shown also, that except for one or two notable cases (2), a l l fungi which are sensitive to griseofulvin, have chitinous c e l l walls (15),  These walls are definitely altered, becoming  frayed and forming thick irregular layers, when sensitive strains are gpown in the presence of the drug.  At the same  time, large l i p i d granules are observed in the cytoplasm (10). The fact that only growing areas of the hyphae became deformed, was confirmed by studies involving the direct application of griseofulvin to various parts of the hyphal elements (7).  It  was shown also that griseofulvin was not translocated within the hyphae, thus i t affects only the area of contact  (2),  Various biochemical studies also have been reported. F i r s t , several workers have carried out studies on oxygen uptake by fungi, in the presence of griseofulvin.  Two of these  (15,44) have reported that griseofulvin has no effect on endogenous respiration, while a third reports that there is a very small percent decrease (63),  One of the authors who reported  that no alterations occurred, listed the Q0 values obtained i n 2  his experiments (15)•  Closer observation of these values  109 indicated that i n almost a l l cases, there was a decrease of approximately 2.0% i n the presence of the drug.  It was sur-  prising at f i r s t , that none of these authors investigated the effects of griseofulvin on the oxidation of a substrate, such as glucose.  The reason became clear though, when i t was found  that the addition of glucose to unstarved c e l l material, resulted i n a decrease i n the endogenous respiration rate ( 6 3 ) . The results of the oxygen uptake studies, which were reported i n this thesis, confirm that a small reduction of endogenous respiration is caused by griseofulvin.  They  further show that glucose oxidation is i n i t i a l l y reduced by the drug, but that with continued starvation of the c e l l material, an increased amount of glucose is oxidized.  In fact, the  amount of glucose oxidized i n the presence of the drug is greater than the amount oxidized i n i t s absence. These oxygen uptake results are d i f f i c u l t to interpret without further correlation with other areas of cellular metabolism.  For example, i t was found that glucose was taken  up by unstarved c e l l material, but that l i t t l e or no oxygen uptake could be measured.  With starvation of the c e l l mater-  i a l , a greater percentage of the glucose was oxidized.  Also,  i t was seen that the effect of griseofulvin on the respiration of unstarved c e l l material was extremely different from i t s effect on the respiration of material which had been starved for five days.  The reasons for this change can only be con-  jectured, because not enough is.known about endogenous respiration i n dermatophytes.  In other words, how would  110 depletion of "endogenous reserves" alter the c e l l , and what are the "endogenous reserves"?  The final conclusions of these  experiments were that griseofulvin may inhibit enzymes involved in synthesis of "endogenous reserves", the cofactors associated with them, or the energy-transport mechanisms which drive these synthetic reactions.  Some confirmation of these conclusions  i s found in the report that the assimilation of phosphate (12) and of nitrogen (71) are inhibited by the action of griseofulvin.  The suggestion was made that griseofulvin uncouples  a phosphorylation i n a respiratory pathway (71). A report by McNall (47) may help to clarify the previously mentioned results.  His investigation involved the  addition of compounds to culture media which also contained griseofulvin.  He reported the partial reversal of the  inhibitory effect of the drug, by the addition of purines, pyrimidines, and their nucleotides.  The most effective com-  pounds i n this partial reversal of inhibition, were guanylic acid and to a lesser degree, adenylic acid.  This possibly  indicates that the purine nucleotides are closely related to the site of action of the drug.  McNall, i n fact, believes  that nucleic acid synthesis is blocked.  It seems l i k e l y  though, that the cellular, energy-transport systems, i n which these compounds play an important role, are also involved. is even possible that griseofulvin i s acting competitively with precursors of these compounds i n energy-transport pathways.  It  Ill A good case can be presented to show that griseofulvin could be a structural analogue of purine nucleosides.  The  bonding properties of griseofulvin (24) are i n many ways similar to those of the nucleosides.  Thus, this drug could enter the  reaction sites of these compounds, but could not carry out their reactions.  