EXTRACELLULAR +23%sz ASSAYS -* or Bmsmmszs nsammms _: ~ AND HeLa ems. m CQMBWED'AND smmg mvmzons -_ '- < . Thesis €0r'th‘eD2me af'_..’s%~;fS.i . . - . gamma mat-e umaasm; —. -_ _. ., mamag-uLLELs-w _ . ‘ mud-fie a? ‘ ;_ HMS & 3]“: f ”0K filmy INC. 3 ,umuvumns Wm". Inc-ml '! ABSTRACT EXTRACELLULAR ENZYME ASSAYS OF BLASTOMYCES DERMATITIDIS AND HeLa CELLS IN COMBINED AND SEPARATE POPULATIONS BY June Dale Hillelson The production of extracellular enzymes by BZastomyces dermatitidis Gilchrist and Stokes, 1898, was investigated while the organism was growing in an environment more nearly comparable to that which supports the infecting yeast. HeLa cells, yeast cells, or a combination of these two cell types were grown in spent (aged 3 days and 11 days) and fresh tissue culture media enriched with 10% fetal calf serum. Exogenous alkaline phosphatase was also placed into the spent material, in a separate experiment. At 12 hour intervals for 60 hours, samples to be assayed were removed from each of the flasks. In another experi- ment, HeLa cells were combined with yeast cells in order to observe the total number of intracellular fungal organisms. Paranitrophenol (PNP) derivatives were chosen as sub- strates for 7 of the 11 enzymes studied. The remaining enzymes were assayed by the Sigma methods. June Dale Hillelson When combining B. dermatitidis yeast cells with HeLa cells, the two cell types might be stimulated to produce a greater quantity of exoenzyme than either of the cell types alone. The overall significance lies not so much in the production of the enzyme, but in its change in activity. The data were based on a comparison of the results of enzyme activity in the medium of the HeLa plus Fungus combination flask to the activity in the medium of each of the separate flasks, and by comparing enzyme activity in the medium of the separate flasks to their respective controls. When utilizing spent tissue culture medium, the data showed immediate increases and/or gradual increases in levels of activity for B-D-glucosidase, N-acetyl-B- glucosaminidase, glutamic-oxalacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT) in the medium of the HeLa flask.’ The results were similar for GPT in the Fungus flask, and with alkaline phosphatase in the HeLa plus Fungus flask medium. Immediate decreases and/or gradual decreases in levels of activity for alkaline phosphatase, exogenous alkaline phosphatase solution, acid phosphatase, and a-hydroxybutyric dehydrogenase (a-HBD) occurred in the medium of the HeLa flask. Similar results were observed with acid phosphatase and a-HBD in the Fungus flask medium, and with B-D-galactosidase, acid phosphatase, GOT, and GPT in the combination flask medium. HeLa cell lysis (60 hours) in the presence of yeasts pro- duced decreased levels of activity for N-acetyl-e- June Dale Hillelson glucosaminidase, alkaline phosphatase, and acid phospha- tase, but an increase in activity occurred with 8-D- galactosidase. When utilizing fresh tissue culture medium, the data showed immediate increases and/or gradual increases in levels of activity for acid phosphatase, GOT, GPT, and a-HBD in the HeLa flask medium. Similar results occurred with GOT in the Fungus flask medium. The combination flask medium at the zero hour demonstrated increased levels of activity for N-acetyl-B-glucosaminidase, alkaline phosphatase, and acid phosphatase. Glutamic-oxalacetic transaminase was increased in the combination flask medium through 48 hours. Immediate decreases and/or gradual decreases in the levels of activity of B-D—glucosidase, N-acetyl-B-glucosaminidase, acid phosphatase, and GPT were observed in the combination flask medium. Similar results occurred with a-HBD in the Fungus flask medium. HeLa cell lysis in the presence of yeasts produced decreased levels of B-D-glucosidase, N-acetyl-B-g1ucosaminidase, B-D-galactosidase, and alkaline phosphatase. Inhibitors produced by the yeasts or HeLa cells, and enzyme half-life, as well as production or destruction of the enzymes may have produced the above changes in enzyme activities. EXTRACELLULAR ENZYME ASSAYS OF BLASTOMYCES DERMATITIDIS AND HeLa CELLS IN COMBINED AND SEPARATE POPULATIONS By June Dale Hillelson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1976 Copyright by JUNE DALE HILLELSON 1976 To my parents and Blastomyces dermatitidis ii ACKNOWLEDGEMENTS The author wishes to thank Dr. A. L. Rogers for his friendship, support, patience, humor, and confidence in his graduate student; Dr. E. S. Beneke ("Father Fungus") for his kindness and helpful suggestions; Mrs. Martha Thomas for her sincere interest, valuable advice, and moral support; Louise Schaub for her friendship, availa- bility, and wisdom of enzyme chemistry; Dr. R. J. Moon for the use of his laboratory, and pearls of wisdom concerning research; Dr. L. Bieber for his interest and helpful sug- gestions during the course of this study; Dr. M. Patterson for serving on my committee; Ruth Vrable for her assistance with the tissue culture techniques; and Frank Buckalew for his warmth and inspiration. Lastly, I am greatly indebted to the Department of Microbiology and Public Health for research funds and graduate assistantships. iii TABLE OF DEDICATION. . . . . . . . ACKNOWLEDGEMENTS. . . . . . . LIST OF TABLES. . . . . . . . INTRODUCTION. . . . . LITERATURE SURVEY . . . . . . Intracellular Enzymes. Glucanase . . . Malate Dehydroge Galactosidases. Phosphatases. . Extracellular Enzymes. Phosphatases. . Urease. . . . . Uricase . . . Glucanases and Xylosidases. Glucosidases. . Galacturonanase Ribonuclease. . Collagenase Multiple Enzymes. Tissue Culture and Fung MATERIALS AND METHODS . . . . Organisms and Cultivati BZastomyces derm HeLa. . . Experiment I - Spent Me CONTENTS nase . o O O O O O i on. O O O atitidis. dium. . . Experiment II - Fresh Medium . . Experiment III - Alkaline Phosphatase. Experiment IV - Phagocy Extracellular Enzyme As Assays. tosis . . says. . . A1pha-—D- glucosidase, B- D- -g1ucosidase, N- ~acety1- B- glucosaminidase, a- -D- galactosidase, B-D-galactosidase, Alkaline Phosphatase, Acid Phosphatase . Glutamic- Oxalacetic Transaminase. (GOT) iv Page ii iii vi 29 30 Page Glutamic-Pyruvic Transaminase (GPT) . 31 Alpha- Hydroxybutyric Dehydrogenase (a- HBD) . . . . . . . 32 Lactic Dehydrogenase (LDH). . . . . . 33 RESULTS . . . . . . . . . . . . . . . . . . . . . . 34 Experiment I - Spent Medium. . . . 34 Alpha- -D- -glucosidase, B- D- glucosidase, N- -acetyl- B- glucosaminidase, a- -D- galactosidase, B-D-galactosidase, Alkaline Phosphatase, Acid Phosphatase . . . . . . . . . . 34 Glutamic- Oxalacetic Transaminase, Glutamic- -Pyruvic Transaminase . . . 37 Alpha- Hydroxybutyric Dehydrogenase, Lactic Dehydrogenase. . . . . . 