Keratinolytic Activity of Microsporum Canis and Microsporum Gypseum*

Vol. 44, No. 5

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

Printed in U.S.A.

1965 by The Williams & Wilkins Co.

KERATINOLYTIC ACTIVITY OF MICROSPORUM CANIS AND MICROSPOR UM GYPSEUM* PEYTON E. WEARY, M.D., CHARLES M. CANBY, MS. AND EDWARD P. CAWLEY, M.D.

Speculation about the ability of various microorganisms to digest naturally-occurring keratin materials has prompted a number of investigations (1—37). Opinions and findings of various

creased growth of several dermatophytes while chemical inducement of additional cross linkages decreases or inhibits growth. Thus, studies in which processed wool fabric (2, 22) is used investigators have varied and much of the as a substrate can hardly be expected to yield earlier experimental work is open to critical information of value in regard to keratinolytic re-evaluation (15, 38, 39) because of several activity of fungi on native keratin. basic defects in the methods of approach and Another major problem is to provide uninterpretation of results. equivocal evidence of digestion of keratin itNoval and Nickerson (15, 16) have obtained self. Native keratin substrates are complex reasonable evidence for keratinolytic activity substances containing, in addition to several of a strain of Streptomyces fradiae which was types of keratin molecules, various non-kerat-

selected because of its impressive ability to digest wool samples in a gross fashion. Their study represents a carefully constructed evaluation of this problem and is used, in part, as a prototype for the investigation reported in this paper. Noval and Nickerson recognized many of the problems inherent in such an

inous organic components capable of pro-

group of substances as native keratin substrates.

tion of the keratinous material itself (hair,

viding substantial nutritive substrate without the occurrence of keratinolysis. Bolliger (41,

42) lists many such water extractable sub-

stances for several hard keratins and in

addition there are others not so easily extractable. Hence, the observation of growth of a investigation, particularly the basic problem of microorganism on a keratin substrate does not preservation of the integrity of such a complex establish keratinolysis nor does visible disrup-

They emphasized the necessity of the gentle nails, etc.) prove that keratinolysis has ochandling of keratin materials in the process of curred, since the disruption may be simply cleaning, finely dividing and sterilizing them. mechanical (39) through growth of organisms The failure to emphasize such details by many in the interstices of the material. of the previous investigators tends to invalidate The most distinctive chemical characteristic some of their observations and conclusions. of keratin, which sets it apart from other There is convincing evidence, for instance, that fibrillar proteins, is its high cystine content. The disulfide linkages of the cystine molecules

ball-milling and autoclaving of wool (27) markedly decreases the cystine content by disrupting disulfide linkages and that even a single passage

through a Wiley mill renders wool substantially less resistant to tryptic digestion (26). There is also evidence that organisms which do not grow on naturally-occurring keratin substrates will show growth on denatured keratins (34). For example, the alteration of native keratin materials (40) by chemical rupture of cross linkages allows significantly inThis investigation was supported by Research Grants No. A-6239 and AM-06239-02 from the National Institutes of Health, United States Public Health Service. Received for publication June 1, 1964.

* From the Department of Dermatology, Uni-

versity of Virginia School of Medicine, Charlottesville, Virginia.

are thought to be largely responsible for providing stability to the keratin molecule and rendering it more resistant to enzymatic diges-

tion. Demonstration of the ability of fungi growing upon keratin to release soluble sulfhydryl-containing amino acids and polypeptides

into the medium in quantities significantly greater than those released by controls or by tryptic digestion of keratin would provide con-

vincing evidence of keratinolysis. It is upon this premise that many of the following observations and conclusions are based. MATERIALS AND METHODS

I. Preparation of Keratin Substrate Freshly shorn wool of medium fiber diameter, obtained from undipped Suffolk sheep, was sorted

300

KERATINOLYTIC ACTIVITY OF fit. CAMS AND fit. GYPSE UM

and the cleanest wool selected for further processing as follows:

A. Single extraction with chloroform followed by air drying and then five washings with distilled water followed by air drying. B. Careful removal of insoluble debris by hand. C. Repeat of Part A.