On the basis of the phosphate assimilation studies  (71), one reaction which might be blocked i s the phosphorylation of the purine nucleosides. The results obtained i n the amino compound studies, proved to be of l i t t l e use i n clarifying the previously obtained results.  They simply indicated that, due to the influence  of griseofulvin, growth was completely inhibited, after one or two days.  No evidence was found i n this investigation to  indicate any barrier i n amino acid metabolism. Investigations into the action of the drug on membrane permeability, showed that there is a slight effect.  In con-  sidering the small alterations observed, i t appears l i k e l y that the site of action is on or near the membrane but is hot the membrane i t s e l f .  Suggested areas are, enzyme systems  associated withi the membrane, or energy mechanisms involved i n maintaining osmotic equilibrium of the c e l l . A large portion of the literature suggests that c e l l wall synthesis is the site of griseofulvin action.  This is certain-  ly supported by electron microscopic observations ( 1 0 ) .  The  use of protoplasts to follow complete c e l l wall synthesis showed that, although there was an i n i t i a l lag i n the formation of this structure, a wall of some type was formed.  This  112 does not necessarily mean that the synthesis of c e l l wall was inhibited.  It appears quite possible that some locus on or  near the membrane i s the object of this compound's activity. The enzymes involved i n c e l l wall synthesis also are probably associated with the membrane, thus they might show the effects of alterations on i t .  On the other hand, the synthesis of one  c e l l wall component could be blocked without inhibiting a l l cell wall formation. This study did not prove that a l l constituents of the c e l l wall were unchanged.  To confirm or disprove the hypoth-  esis that c e l l wall synthesis was the site of the drug's action, a qualitative analysis of c e l l wall was undertaken. showed that no qualitative or relative quantitative  The results differences  in amino acid, amino sugar, and sugar content, occurred between cells grown i n the presence and the absence of griseofulvin. Finally, attempts to demonstrate griseofulvin breakdown products in c e l l wall hydrolysates, point to the fact that griseofulvin is not incorporated into c e l l walls to give a modified c h i t i n , as has been suggested (33) • The general conclusions of this study are that the site of griseofulvin action is related to enzymes involved i n synthesis of endogenous substrates, or to mechanisms controlling these enzymes and their a c t i v i t i e s , ,  It is further concluded  that these systems are located on or near the cytoplasmic membrane.  113 From the results obtained i n these investigations,  it  appears that further work should be carried out to confirm the results of McNall (47), on the partial reversal of griseofulvin inhibition, using purines, pyrimidines, and their nucleotides. It also is suggested that such results be correlated with oxygen uptake studies.  114 SUMMARY  Several mine t h e  series  effects  of the  dermatophytes. determine clearly both  whether  endogenous the  starvation of  throughout  presence  With  of the  study these  not  under  success,  after  one  o r two d a y s , earlier  inhibition.  It  great  for  increase The l a c k  It  also  was  inhibiting  the  effect  glucose occurred  in  reports  manometric methods,  excreted  a l s o was n o t e d  that  with  to  was  this  cell-free  fungistat  inhibited thus  before caused  medium. extracts  while  demon-  of griseofulvin,  some g r o w t h o c c u r s that  but  determine  cells,  g r o w t h was  i n soluble p r o t e i n i n the of results  by the  tests  measurements.  No s u c h r e s u l t  presence  that  These  was u n d e r t a k e n ,  drug.  i n the  alterations,  dehydrogenase  was o b s e r v e d t h o u g h ,  confirming  demonstrat-  endogenous  cell-free extracts.  i n v e s t i g a t i o n next  influence of the It  respiration,  glucose oxidation,  only with  Thunberg techniques  strated.  to'  show more  The r e s u l t s  physiological  any amino compounds were  the  on  drug.  were w i t h o u t  whether  on c e l l  oxidation of  utilize  A short  m a t e r i a l would  continued s t a r v a t i o n though,  a t t e m p t s w e r e made t o  with  e x p e r i m e n t s was  starvation period.  affected  deter-  first  slight reduction of  and i n c r e a s e d  To f u r t h e r  also  cell  to  griseofulvin,  had any e f f e c t  the  griseofulvin  was r e v e r s e d ,  of the  c a r r i e d out  drug,  and g l u c o s e o x i d a t i o n .  initially.  