39 Hydrogen Ion Concentrations and Cell Counts. . . . . . . . . . . 41 Experiment II - Fresh Medium . . . 43 Alpha- -D- -glucosidase, B- D- glucosidase, N- acetyl- B- glucosaminidase, a- -D- galactosidase, B-D-galactosidase, Alkaline Phosphatase, Acid Phosphatase . . . . . . . . . . 43 Glutamic- Oxalacetic Transaminase, Glutamic- -Pyruvic Transaminase . . . 47 Alpha- Hydroxybutyric Dehydrogenase, Lactic Dehydrogenase. . . . . 48 Hydrogen Ion Concentrations and Cell Counts. . . . . . 49 Experiment III - Alkaline Phosphatase. . . . Sl Alkaline Phosphatase. . . 51 Hydrogen Ion Concentrations and Cell Counts. . . . . . . . . . . . . SZ Experiment IV - Phagocytosis . . . . . . . . 52 DISCUSSION. . . . . . . . . . . . . . . . . . . . . SS LITERATURE CITED. . . . . . . . . . . . . . . . . . 68 LIST OF TABLES Table Page 1 Extracellular B-D-glucosidase activities in spent tissue culture medium . . . . . . . 35 2 Extracellular N-acetyl-B-glucosaminidase activities in spent tissue culture medium. . 36 3 Extracellular B-D-galactosidase activities in spent tissue culture medium . . . . . . . 36 4 Extracellular alkaline phosphatase activi- ties in spent tissue culture medium. . . . . 38 5 Extracellular acid phosphatase activities in spent tissue culture medium . . . . . . . 38 6 Extracellular glutamic-oxalacetic transamin- ase activities in spent tissue culture medium 0 O O O O O O O O O O I O O O O O O O 39 7 Extracellular glutamic-pyruvic transaminase activities in spent tissue culture medium. . 4O 8 Extracellular a-hydroxybutyric dehydrogen- ase activities in spent tissue culture medium 0 O O O O O O O O O O O O O O O C O O 40 9 Cell counts and hydrogen ion concentrations in spent tissue culture medium . . . . . . . 42 10 Extracellular B-D-glucosidase activities in fresh tissue culture medium. . . . . . . . . 44 ll Extracellular N-acetyl-B-glucosaminidase activities in fresh tissue culture medium. . 44 12 Extracellular B-D-galactosidase activities in fresh tissue culture medium . . . . . . . 45 13 Extracellular alkaline phosphatase activi- ties in fresh tissue culture medium. . . . . 46 14 Extracellular acid phosphatase activities in fresh tissue culture medium . . . . . . . 46 vi Table 15 16 17 18 19 20 Page Extracellular glutamic-oxalacetic transamin- ase activities in fresh tissue culture medium“ 0 O O O O I O O O O O O O O O I O O O 47 Extracellular glutamic-pyruvic transaminase activities in fresh tissue culture medium. . 48 Extracellular a-hydroxybutyric dehydrogen- ase activities in fresh tissue culture medium . . . . . . . . . . . . . . . . . . . 49 Cell counts and hydrogen ion concentrations in fresh tissue culture medium . . . . . . . 50 Extracellular alkaline phosphatase activi- ties in spent tissue culture medium. . . . . 51 Cell counts and hydrogen ion concentrations in spent tissue culture medium . . . . . . . 53 vii INTRODUCTION The investigation of extracellular enzyme production by any organism is enhanced by cellular enzyme localiza- tion. In studies of various fungal cells other than BZastomyces dermatitidis, vacuoles have been the major site of cytochemical localization of acid hydrolases (2,37). Both acid and alkaline phosphatases have been reported intracellularly. In Candida aZbicans, acid phosphatase was observed during different phases of cell growth (58). The activity increased up to 15-18 hours and then decreased. Sites of acid phosphatase activity were found in intracellular granules similar to lysosomes or in cytoplasmic vacuoles. In Schizophyllum commune, acid phosphatase activity was found to be associated with vacuoles, endoplasmic reticulum, lipid bodies, small mitochondrial granules, and the nuclear envelope (70). Acid phosphatase activity has also been demonstrated in fungi during lytic processes, such as the release of (ascospores from asci (105) and during the autolysis of fruiting bodies (44). Yeast protoplasts have been demonstrated to contain find have released acid phosphatase. Whether or not the (Enzyme was located within the plasmalemma or outside the ITrotoplast was studied by cytochemical means, in order to 1 2 gain more insight into the process of secretion of the exoenzyme. In Saccharomyces cerevisiae, acid phosphatase was found localized over the cell wall of both the mother cell and its bud, and over the forming septum (7). Adja- cent cell wall vesicles also showed acid phosphatase activity. Prior to cytochemical studies, evidence for conclud- ing that certain phosphatase enzymes were on the cell surface was that phosphorylated substrates considered incapable of penetrating the cell membrane were hydrolyzed by intact cells. Later scientists began to isolate cell walls that were enzymatically active or converted yeast cells into protoplasts and showed that the enzyme was released from the cell as the cell wall was digested. Support for the study of extracellular enzymes is evidence which relates the importance of specific enzyme to the development of the organism. Extracellular enzymes cleaving B-glucosidic linkages have been suggested to play a role in the maturation of certain fungi (104). The sig- nificance of these enzymes cleaving B-glucosidic linkages may be that most of the higher fungi have cell walls rich in the various B-glucans. These enzymes are probably involved in cell wall alteration during various morpho- genetic events. Other studies with this enzyme deal with the role of B-glucosidase in the metabolism of cellulose and B-glucosides (25). Biochemical and genetic studies have described certain enzymes whose expression is closely related to the control of progress through the developmental 3 phase of the life cycle of Dictyostelium discoideum (21). These studies indicated that the expression of B-glucosidase was directly related to the control of development. It can further be described as stage specific or developmen- tally controlled. An increase in both a-D-galactosidase and 8-D- glucosidase activity as the basidiocarp of Pleurotus ostreatus reaches maturity suggests that these enzymes could be related to the fruiting process (55). Higher fungi may be expected to have a-galactosidase in order to utilize the a-galactosides present in plant materials. The exact role of acid and alkaline phosphatase in metabolism is unknown. Acid phosphatase activity is associated with the meiotic process in Calvatia cyathi- formis because of the increased activity found in meiotic tissue, as opposed to that found in post-meiotic tissue (12). The loss of alkaline phosphatase activity, on the other hand, is correlated with the inability of certain mutants of Aspergillus niduZans lacking alkaline phospha- tase to cleave organic phosphates (67). Several enzymes have been