301

A. Keratin Controls—to which were added 2

gms of sterile wool and 10 cc of basal salts solution containing Kanarnycin (40 mgm/cc). B. Fungus Controls—to which were added 10 cc of the fungal inoculum.

C. Test Samples—to which were added 2 gms of sterile wool plus 10 cc of the fungal

D. Cutting by scissors into short fragments inoculum.

and sifting through a plain copper screen.

The flasks were plugged with cotton and

gauze, weighed and incubated as static cultures. E. Repeat of Part A. The final rinse water was found on preliminary Previous pilot studies had indicated no advantage

studies to be ninhydrin negative and to show a from the use of agitated cultures in this study. negligible absorption at 280 millimicron (protein) This is probably because the Fernbach flasks using a Beckman DU spectrophotometer. provided a large surface area for gas exchange The wool was then weighed into two gram in relation to fluid volume (the depth of the aliquots which were sterilized in plugged Erlen- medium is about 1 cm). Three temperatures were meyer flasks by ethylene oxide in a Brewer used for incubation: 35° C., 30° C. and room anaerobic jar at room temperature for 48 hours temperature which ranged from 22.2° C. to (modified method of Noval and Nickprson) (15), 29.5° C.

after which the sterility of the samples was checked by the addition of aliquots of the wool

to both thioglycollate broth and Sabouraud's Dextrose agar slants.

V. Removal of Samples

Aliquots (10 cc) were removed from all flasks

at the end of 24 hours, at 7 weeks and at 10

weeks. Prior to removal of samples, the flasks were reweighed and appropriate amounts of A basal salts solution was employed which sterile basal salts solution added to each flask contained the following ingredients per liter of to restore the volume lost by evaporation since the previous sampling in order to eliminate this glass distilled water: II. Preparation of Flasks for Inoculation

K211P01—1.5 Gnm

MgSOe7H2O—.025 Gms CaCh—.025 Gms Fe50e71120—.015 Gms Zn504 71120—005 Gms

concentrating effect.

Portions of each sample were added to thioglycollate broth and Sabouraud's dextrose agar to test for bacterial or fungal contamination.

Prior to the use of Kanamycin much difficulty had Fernbach flasks containing 390 cc of the basal been encountered with bacterial contamination of salts solution were autoclaved at 15 pounds for those flasks containing wool samples (presumably 15 minutes. The final pH after autoclaving was the result of spore forms resistant to ethylene

oxide) but this was largely eliminated by the

8.3—8.4.

use of this antibiotic. III. Preparation of Fun gal Inoculum

Two strains of Microsporum canis (American

VI. Determination of Soluble SulfhydrylContaining Compounds

Type Culture Collection Strains 10214 and 11621) and two strains of Microsporum gypseum (Ameri-

Portions of the supernate from the centrifuged

10215) were grown on Sabouraud's dextrose agar

sulfhydryl-containing compounds by the spectro-

can Type Culture Collection Strains 9083 and aliquots were analyzed for the content of soluble

slants. The mycelial mats were removed and photometric method of Boyer (43). Cysteine homogenized in a small quantity of basal salts was used as a standard and soluble sulfhydryl-

solution under sterile conditions. This suspension containing compounds are expressed in terms of was used to inoculate flasks containing Czapek- equivalence to cysteine (micrograms per cc of Dox broth (1000 cc) which were then continuously medium with an average experimental variation of micrograms of cysteine/cc). agitated on a gyrorotary shaker. The fungi grew Amperometric titrations of soluble sulfhydryl— slowly as discrete hyphal pellets over a period of several weeks. When the average pellet diameter approximated ½ centimeter, samples were removed

containing compounds were conducted by the

cess medium discarded. The fungi were resuspended

methods were in substantial agreement and, because the spectrophotometric method was more rapid and more sensitive it was used routinely

method of Kolthoff (44, 45) on the aliquots to check for bacterial contamination and the removed from time to time to confirm the specificcontents of the flasks were centrifuged and the ex- ity of the spectrophotometric method. The two in a solution containing Kanamycin (40 mg/cc) in basal salts solution. Thus 10 cc of this inoculurn

yielded a final concentration in the Fernbach for the determinations. flasks of 1 mgm of Kanamycin per cc. IV. Inoculation of Fernbach Flasks The Fernbach flasks containing 390 cc of sterile basal salts were divided into the following groups:

VI]. Determination of Protein The supernate of the centrifuged aliquots was analyzed for protein content using the method

of Lowry et al. (46). Freshly prepared bovine

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE I Keratin controls at various temperatures Protein Determinations in Micrograms Protein per cc Medium

Sulfhydryl Determinations in Micrograms Cysteine per cc Medium

24 hrs.

7 wks.

10 wks.

9.0

40.0

45.0

9.0 9.0

40.0 40.0

39.0

6.3 6.5 6.4

13.5

35.5

9.0 11.2

39.0 37.2

33.0 35.0 34.0

3.9 6.5 5.2

16.5 13.5 15.0

25.0 28.5 26.8

25.0 27.0 26.0

24 hrs.

7 wks.

10 wks.

1.0

7.9

8.6

0.0

9.0 8.5

9.8 9.2

5.7 5.9 5.8

3.5 6.0

35° C.

Keratin Controls Average

0.5

42.0

30° C.

Keratin Controls

0.0 0.3

Average Room temp. Keratin Controls Average

0.1 0.7

0.0 0.3

4.7

TABLE II

VIII. Tryptic Digestion of Wool Samples

Two strains of Microsporum canis—fungus controls and test samples at various temperatures Sulfhydryl Determinations in Micrograms Cysteine per cc Medium

Protein Determinations in Micrograms

Protein per cc Medium

Samples of both sterilized and unsterilized

wool were digested with 0.025% trypsin (Parenzyme) in basal salts solution. The digestion was carried out for one 72 hour period followed by two 48 hour periods at 30° C. using freshly prepared trypsin solution for each digestion period. Soluble sulfhydryl—containing compounds and pro-

teins were measured before and after each diges-

24

7

10

hrs. wks. wks.

hrs. wks.

tion period and compared with control wool

samples incubated in basal salts solution.

Strain 10214 350 C.

Fungus control Test sample 300 C.

Fungus control Test sample Room temp. Fungus control Test sample

RESULTS

0.0 0.0 0.0 5.0 0.0 8.0 1.0 15.5 19.0 15.0 11.0 56.0

0.0 0.3 0.0 4.0 0.0 4.5 and in Figures 1—8. In all figures (1—8) the 0.0 23.9 31.7 14.5 94.0 119.5

values for the keratin control flasks (Table I) are added to those of the fungus control flasks

0.0 0.0 0.5 5.0 0.0 0.0

(Tables II and III) to provide the basis for comparison with the test samples (Tables II and III) containing both keratin and fungal

0.5 15.7

21.0 10.5 79.0 105.5

Strain 11621 350 C.

Fungus control Test sample

1.7

2.1 2.2 9.0 0.0 9.0

2.4 13.816.415.044.0

44.0

30° C.

Fungus control Test sample Room temp. Fungus control Test sample

The results of the evaluation of two strains of Micros porum canis and two strains of Microsporum gypseum are presented in Tables I—ITT

1.4 2.4 2.4

4.0 0.0 12.5

organisms.

Correct interpretation of the data presented depends upon realization that the significant

factor is not an absolute value for soluble

35.0

sulfhydryl-containing compounds or for protein but, rather, the difference in values between the

0.7 2.8 1.5 6.0 0.0 7.0

sum of the controls and the test samples. The values for soluble sulfhydryl-containing compounds and protein presented in the tables are

2.6 10.0 12.2

12.0 24.0

1.7 6.3 8.1 15.0 11.0 23.0

albumin was used as a standard and values are expressed as micrograms of protein per cc of medium. (Average experimental variation crograms of protein/cc.)

mi-

only a proportionate fraction of the actual total release of such substances during the entire period of incubation and, in fact, represent indicators of the metabolic balance be-