the  fungistatic  d r u g causjed a  respiration shown t h a t  experiments were  The p u r p o s e  i f griseofulvin  ed t h a t  it  of  was  a  115considered  due t o t h e r a t h e r  o b t a i n them. enzymes,  specifically  cell walls  procedure. if  chitinase  a s t o make  I t was a l s o  Results  a better  protoplast A  here,  study  Neurospora  whether  enzymes,  study  but that  would  simple  to find  from dermatophytes  indicated that  chance  would  out  be  partially purified  crude  s n a i l gut  chitinase  f o r both c e l l w a l l weakening and  now w a s i n i t i a t e d t o f o r m p r o t o p l a s t s As a preparatory  s p e c i e s were  dermatophytes  cedure  of this  to  formation,  dermatophytes.  with  and r e l a t e d  the purpose  c h i t i n a s e was n o t e f f e c t i v e , provided  necessary  ce.ll breakage a very  the formation of protoplasts  possible.  conditions  A s t u d y was u n d e r t a k e n t o d e t e r m i n e  more  so w e a k e n  rigorous  also  which would give  step,  protoplasts  formed and s t u d i e d . were  successful,  higher  yields  of of  Further  attempts  and a m o d i f i e d  of protoplasts  pro-  was  discovered. Having out  obtained  to determine  protoplast  the effects  cytoplasmic  membrane was a l t e r e d addition,  on c e l l w a l l  was c a r r i e d  I t was found t h a t  s l i g h t l y by the drug's employed t o study synthesis,  this  action.  In  the influence  A small effect,  o f some  type  was  T h e r e w a s some which formed  but  formed.  question as t o whether  i n the presence  w h i c h was formed  of  i n the  o f a t i m e l a g i n c e l l w a l l p r o d u c t i o n , was o b s e r v e d ,  a wall  that  were  an experiment  of g r i s e o f u l v i n d i r e c t l y on the  membrane.  only  protoplasts  griseofulvin form  protoplasts,  of griseofulvin,  i n i t s absence.  the c e l l  wall  w a s t h e same  I n other words,  as  were  116  any c e l l wall components deleted or was griseofulvin possibly incorporated into the c e l l wall structure.  It was concluded  from experimental results that no alterations i n amino acid, amino sugar, or sugar content could be observed.  It was  further demonstrated that griseofulvin was not incorporated into the c e l l wall, A general conclusion was drawn that the biochemical site of griseofulvin action is related to the enzymes involved i n synthesis of endogenous substrates, or to the mechanisms cont r o l l i n g these enzymes.  It also was concluded that the  morphological site of action of the drug was located on or near the cytoplasmic membrane. The suggestion was made by this author, that the reported partial reversal of griseofulvin activity by purines, pyrimidines, and their nucleotides, be confirmed and correlated with oxygen uptake resuits  0  117 APPENDIX I . Percent reduction of Endogenous Respiration The calculation used to determine the percent reduction of the endogenous oxygen uptake by griseofulvin action is as follows: (E - E G r ) X 100 % R E  e  E - Endogenous oxygen uptake. EGr - Endogenous plus griseofulvin oxygen uptake. fo Re - Percent reduction of endogenous respiration.  I I . Percent Reduction or Increase*of Glucose Oxidation The calculation used to determine the percent reduction or increase of the glucose oxygen uptake due to griseofulvin activity, is as follows: (G - E) - GOx  ;  (GOx - GOxGr) GOx  X 100  (GGr - EGr) - GOxGr - f R* 0  g  G - Glucose oxygen uptake. E - Endogenous oxygen uptake. GGr - Glucose oxygen uptake in the presence of griseofulvin. EGr - Endogenous oxygen uptake i n the presence of griseofulvin. GOx - Oxygen uptake due to the oxidation of glucose, .*. (If this value was a negative one, i t was described as percent increase).  118 GOxGr - Oxygen uptake due to the oxidation of Glucose i n the presence of griseofulvin. % Rg - Percent reduction of glucose oxidation. III.  Percent of Total Theoretical Glucose Oxidation The calculation used to determine the percent of total  theoretical oxygen uptake due to oxidation of glucose is as follows: (GOx) I 100 - f TGOx (No. of uM glucose i n cup) X (134.4*) 0  GOx - Oxygen uptake due to the oxidation of glucose. %TG0x - Percent of total oxygen uptake expected i f glucose was totally oxidized.  *(This value i s the number of u l . of oxygen required to totally oxidize 1.0 }M of glucose to C0£ and H 0). 2  119  BIBLIOGRAPHY  1.  Abbot, M . T . J , and Grove, J . F . 1 9 5 9 . Uptake and translocation of organic compounds by fungi. I Microspectrophotometry i n the study of translocation. Exptl. Cell Res., 12,  95-104.  2.  Abbot, M . T . J , and Grove, J . F . 1959. 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