localized and have been shown to be important in the development of several fungi, but relatively few have been demonstrated extracellularly. This is particularly true for those fungi causing systemic disease. Beneke et a1. (9) investigated the presence of several extracellular enzymes of B. dermatitidis. The yeast phase (37 C) was reported to produce alkaline and acid phosphatase when grown in both liquid and solid 4 casein-peptone-yeast extract-glucose (CPYG) agar. Unpub- lished data showed that lower phosphatase levels were produced when the yeast phase was grown in brain-heart- infusion medium. No a- or B-glucosidases, a- or 3- galactosidases, N-acetylglucosaminidases, or fatty acid esterases were detected. The objective of this research is to investigate the production of exoenzymes by B. dermatitidis, and to study the enzyme activities in an environment more nearly com- parable to that which supports the infecting yeast. BZastomyces dermatitidis can be maintained more readily in the yeast phase in tissue culture medium with HeLa cells than in artificial medium. By placing yeast cells together with HeLa cells, both in spent and in fresh tissue culture media, one might expect that the two cell types in combination will be stimulated to produce a greater amount of exoenzyme than either of the individual cell types alone. Exogenous enzyme will also be placed into the spent medium experiment, in order to observe its activity over a period of time. Finally, parasitism of the yeast cells by the HeLa cells will be investigated. LITERATURE SURVEY Blastomyces dermatitidis Gilchrist and Stokes, 1898, the imperfect stage of an Ascomycete, AjeZZomyces derma- titidis McDonough and Lewis, 1968, is the causative agent of North American blastomycosis. It is a dimorphic fungus, growing in tissues in culture at 37 C as a round or oval, 8-15 u thick-walled budding yeast and on conventional media (25 C) as mycelium giving rise to pear-shaped or exogenous spores. Conversion from mycelial to yeast growth, and vice versa, is easily accomplished in vitro by varying the temperature. Ricketts (73) and Hamburger (31) were among the first to observe in vitro fungal growth and indicated temperature as an influential factor in differentiating between the two morphological types. This finding has since been confirmed by Michelson (56), DeMonbreun (l8), and Levine and Ordal (52). Salvin (83) concluded that no specific carbohydrate, amino acid, or other "growth substance" was implicated as an essential factor for yeast-like growth. The organism grew best in a medium containing only serine or hydroxyproline as sole amino acids, or in a medium containing twenty different amino acids. The amount of growth produced, however, did vary according to the medium. 6 The fungus of North American blastomycosis is found mostly in the central and southeastern portions of the United States and, less frequently, in Canada and Africa. Defining the geographic distribution of blastomycosis is difficult due to the lack of availability of an acceptable method to detect infection, and the inability to recover the etiological agent from its saprophytic environment. The natural habitat of B. dermatitidis remains problematic. Since infections appear to be acquired by inhalation of spores, the organism is considered to be a soil saprophyte. However, only a few attempts at isolating the organism from soil in endemic areas have been successful. Spores have been isolated from the soil in Lexington, Kentucky, and in Georgia (19,20). BZastomyces dermatitidis has been cultivated in the laboratory on sterilized soil (6,27), but the basic problem with soil isolation was that repeated sampling for the organism from previously positive loca- tions turned out to be negative. Dogs, along with other animals, have frequently acquired this disease, but are not known to transmit the infection to man. North American blastomycosis, or Gilchrist's disease, named for the man who first reported the skin disease in 1894 (76), is divided into three clinical patterns of disease, cutaneous blastomycosis, pulmonary blastomycosis, and systemic dissemination (8). Cutaneous blastomycosis is acquired by spore inhalation and spread to the skin, or by implantation of spores into broken skin. The lesions occur on the more exposed parts of the body, namely hands, 7 wrists, face, feet, and ankles. The initial lesion may spread slowly during the course of the disease, which may be for months or years. This may be followed by satellite lesions occurring in more distant areas. Pulmonary blastomycosis, developing from spore inhala- tion, is manifested by miliary abscesses in the lung. This is usually accompanied by granulomatous reactions. The yeast cells are visible within the abscesses and granulo- mata. From the lungs, infection can spread hematogenously to establish focal lesions in skin, bones, prostate and other viscera, except for the gastrointestinal tract, to produce systemic blastomycosis. The lesions of North American blastomycosis are not unique to man. Bowen and Wolbach (ll) inoculated four mice intraperitoneally and observed abdominal and pulmonary lesions. They described the pathologic process as "filling of the alveoli with large cells with little other reaction." After having injected the yeast form intraperitoneally, DeMonbreun (18) found that "each of six mice died in from three to five weeks after inoculation, and numerous small abscesses containing the fungus cells were found in the lungs, liver, spleen and kidneys." In mice inoculated intraperitoneally with yeast by Bergstrom et a1. (10), lesions developed in five weeks, but the animals did not die until ten weeks following inoculation. Davis (16) observed that the organisms were taken up by leukocytes and macrophages following injection into the peritoneal cavity of guinea pigs. 8 Salfelder (82) found that subcutaneous injections of the yeast form of B. dermatitidis produced a non-ulcerative active skin lesion in animals, which healed without dissemination. During their laboratory induced disease study, Ebert and Jones (24) found that 105 yeast cells injected intra- peritoneally were sufficient to produce blastomycosis in dogs. Treatment with amphotericin B, the drug of choice, prevented the death of some of the injected animals. Baker (5), working with yeast cells, injected mice intraperitoneally with living B. dermatitidis, killed suspensions of the organism, and a phosphatide fraction of this fungus. He found that repeated injections of killed yeasts were toxic and often lethal for mice. The lesions, in mice receiving the live yeast cells, were mostly composed of organisms, as opposed to other infec- tious diseases in which the lesions primarily consist of the reacting host cells. The phosphatide fraction brought about a monocytic response, and was therefore not impli- cated in the necrotizing effect produced by the living organisms. Baker did not continue studying the phospha- tide fraction because he was particularly interested in the suppurative response. Peck and Hauser (65) isolated minute amounts of a phospholipid-containing carbohydrate fraction from the yeast form of B. dermatitidis. Later, Peck (64) found that this constituent was associated only with pathogenic fungi. 