KERATINOLYTIC ACTIVITY OF lit. CANIS AND lit. GYPSEUM

tween release of, and destruction or utilization of, these substances. This is particularly important in consideration of values for soluble sulfhydryl-containing compounds, the chief representative of which is presumably cysteine, because of the findings of Stahl and coworkers (25) that cysteine is quite unstable in a similar

303

of soluble sulfhydryl-containing compounds

from the wool may be the result of either

absence of keratinolytic activity or, more likely, a much smaller fungal inoculum than was used

system.

for investigation of the other strains. Figures 5 and 6: Strain 9083 of Microsporum gypseum shows significantly greater release of sulfhydryl-containing compounds than does the

spo rum canis shows a significantly greater re-

greatly different.

Figures 1 and 2: Strain 10214 of Micro-

sum of the controls. Protein values are not

lease of soluble sulfhydryl-containing com-

Figures 7 and 8: Strain 10215 of Micro-

pounds from ethylene oxide-sterilized wool than

sporum gypseum shows significantly greater

shown by the controls. Maximum act:ivity is release of sulfhydryl-containing compounds than at 300 C. Protein values are higher in the test the sum of the controls. Temperature dif-

sample than in the control flasks but these ferences are slight and probably not significant. values are far less reliable as indicators of The protein release in the test samples is also keratinolysis.

Figures 3 and 4:

significantly greater than that in the sum of the Strain 11621 of Micro-

sporum canis shows no significant difference between the controls and the test sample by either measurement under the particular conditions of this experiment. Failure of this organism to release significantly greater amounts TABLE III Two strains of Microsporum gypseum—fungus controls and test samples at various temperatures Sulfhydryl Determinations in Micrograms Cysteine per cc Medium 24

7

10

Protein

Determinations in Micrograms Protein per cc Medium 24

7

10

hrs. wks. wks. hrs. wks. wks.

Strain 9083 350 C.

Fungus control Test sample 300 C.

Fungus control Test sample Room temp. Fungus control Test sample Strain 10215

0.3 0.0 0.0 0.0 0.0 0.0 2.2 20.8 24.3 0041.0 58.5 0.8 0.0 0.0 0.0 0.0 0.0 1.7 17.0 50.0 61.5

22.15.0 0.7 0.0 0.0 D.0 0.0 0.0 1.4 8.6 12.4 5.0 40.0 57.0

30° C.

Fungus control Test sample Room temp. Fungus control Test sample

Figures 9 and 10: Tryptic digestion of both sterilized and unsterilized wool is analyzed in these two figures.

Since the same wool sample (5 mgrn/cc) in

each case was resuspended (5 mgrn/cc) in fresh .025% trypsin solution at the end of three days and again at the end of five days, the values presented in Figures 9 and 10 as points are not cumulative values but represent release of the substances during each period of digestion.

We interpret the data as indicating a somewhat greater release of soluble sulfhydryl-contaming compounds from wool sterilized with ethylene oxide than from unsterilized wool by .025% trypsin solution. The maximum release was observed to occur during the first period of incubation and did not exceed 11 micrograms per cc. The release was not sustained and the total release was 16 micrograms per cc. These

figures are not of an order of magnitude to result in serious depletion of the total content of soluble sulfhydryl-containing compounds of the wool samples used (prior determinations reveal that a 5 mgm sample of our wool per cc releases 555 micrograms of soluble sulfhydryl-

350 C°

Fungus control Test sample

controls.

0.7 0.0 0.50.0 0.0 10.0 1.2 18.0 25.55.0 78.0 105.5

0.3 0.3 0.0 0.0 4.5 12.5 1.7 15.9 19.8 SO 19.0 95.5

1.0 0.0 0.0 0.0 0.0 7.0 1.7 14.6 17.9 0.5 82.5 118.5

containing compounds upon complete solubilization following refluxing with a mixture of hydrochloric acid 20% and formic acid 50%). Nevertheless we must conclude, in contrast to observations of Noval and Nickerson (15), ethylene oxide sterilization does alter wool to

a limited extent as far as its digestibility by trypsin is concerned and this alteration must

SUM OF KERATIN AND FUNGUS CONTROLS —TEST SAMPLES (KERATIN+ FUNGUS)

3230-

30°C

35°C

ROOM TEMP.