9 Prior to the work of DiSalvo and Denton (22) in 1963, relatively few attempts had been made to determine cellular constituents responsible for the virulence of B. dermati- tidis. These authors looked at four strains of B. der- matitidis and observed a greater abundance of lipid in the more virulent of the four strains. 7 Having demonstrated in 1972 (14) that two strains of B. dermatitidis with different virulence for mice also differed in the amount of bound phospholipid present in the cell wall, Cox et a1. (15) later attempted to clarify certain aspects of the pathogenesis of blastomycosis by looking at host resistance and the factors relating to the virulence of the infecting agent. Mice were inoculated with cell-wall fractions from the two above strains, to compare the histological responses and the differences in virulence. The same pathologic response resulted when either whole cells or just cell walls were injected. Infarct-like tissue response was common to both groups, and the necrosis seemed limited to the areas of accumulated inocula. From this, the authors suggested that the cell walls might possess endotoxin activity, but made no con- clusions as to the relationship between toxicity and necrotic lesions. In 1952, Salvin (84) demonstrated specific endotoxin by injecting acetone-dried yeast cells of B. dermatitidis and tubercle bacilli intraperitoneally into mice. Death occurred in about 80% of the mice in 48 hours when com- ponents from 80 mg of acetone-dried cells were injected. 10 Taylor (98) found that a similar toxin was released when B. dermatitidis yeast cells were treated with trypsin or with 1% HCl. Lethality was enhanced when a suspension prepared from disrupted yeast cells, containing the spe- cific endotoxin, was injected intraperitoneally into mice previously inoculated with Newcastle disease virus (43). The specific factors implicated in fungal infection have been less investigated than those mechanisms in bacteria. Attempting to correlate virulence with an endotoxin is as much an enigma as trying to correlate virulence with an enzyme or a group of enzymes. Enzyme production alone by an organism does not seem sufficient for pathogenesis. For example, Trichophyton evolceanui and T. indicum are two non-pathogens which produce elastase (38). Rippon and Varadi (78), on the other hand, demonstrated elastase production by a relatively old culture of Streptomyces madurae, a pathogen, and no production of this enzyme by any of the other actinomycetes tested. Purnell and Martin (67) were the first to associate alkaline phosphatase activity with virulence in any patho- genic organism. They found that certain mutants of Asper- giZZua nidulans, lacking alkaline phosphatase and unable to grow on medium with beta-glycerophosphate, had a decreased virulence for mice. Mutants without alkaline phosphatase that were able to grow on the medium were virulent. Also, a mutant without alkaline phosphatase which could grow on the beta-glycerophosphate medium was ll virulent. The authors reported a definite decrease in virulence of certain strains associated with the presence of specific mutations affecting alkaline phosphatase activity. Decreased virulence in those strains without alkaline phosphatase was associated with an inability to split certain organic phosphates. Montes and Wilborn (58) investigated Candida aZbicans cytochemically throughout the logarithmic and stationary growth phases. Intracellular activity increased up to 15-18 hours of growth and then began to drop. Acid phosphatase activity was located in small intracellular granules or in yeast vacuoles. The authors suggest that this enzyme activity correlates directly with fungal virulence, but this relationship needs to be clarified through further experimentation. In C. aZbicane, toxic activity has been reported to be associated with hydrolytic enzyme activity and the presence of an intracellular alkaline phosphatase by Kurup (48), a leucine aminopeptidase in cell extracts by Kim et al. (46), an excreted peptidase by Staib (92), and a plasma coagulase by Zaikina and Elinov (108). In media containing protein as the nitrogen source, proteo- lytic enzymes are excreted by certain strains of C. albicans (72). Only the proteolyzing strains caused severe infection when injected into mice. 12 Intracellular Enzymes Much of the fungal enzyme work to date has been done using intracellular enzymes produced by fungi other than B. dermatitidis. Glucanase Glucanase production by Histoplasma capsulatum and B. dermatitidis was investigated by Davis and Domer (17) with the intention of locating autolytic enzymes capable of degrading cell wall carbohydrates from these two organisms. Glucanases were found in cellular extracts and in culture filtrates from the yeast and mycelial phases of both organisms. The fungal enzymes appreciably hydrolyzed only the glycoprotein fraction of the cell walls. It was suggested that a relationship between cell wall growth and these autolytic enzymes may exist. Malate Dehydrogenase Isoenzymes of malate dehydrogenase (80) were studied in cell-free extracts, mitochondrial fractions, and cyto- plasmic portions of both the mycelial and yeast phases of B. dermatitidis. .Through the use of electrophoresis on polyacrylamide gels, isoenzyme patterns of both fungal phases were apparent in several bands. The mitochondrial fraction of the yeast phase demonstrated two bands, whereas five bands were present in the cytoplasmic portion of the same phase. 13 Galactosidases Intracellular B-D-galactosidase activity in single yeast cells of Saccharomyces Zactis taken from a random population was measured (106) using a fluorogenic assay. Photographs were taken to establish specific stages of the growth cycle of each yeast cell. Similar enzyme levels were found in cells without buds and in cells with intermediate-size buds. Mature cells, or nearly mature cells, possessed approximately double the level of this enzyme. Alpha-galactosidase received attention from Arnaud et a1. (3). They grew Penicillium duponti, a thermo- philic fungus, in medium containing either raffinose or stachyose. This particular enzyme was selected because of its ability to hydrolyze raffinose in beet molasses and to hydrolyze oligosaccharides in vegetable seeds. Their experiment showed that a-galactosidase from P. duponti exhibited thermostability characteristics dif- ferent from other fungal a-galactosidases. Phosphatases In 1974 (61) acid phosphatase was used to investi- gate the relationship of enzyme levels in the identifica- tion and the taxonomy of pathogenic species of Candida. It is known that nonspecific acid phosphatase is localized on the outer surface of the cell membrane in S. cerevisiae (86,96). Ueda and Oshima (100) described a recessive constitutive mutant, phoT, with decreased 14 ability to assimilate inorganic phosphate (Pi). Reces- sive constitutive mutants, phoR and phoU, were unable to accumulate Pi. Komano (47) related the culture age of A. niger to the formation of multiple forms of acid and alkaline phosphatases by first extracting the mycelium, separating and purifying into four fractions, and then observing the changes in activity of these phosphatases in peptone medium during growth. Extracellular Enzymes The pathogenic significance of enzymes, if any, depend partly on whether or not they are excreted from the organism or if they are associated with the fungal cell surface so they may attack host substrates. Phosphatases Differentiating between the products of secretion and autolysis is often difficult. By using alkaline phosphatase as a cell marker, proteases of Microeporum canis (a dermatophyte) were determined to be truly extra- cellular (63). Since the autolytic marker enzyme was present in minute amounts when the proteases appeared, the process was thought to be true secretion. Beneke et al. (9) reported the production of extra- cellular acid and alkaline phosphatases by species of B. dermatitidis in a single culture medium. The yeast and mycelial phases were shown to secrete different amounts of enzyme into the culture medium. Enzymes displaying no 15 activity in the culture medium were: B-glucosidase, a- and B-galactosidases, N-acetylglucosaminidase, and butyrate, palmitate, acetate, laurylate, and stearate esterases . Urease Urease is particularly interesting because it was the first enzyme to be isolated in crystalline form and the first example of an enzyme shown to contain sulfhydryl groups (95). Shibata (87) found urease in A. niger mycelium, whereas Kiesel (45) found both urea and urease in the same organism. Much later, Rosenthal (79) incorporated a specific substrate into the medium, in order to enhance specific identification of pathogenic fungi. An application of this is seen today in the differentiation of two similar species of dermatophytes. Trichophyton rubrum does not hydrolyze the urea in 7 days, as opposed to T. mentagro- phytes, which does hydrolyze the substrate. In 1962, urease was demonstrated as a specific sub- strate in B. dermatitidis (99). In growing cultures with urea as the sole nitrogen source, and in cell extracts, both the mycelial and yeast phases demonstrated a consti- tutive urease activity. This activity was shown to be both intracellular and extracellular. Uricase In 1962, Taylor (97) found uricase in the mycelial phase of B. dermatitidis, but not in the yeast phase of 16 the organism. Under alkaline conditions (pH 9.0), this activity was inhibited, but in the mycelial form the organism continued to grow well and to utilize uric acid acid at pH 6.5, 7.0, and 8.0, at 25 C. Glucanases and Xylosidases Within the last two years, extracellular glucanases have been studied extensively in fungi. Beta-glucanases have been produced and separated in asynchronous cultures of Cryptocoacus albidus var. aerius (59). In Schizosac- charomyces japonicus var. versatilis, an exo-beta-glucanase was isolated (28). Eriksson and Pettersson (1,26,94) worked extensively with exo- and endo-l,4-beta-g1ucanases used by Sporotrichum pulverulentum for cellulose breakdown. In 1975, they began by purifying, characterizing, and separating five endo-l,4-beta-glucanases (94). In their next publication, the activities of the five enzymes towards carboxymethylcellulose were compared (1). Follow- ing this, purification and characterization of an exo-l,4- beta-glucanase were performed (26). Beta-xylosidases were also detected by Notario et a1. (60) in Cryptococcus albidus var. aerius, provided the medium contained glucose as the sole carbon source. Friebe and Holldorf (29) worked on the inactivation of 1,3-beta-glucanase activity in the Basidiomycete species QM806. Glucosidases A large molecular weight glucosidase of A. fumigatus was purified and, by polyacrylamide gel electrophoresis 17 followed by carbohydrate and protein staining, was shown to be a glycoprotein (81). Wilson and Niederpruem (102) examined the regulation and nature of enzymes cleaving beta-glucosidic linkages in Schizophyllum commune in order to relate these par- ticular enzymes to mycelial growth and basidiospore germination. They found the enzymes to be present in low amounts during growth and germination. Galacturonanase Through salting-out and two different types of chroma- tography procedures, an exo-D-galacturonanase was purified from a culture filtrate of A. niger (32). This enzyme not only catalyzes the degradation of D-galacturonans by terminal action but also acts on other substrates of medium and high molecular weights with the same terminal ends. Ribonuclease Cryptococcus Zaurentii and its haploid Basidiomycete relative, TremeZZa foliacea, exhibited ribonuclease pro- duction in liquid medium (13), which was completely repressed in both fungi by the addition of orthophosphate. These organisms have a close affinity to the Basidiomycetes and certain Ascomycetes which were shown to have extra- cellular ribonuclease activity. Other Ascomycetes tested did not have this actiVity. These results suggested a close relationship between the two previously mentioned fungi. 