E 2826-

210-

E

8-

0 C-,

42-

0-

I

I

I

2O30405O6O70

I

I

I

I

I

boooooo

10203040506010 Time n Days

Time in Days

Time in Days

FIG. 3 SUM OF KERATIN AND FUNGUS CONTROLS

E

TEST SAMPLES (KERATIN + FUNGUS)

130-

30°C

35° C

. 20-

ROOM

TEMP.

hO100-

9080-

. TO60-

30-

g' zoC-,

I

I

I

I

I

I

0203040506070 Time in Days

I

I

I

I

0203040

Time in Days

FIG.

I

6070

I

I

I

I

Time n Days

4

-o

0

a (—)

0 E 0

C, 0 C-,

Time in Days

Time in Days

FIG. 5 305

I

h02030405060 0

Time in Days

3

307

KERATINOLYTIC ACTIVITY OF K. CAMS AND K. GYPSEUK

which render the keratin substrate more readily digestible.

Wool in Basal Salts Sterilized Wool in .025% Trypsin Unsterilized Wool in .025%Trypsin

8 DISCUSSION

The ability of dermatophytes to grow on native keratin substrates has been recognized

for many years and has led various investigators to conclude that growth alone is proof of keratinolytic activity (2, 5, 11, 17, 20, 22, 31, 32, 35, 36, 37). The fallacy of this conclusion was pointed out by Raubitschek (39) who decided in 1961 that no evidence of chemical keratinolysis for any of the dermatophytes had been uncovered up to the time of publication of his article. In 1961, Chattaway et al. (38)

'4-

as a 210U,

0 S

6-

U)

8 a

4-

0'

o 2-

also expressed skepticism about keratinase activity of dermatophytes. The evidence for keratinolytic activity of dermatophytes other than that of visible growth on keratin will be

01234561 I

1

I

I

I

I

I

Time in Days

briefly presented in the remainder of this sec-

FIG. 9

tion.

Daniels (3), in addition to noting penetration and disruption of human hair by Microsporum canis, also qualitatively determined the release of amino acids into the medium similar to amino acids found after hydrolysis of the hair. However, conclusions based on the use of human hair may be subject to error unless it is certain

8

0

that the hair has not be subjected to heat or damaging chemicals (waving lotions for instance). Unfortunately in Daniel's study the hair was also damaged by heat sterilization.

a-

Furthermore, qualitative release of amino acids

0

provides no assurance of keratinolysis since Bolliger (41) found amino acids in simple

8 a a, 0

aqueous extracts of hair.

a C)

C

0

The most elaborate investigations of the ability of a dermatophyte (Microsporum gypseum) to digest wool keratin was that of Stahl and coworkers (12, 25, 26, 27, 28, 34). They measured the cystine, cysteine, sulfate, and ammonia nitrogen contents of the residue remaining after incubation, consisting of mycelium plus wool. They observed a fall in

Time in Days

FIG. 10

cysteine and nitrogen and a rise in cystine and

insoluble residues necessitating some attempt to separate chemical values for wool from those for the mycelium, a task not possible by direct backs, several of which the authors recognized. means and not entirely acceptable as accurate 1. The wool was subjected to both milling by the indirect methods used in their study. 3. The experiments were discontinued too and autoclaving and, hence, was partially denatured. early, probably before true keratinolysis actu2. Chemical analyses were performed on the ally occurred, and this, perhaps more than any sulfate which lasted only 14—21 days and which they interpreted as indicating limited keratinolysis. Their investigation had some inherent draw-

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

308

chemically. This work, however, does not provide biochemical data to help settle the issue of whether dermatophytes are capable of keratinolysis of unaltered keratin. Chattaway et al. (38) investigated cell-free peptidase extracts from two trichophyton strains (T. rubrum and T. verrucosum) for keratinase activity using essentially the same criteria as we