18 Collagenase An extracellular collagenase from T. schoenleinii was observed (74) to be different from bacterial collagenase in having a smaller molecular weight and requiring an acid pH. This fungal enzyme proved to be inhibited by ethylene- diaminetetraacetic acid (EDTA) as opposed to bacterial enzymes. The activity of the bacterial enzyme can be restored by magnesium and calcium. Multiple Enzymes In 1969, Rippon and Garber (77) investigated dermato- phyte pathogenicity as a function of mating type and related enzymes. Enzyme profiles of several dermatophytes and B. dermatitidis exhibiting enzymatic differences in sexual stages were demonstrated in 1971 (75). In the yeast phase of B. dermatitidis, the plus mating type produced elastase, urease, and hydrolyzed casein, while the minus mating type produced urease, and hydrolyzed casein, but no elastase. One mycelial strain (M784) tested produced alkaline phosphatase, acid phosphatase, elastase, leucine amino peptidase, urease, and hydrolyzed casein. The other mycelial strain (M788) produced gela- .tinase, alkaline phosphatase, acid phosphatase, leucine amino peptidase, urease, and also hydrolyzed casein. Tissue Culture and Fungi Tissue culture techniques have been employed to a limited extent in the study of pathogenic diphasic fungi. Friedheim and Baroni (30) were the first to look at the 19 effects of fungi on cultured animal cells in vitro. By inoculating guinea pig fibroblast cultures and mouse embryonic tissue, and splenic leukocytes of mice and guinea pigs with Nocardia asteroides, they observed dis- integration of the fibroblasts and leukocytes. They attributed this disintegration to the exhaustion of nutrients required by the animal tissue cells to the rapid growth of the fungus. Pathological information concerning work on this inoculation of chicken and rat fibroblasts with B. der- matitidis, Coccidioidee immitie, H. capeulatum, and M. Zanoeum was recorded by Duque (23). Blaetomyces dermati- tidis was shown to have no effect in his tissue culture system. Randall and McVikar (93) observed the development of yeast cells of H. capsulatum in fibroblast cells of tissue cultures prepared from horse placental tissue and chick embryos. Histoplasma capsulatum has also been cultured in human tissues (68), in HeLa cells (51), and in Earle's L-strain mouse cells (69). Upon subsequent inoculation of mice with H. capsulatum, Hill and Marcus (35) demonstrated that cultured mononuclear cells from the animals were able to restrict the growth of this yeast. Larsh et a1. (49) demonstrated the value of HeLa cells in culture for converting the mycelial phases of B. der- matitidis, H. capsulatum, C. immitie, and Sporothrix schenckii to their yeast phases. Previously, Lubarsky and Plunkett (53) noted that with some strains of C. immitis 20 tissue phase conversion occurred in animal tissue culture medium alone. HeLa cell culture techniques were found to be valuable in the laboratory identification of the diphasic fungi (36). Larsh et al. (50) expanded their earlier report to include tissue culture as providing an efficient tool for the evaluation of antifungal drugs. In 1959, Wang and Schwarz (102) studied phagocytosis of several pathogenic fungi and yeasts by human white blood cells. This report described the general features of phagocytized yeast-like organisms in vitro. Howard and Herndon (42) compared cultured peritoneal exudate cells inoculated with the mycelial phase of B. dermatitidie to those inoculated with the mycelial phase of H. capsulatum. Both fungi converted to the yeast phase. It was found that B. dermatitidis destroyed cell cultures to a degree directly proportional to the number of inocu- lated fungal cells. Also, the fact that B. dermatitidis had a strong tendency to form aggregates during prolifera- tion greatly hindered any study of effective intracellular parasitism. From this work, the authors concluded that B. dermatitidis was less suited to animal tissue culture studies involving peritoneal exudates. In 1964 Howard (39) reported an intracellular genera- tion time of 10.3 i 1.5 hr for H. capsulatum in mouse histiocytes, and said that this generation time was not affected by the age of the histiocytes. From these earlier data, Howard (40) published findings on the !- 5-1;. “A“...a... a? V i '1 ___.i-—. _ _ 21 intracellular growth of five strains of H. capeuZatum in guinea pig and mouse histiocytes. The intracellular generation time was not changed when yeast cells were exposed to specific antibodies and complement. No dif- ferences were noted in cells from immunized and normal animals, with or without previous exposure of the Histo- pZasma cells to complement and hyperimmune sera. Wagoner et al. (101) undertook a study of conversion to the yeast phase of various H. capsulatum isolates in stationary HeLa cell cultures. Conversion took place in Hely or Medium 199, with fresh guinea pig serum. When calf, human, horse, and chicken sera were used, conversion took place to a lesser extent. Mycelium two, four, and eight weeks of age displayed no apparent differences in their ability to convert to the yeast form. Hempel and Goodman (33) described a convenient method for the positive identification of H. capsulatum, B. der- matitidis, and S. schenckii whereby the time required for conversion to the yeast phase is considerably shortened. Mycelial phases of each of these organisms were used to inoculate primary cell cultures of guinea pig peritoneal macrophages. These tissue cultures were stained after 24 hours and the characteristic yeast cells were observed microscopically. The effect of different temperatures (25, 30, 37 C) on the intracellular growth of H. capsuZatum was questioned by Howard (41). Specifically, data concerning blastospore formation, germ tube development, and yeast-phase growth 22 within mouse, frog, and fish histiocytes was reported. In 1967, Stanley and Hurley (93) compared the growth of various species of Candida in mouse renal epithelial cell cultures with that of a control yeast, S. cerevisiae. Cultures of murine renal epithelial cells were destroyed at different rates by the various species of Candida and were partially destroyed by the non-pathogenic control. The rate of destruction was directly proportional to the size of the fungal inoculum. From this, the authors suggested that pathogenic mechanisms are an expression of the effects of fungi on mammalian cells. MATERIALS AND METHODS Organisms and Cultivation Blastomyces dermatitidis ’ The Oklahoma strain, isolated from a patient in the Oklahoma Medical Center, The University of Oklahoma, Oklahoma City, Oklahoma, was obtained from the laboratory of Dr. E. S. Beneke and Dr. A. L. Rogers, Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan. The mycelial phase, initially grown on Sabouraud's glucose agar, was transferred to brain-heart—infusion agar (BHIA) and placed at 37 C in order to obtain conversion to the yeast phase. The mycelial phase was not investigated. After conversion to the yeast phase on BHIA, the cells were subsequently washed in sterile physiological saline, centrifuged, and placed into Joklik- Modified minimum essential medium (MEM) without phenol red (GIBCO). Sterilized fetal calf serum (GIBCO) was added to obtain a ten percent solution. This stock culture was maintained on a rotator shaker (New Brunswick Scientific Company, model G2), at 200 revolutions per minute. Every ten days the yeast suspension was resuspended in fresh MEM with 10% fetal calf serum, and returned to the shaker. 