II¼, 0—

have used for determining keratinolysis and were unable to find such activity. This is not conclusive evidence of lack of such activity by the organisms except under the highly artificial conditions imposed by their experimental procedure. Mercer and Verma (14) in an electron micro0—

48

I

I

I

I

I

I

6 20 24 28 32 36

scopic study of hair penetration by T. men-

tagrophytes give additional indirect support to the concept that true keratinolysis is within the capability of the dermatophytes. FIG. 11 Additional suggestive evidence for keratinoother, is the most troublesome feature of their lytic activity by dermatophyte organisms is presented by Evolceanu and Lazar (47). They study. The authors interpreted a lack of change of used the simple device of a two-chambered the chemical analyses (cystine, cysteine, sulfate, system permitting free flow of fluid from one ammonia nitrogen) occurring between the 14th compartment to the other but not permitting and 21st day as the result of cessation of kera- fungal penetration from one compartment to tinolytic activity, when, in fact, it is probable the other. They obtained some evidence of that true keratinolysis had not yet occurred to digestion of wool, hair and horn by cell-free a significant degree. For instance, their values for fluid from the adjoining compartment in which cystine content of the wool residues showed a fungi were allowed to grow on keratin substrate. gradual increase (8.55% to 10.29%) from the The fungi investigated were Micros porum canis, start of the experiment to the fourteenth day Microsporum gypseum, Clenomyces inter(28). Rather than indicating keratinolysis this digitale, and Clenomyces mentagrophytes. would point to a gradual dissolution of the non- Failure of the authors to mention what measures keratinous material, leaving increasingly higher were used to sterilize their keratin substrates concentrations of cystine-rich keratin. makes it very difficult to evaluate the signifiOne of our preliminary studies on a strain of cance of their observations. Micros po.rum gypseum involved removal of We interpret our results as clearly demonaliquots of the test sample at four day intervals strating a significant keratinolytic activity for the determination of soluble sulfhydryl-con- attributable to three of the four organisms taming compounds. Figure 11 illustrates the under investigation. It is evident that the kerafact that a fall in values occurred from the tinolytic process observed in the case of these eighth to the twentieth day after which a slow dermatophytes is a very slow one in contrast sustained rise occurred. We interpret this find- to the rapid keratinolysis observed by Noval ing as indicating that the organism will utilize and Nickerson (15, 16) with their strain of the more readily digestible material first and Streptomyces fradiae. It is possible that the that, until this is exhausted, true keratinolysis experimental conditions (temperature, PH, salt will not occur to any measurable degree. or trace element concentration, size of inoculum, Barlow and Chattaway (40) noted decreased degree of oxygenation, etc.) may not be optiresistance of keratin to digestion by certain mum for the keratinolytic process and further dermatophytes when disulfide linkages were evaluation may help to define more clearly the chemically disrupted and conversely increased optimum conditions. resistance with creation of additional linkages The existence of an extracellular keratinase I

12

TUne in Days

309

KERATINOLYTIC ACTIVITY OF M. CANIS AND M. GYPSEUM

elaborated by the dermatophytes remains to 10. Kapica, L. and Blank, F.: Formation of ammonium magnesium phosphate crystals in be established by further investigation. The cultures of fungi growing on keratin. Mycopresence of keratinolytic activity does not imply

pathologia, 18: 119,

1962.

the presence of one enzyme capable of such activity. Keratinolysis may be the result of a

11. MacFayden, A.: A contribution to the biology of the ringworm organism. J. Path. Bact., 3:

complex chain of reactions among which reduction of disulfide linkages occurs at some point

12. Mandels, G. R., Stahl, W. H. and Levinson, H. S.: Structural changes in wool degraded

but not necessarily as the primary step. The ultimate significance of the observations presented in this study awaits clarification. SUMMARY

1. Evidence

176, 1896.

by the ringworm fungus microsporum gyp-

seum and other microorganisms. Textile Res.

J., 18: 224, 1948. W., Thianprasit, M. and Rieth, H.: Demonstration, isolation and identification of keratin utilizing soil fungi pathogenic for

13. Meinhof,

the skin. Arch. Klin. Exp. Derm., 212, 1960.

is presented that one strain of

Micros porum canis and two strains of Micro-

sporum gypseum are capable of exerting a keratinolytic effect on ethylene oxide-sterilized wool.