23 24 All tissue culture media refrigerated longer than one month were supplemented with 29.2 mg of L-glutamine (GIBCO) per 100 ml and streptomycin sulfate (Pfizer) at 100 mcg per m1 plus penicillin G (Pfizer) at 100 Units per m1 prior to use. Throughout the entire experiment, sterility checks using sheep blood agar and Sabouraud's glucose agar were done whenever the contents of any flask were manually 1.9.1.1 9.. ml? V disturbed. [I r. 4 HeLa Stock cultures of the HeLa (human epithelioid carcinoma),originally from the American Type Culture Collection Cell Repository, were obtained from Wayne Roberts, Clinical Microbiology Laboratory, Michigan State University, East Lansing, Michigan. The serial line in monolayer was aseptically maintained at 37 C in 75 cm2 plastic Falcon tissue culture flasks. Joklik-Modified MEM without phenol red was used with 10% fetal calf serum for culturing HeLa cells. A humidified mixture of 5-108 CO2 with air was maintained to adjust the pH to circa 7.2-7.4. The cultures were routinely transferred under a laminar flow hood when the monolayer became dense (about four days). Culture transfer techniques, utilizing Hank's balanced salt solution (pH 7.2) and trypsin-EDTA (GIBCO), described by Merchant, Kahn and Murphy (54), were followed in this investigation.‘ 25 Experiment I - Spent Medium Growth curves of B. dermatitidis were done in order to ascertain a period of growth referred to as late log phase. Log phase occurred on the second day of growth and continued through the twelfth day. The eleventh day of yeast growth was chosen for investigation as a period during late log phase. Two Falcon tissue culture flasks, each containing 100 m1 of 106 fungal cells/ml MEM with 10% fetal calf serum, were placed at 37 C. (All cell counts were per- formed by placing one drop of a 1:3 dilution of fungal cells in phosphate buffered saline and 0.4% trypan blue vital stain in normal saline (GIBCO) onto a Neubauer hemacytometer.) A control flask, containing all sub- stances except the organism, was placed at 37 C. The three flasks were incubated for 11 days. On the eighth day, stock HeLa monolayers were trans- ferred. These two tissue culture flasks, each containing 100 ml of 10S HeLa cells/ml MEM with 10% fetal calf serum, were placed at 37 C. One control flask with all substances except cells 'was placed at 37 C. These flasks were kept in the incubator for three days. On the eleventh day of fungal growth and on the third day of tissue culture growth, the medium from one of the test monolayers was removed and replaced with the contents of one of the test fungal flasks. The five flasks con- taining spent media (3 day old and 11 day old media) and sera were labeled and tested as: HeLa, Fungus, HeLa plus 26 Fungus, Fungus Medium Control (11 day old medium with serum), and HeLa Medium Control (3 day old medium with serum). Immediately before combining the HeLa cells with the B. dermatitidis cells, the hydrogen ion concentrations of all five flasks were adjusted to pH 7.2-7.4. The addi- tion of B. dermatitidis with its growth medium to a HeLa monolayer marked the zero hour in the first three experi- ments. At this time, 0.5 m1 from each of the test flasks containing cells was removed sterilely for viability counts. Ten milliliters from each of the five flasks were removed and placed into appropriately labeled sterile screw cap tubes. These sampler tubes were then centri- fuged for 10 min at 1000 rpm, followed by the removal of the cell-free supernatants and the placement of the tubes onto ice. From these tubes, volumes corresponding to the amount of test material required for each enzyme assay were aliquoted into separate tubes. Depending on the survival conditions of a particular enzyme, the aliquots were either placed on ice for immediate testing, or placed at 4 C or at -20 C for future testing. At twelve hour intervals after the zero hour, 10 ml samples to be assayed and separate 0.5 ml samples to be counted were removed from each of the five flasks, prior to adjusting the pH. The samples were then centrifuged and aliquoted in the same manner as previously described zero hour samples. 27 All phases of all experiments were done in duplicate. The experiments were terminated when dead HeLa cells, which incorporated the intense blue color of the vital stain, were lysed. The lysis occurred between 50 and 60 hours. Experiment II - Fresh Medium The contents of a stock tissue culture flask adjusted to 100 ml of 2 x 108 fungal cells/ml MEM with 10% fetal calf serum were washed in sterile normal saline. Follow- ing centrifugation for 10 min at 1000 rpm, 106 yeast cells/ ml were placed into two tissue culture flasks containing 100 ml of fresh MEM with 10% fetal calf serum. At the same time, media from two flasks, each con- taining a three day old monolayer, were discarded. One of these flasks received 100 ml of fresh MEM with 10% fetal calf serum. The other HeLa flask received the con- tents of one of the above fungal flasks, again in fresh medium. The three flasks and a single general medium control (without organism of any kind) were incubated at 37 C. The four flasks containing fresh medium and serum were labeled and tested as: HeLa, Fungus, HeLa plus Fungus, and General Medium Control. The procedures concerning pH, cell counting, sampling, and aliquoting at the zero hour and subsequent 12 hour intervals until 60 hours, were performed as in Experiment I. 28 Experiment III - Alkaline Phosphatase Experiment III followed the same basic protocol as Experiment I, with the following modifications. One of the fungal flasks containing 106 cells/ml was added to a monolayer tissue culture flask, as in Experiment I. The first modification was at the zero hour, when 5 ml of an alkaline phosphatase solution (500 ug/ml in MEM with 10% fetal calf serum, followed by millipore filtration) were added sterilely to all flasks except the one mono- layer medium to be discarded. Each flask before the addition contained 45 ml of experimental material. The final total volume in each of the 5 flasks was 50 m1. All of the flasks, including the media controls, contained 50 ug/ml of alkaline phosphatase. Another modification was the removal of only 3 ml of test material per 12 hour interval from all of the flasks, since only the alkaline phosphatase assay was to be performed. Experiment IV - Phagocyxosis Ten milliliters of 1.56 x 105 HeLa cells per m1 of MEM, supplemented with 10% fetal calf serum,‘were trans. ferred to each of 8 sterile plastic petri dishes. Each petri dish contained a 22 x 50 mm coverglass (Sargent). Twenty-four hours later, 106 yeast cells were added to each dish. One coverglass was removed, fixed, and