14. Mercer, E. H. and Verma, B. S.: Hair digested by

trichophyton mentagrophytes. Arch.

Derm. (Chicago),87: 357, 1963. 15. Noval, J. J. and Nickerson, W. J.: Decomposi-

tion of native keratin by streptomyces

fradiae. J. Bact., 77: 251, 1959. 16. Noval, J. J. and Nickerson, W. J.:

Exocellular

2. Ethylene oxide sterilization, in contrast

keratinase from a streptomyces sp. Bact.

to the opinions expressed by previous workers, does alter wool to a limited extent in respect to enhancing the release of soluble sulfhydrylcontaining compounds by trypsin. Nevertheless, it is the most satisfactory method for sterilization of wool for such as investigation. 3. The keratinolytic activity, as measured by ability to release soluble sulfhydryl-coiitaining compounds from the wool, was found to exceed

17. Page, R. M.: Obsenations on keratin digestion by microsporum gypseum. Mycologia, 42:

that of trypsin but as yet the existence of an extracellular keratinase elaborated by these organisms remains unproven. REFERENCES

Proc., 125, 1956. 591, 1950.

18. Raubitschek, F.: In vitro invasion of hair by dermatophytes. Sabouraudia, 1: 87, 1961.

19. Raubitschek, F. and Maoz, R.: Invasion of nails in vitro by certain dermatophytes. J. Invest. Derm., 28: 261, 1957.

20. Roberts, L.: The physiology of the trichophytons. J. Path. Bact., 3: 300, 1894.

21. Roberts, L.: Experimental note on the fer-

ments of the ringworm fungi. Brit. Med. J., 1: 13, 1899.

22. Rogers, R. E., Hirschman, D. J. and Humfeld, H.: Comparison of growth of trichophyton interdigitale on wool fabric with and without additional nutritive media. Proc. Soc. Exp. Biol. Med., 45: 729, 1940.

1. Ajello, L.: The dermatophyte microsporurn 23. Schatz, A., Karlson, K. E. and Martin, J. J.: Trace element stimulation of keratin (hair) gypseum as a saprophyte and parasite. J. Invest.

Derm., 21: 157, 1953.

2. Bonar, L. and Dreyer, A. D.: Studies on ring-

worm funguses with reference to public health problems. Amer. J. Public Health, 22:

909,

1932.

3. Daniels, G.:

The digestion of human hair kera-

tin by microsporum canis bodin. J.

4.

Gen.

Microbiol., 8: 289, 1953. English2

M. P.: The saprophytic growth of

keratinophilic fungi on keratin. Sabouraudia,

2: 115, 1963. 5. Felsher, Z.; Observations on the fluorescent

material in hairs infected by microsporon in tinea capitis. J. Invest. Derm., 12: 139, 1949. 6. Hirschman, D. J., Zametkin, J. M. and Rogers, R. C;: The utilization of wool by four sapro-

phytic microorganisms in the presence of added nutrients. Am. Dyestuff Reptr., 33: 353, 1944.

degradation by oral keratinolytic microflora. Experientia, 12: 308, 1956.

24. Sen Gupta, S. R., Nigam, S. S. and Tandan, R. N.: A new wool-destroying fungus ctenomyces species. Textile Research J., 20: 671, 1950.

25. Stahl, W. H., McQue, B., Mandels, G. R. and Siu, R. G. H.: Studies on the microbiological

degradation of wool. I. Sulfur metabolism. Arch. Biochem., 20: 422, 1949.

26. Stahl, W. H., McQue, B., Mandels, G. R. and Siu, R. G. H.: Studies on the microbiological degradation of wool. Digestion of normal and modified fibrillar proteins. Textile Research J., 20: 570, 1950.

27. Stahl, W. H., McQue, B. and Siu, R. G. H.:

Decomposition of cystine and wool by treatment in the ball mill and autoclave. J. Biol. Chem., 177: 69, 1949.

7. Jensen, H. L.: Decomposition of keratin by 28. Stahl, W. H., McQue, B. and Siu, R. G. H.: Studies on the microbiological degradation soil microorganisms. J. Agr. Sci., 20: 390, of wool. II. Nitrogen metabolism, Arch. Bio1939. chim. 27: 211, 1950. 8. Kapica, L. and Blank, F.: Growth of candida albicans on keratin as sole source of nitrogen. 29. Tate, P.: On the enzymes of certain dermatoDermatologica, 115: 81,

9.

1957.

Kapica, L. and Blank, F.: Growth of candida

parpsilosis with keratin as sole source of nitrogen. Dermatologica, 117: 433,

1958.

phytes or ringworm fungi. Parasitology, 21:

31, 1929.

30. Tate, P.: The dermatophytes or ringworm fungi. Biol. Rev., 4:

41, 1929.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

310

31. Van Breusgehern, R.: La culture des dermatophytes in vitro sur des cheveux isoles. Ann. Parasitol., 24: 559, 1949.

40. Barlow, A. J. E. and Chattaway, F. W.: The attack of chemically modified keratin by certain dermatophytes. J. Invest. Derm., 24:

dermatophytes: A specific diagnostic method. Mycologia, 44: 176, 1952.

41. Bolliger, A.: Water extractable constituents of

32. Van Breusgehem, R.: Keratin digestion by

65, 1955.

hair. J. Invest. Derm., 17: 79, 1951.

33. deVries, G. A.: Keratinophilic fungi and their 42. Bolliger, A. and Gross, R.: Non-keratins of human toe nails. Aust. J. Exp. Biol. Med. action. Antonie Leeuwenhoek, 28: 121, 1962.

34. White, W. L., Mandels, G. R. and Siu, R. 0. H.: Fungi in relation to the degradation of woolen fabrics. Mycologia, 42: 199, 1950.

Sci., 31: 127, 1953.

43. Boyer, P. D.: Spectrophotometric study of the

reaction of protein sulfhydryl groups with organic mercurials. Amer. Chem. Soc. J., 76:

35. Williams, J. W.: Scalp products and hair as a culture medium for certain pathogenic fungi. Proc. Soc. Exp. Biol. Med., 31: 586, 1934. 36. Williams, J. MT.: Scalp products and hair before

44. Kolthoff, I. M. and Harris, W. E.: Amperometric titration of mercaptans with silver

pathogenic fungi. Proc. Soc. Exp. Biol. Med., 31: 944, 1934.

45. Benesh, R. and Benesh, R. E.: Amperornetric titration of sulfhydryl groups in amino acids

men and women as culture media for certain pathogenic fungi. Proc. Soc. Exp. Biol. Med.,

46. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurements

puberty as a culture medium for certain

37. Williams, J. W.: Scalp products and hair of 32: 624, 1935. 38. Chattaway, F. W., Ellis, D. A. A.

and Barlow,

J. E.: Peptidases of dermatophytes. J.

Invest. Derm., 41: 31, 1963.

39. Raubitschek, F.: Mechanical versus chemical keratolysis by dermatophytes. Sabouraudia, 1:87,1961.

4331, 1954.

nitrate using the rotating platinum electrode. md. Eng. Chem. Anal. Ed., 18: 161, 1946.

and proteins. Arch. Biochem., 19: 35, 1948.

with the folin phenol reagent. J. Biol. Chem., 193: 265,

1951.

47. Evolceanu, R. and

Lazar, M.: Le probleme de

l'existence et de la mise en evidence des

ferments Keratinolytiques chez les dermatophytes. Mycopathologia, 12: 216, 1960.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

24. Wynn, C. H. and Iqbal, M.: Isolation of rat

skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.

human epidermis, p. 511, The Epidermis

Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-

No warranty is given about the accuracy of the copy.

Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.

Cell Biol., 27: 603, 1965.

27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-

C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.

30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.

31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-

lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.

32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.

33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.

34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:

Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.

43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.

44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.

45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.

46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,

Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by

Res., 33: 176, 1964.