stained with Wright's stain at l, 2, 3, S, 9, 24, 51 and 77 hours after exposure to the yeasts. In the HeLa cell culture, 29 the total number of intracellular B. dermatitidis bodies per 43X field was counted. Extracellular Enzyme Assays Assays All enzyme assays were monitored colorimetrically using a Bausch and Lamb Spectronic 20 spectrophotometer. Enzyme activity is expressed in umol per unit time. Readings were made at 30 second intervals. Whenever available, positive controls (Sigma 2-N) were incorporated into the assay procedures. Al ha-D- lucosidase, B-D:g1ucosidase, 'N-acetyI-B-glucosaminiH§se,'a-D- alac- tosidase, B-DfigaIactosidase, A1 a 1ne Phosphatase, Acid Phosphafase To detect the above various extracellular enzymes produced in the culture filtrate, assays were performed utilizing the appropriate chromogenic substrates (Sigma). Free paranitrOphenol (PNP) is released (104) as the enzyme reacts with the substrate. Seven PNP derivatives were chosen as substrates for the above enzymes studied. Assays were performed every 12 hours for 60 hours. The following substrates and buffers were prepared according to the method described by Beneke et a1. (9), with modifications made in some of the buffer concentrations: PNP-a-D-glucoside (0.5 mg/ml), PNP-B-D- glucoside (0.5 mg/ml), PNP-N-acetyl-B-glucosaminide (0.15 mg/ml) all in sodium acetate buffer (0.1 M), pH 5.4; PNP-a-D-galactoside and PNP-B-D-galactoside, both 1.0 30 mg/ml in citrate-phosphate buffer (0.05 M), pH 7.0; PNP- phosphate (1.0 mg/ml) in 0.05 M Tris-HC1 buffer, pH 8.6 for alkaline phosphatase. No change was made in the preparation of PNP-phosphate (1.0 mg/ml) in 0.1 M sodium acetate buffer, pH 5.0, for acid phosphatase. The assay for the hydrolysis of the above substrates was modified from the method of Beneke et al. (9). One— half milliliter of the medium supernatant (enzyme source) was added to 0.8 ml of the substrate solution. The reac- tion was immediately stopped by the addition of 1.7 ml of 0.5 M Tris buffer (pH 9.8), which develOps the yellow color of free PNP. A duplicate set of cuvettes contain- ing the test material plus substrate solutions was incu- bated for two hours at 37 C prior to the addition of Tris buffer. Enzyme activity was initially determined as a change in absorbance units at 410 nanometers over a 2 hour period during incubation at 37 C. Standard curves were made by preparing serial dilutions of free PNP in each of the buffers. To obtain a volume of 3 ml, 0.8 m1 of the PNP dilution was combined with 2.2 ml Tris buffer. The absorbance at 410 nanometers was graphed against umol of liberated PNP. Glutamic-Oxalacetic Transaminase (GOT) The method used for assaying GOT was that of The Sigma Chemical Company (91). No modifications of the actual method were instituted. The reaction mixture con- tained 1.0 ml substrate and 0.2 ml test material. One 31 milliliter of color reagent was added after an incubation period of one hour at 37 C, followed by the addition of 10 ml of 0.4 N NaOH. Media controls were treated as test samples. As recommended by Sigma, those samples not being tested immediately may be kept at 4 C for at least 2 weeks. The GOT samples from each major experiment were assayed as a group after two days of refrigeration. Glutamic-oxalacetic transaminase catalyzes the reac- tion between a-ketoglutarate and L-aspartate which yields glutamate plus oxalacetate. The amount of oxalacetate formed in one hour is determined by the formation of a hydrazone from oxalacetate which was measured at 505 nonometers. Standard curves were prepared using a standardized calibration solution containing pyruvic acid. One Sigma- Frankel Unit of GOT forms 4.82 x 10-4 umol of glutamate/min at pH 7.5 and 25 C. Results of GOT activity were expressed in umol x 104/min at pH 7.5 and 25 C. Glutamic-Pyxuvic Transaminase (GPT) The treatment of the media controls as tests, the reaction mixture, and the storage of samples to be tested as one major group from each experiment were handled as in the GOT assay. The activity of GPT was measured in the same manner as the GOT. This enzyme catalyzes the reaction between 32 a-ketoglutarate and alanine forming glutamate plus pyruvic acid. Alpha-Hydroxybutyric Dehydrogenase (a-HBD) Alpha-hydroxybutyric dehydrogenase was assayed by the method of The Sigma Chemical Company (89). The enzyme catalyzes the reduction of a-ketobutyric acid to a-hydroxy- butyric acid in the presence of NADH. The amount of l a-ketobutyric acid remaining after the incubation period , _...._.__.___ .|\¢ , l I {In—_- _. J—___ is determined by forming its colored dinitrophenylhydra- zone in alkaline solution. The activity of a-HBD is inversely proportional to the intensity of this color, measured at 440 nanometers. No modifications in the actual procedure were made. The reaction mixture was made by mixing 0.5 ml substrate and 0.5 mg NADH with 0.05 test material. One milliliter of color reagent was added after an incubation period of one hour at 37 C, followed by the addition of 5.0 ml of 0.4 N NaOH. As recommended by Sigma, those samples not being tested immediately may be stored at -25 C for two weeks. The a-HBD samples from each major experiment were assayed as a group after two days at -20 C. Media controls were treated in the same manner as test samples. Standard curves were prepared using a stock HBD substrate. One Sigma Unit of a-HBD will reduce one millimicromole of a-ketobutyric acid per minute at 25 C. 33 Results of a-HBD activity were expressed in umol x 104/ min at 25 C. Lactic Dehydrogenase (LDH) Attempts were made to detect extracellular LDH. The enzyme was assayed by the method of The Sigma Chemical Company (90). RESULTS Experiment I - Spent Medium Alpha-D-gluc0§idase, B-nglucosidase, NFacetylefifgIEcosaminidase, a- 4'alac- ‘ tosidase, B-DF alactOSidasex'AIka ine Phosphatase, _cid’PhosphataSe The extracellular activities of the above enzymes in spent tissue culture medium with the exceptions of a-D-glucosidase and a-D-galactosidase are given in Tables 1 through 5. Standard curves, made by preparing serial dilutions of free PNP in each of the buffers, produced a linear curve with absorbance being directly proportional to umol PNP. In the first experiment, a change in absorbance from zero to two hours yielded no measurable a-D-glucosidase or a-D-galactosidase activity in any of the flasks through- out the 60 hour testing period. The media of the flasks with the Fungus and the Fungus Medium Control demonstrated no enzyme activity in either of the glucosidase assays (Table l). The HeLa plus Fungus medium showed no B-D-glucosidase activity. From the zero hour to the 60th hour, the HeLa Medium Control did not change in B-D-glucosidase activity. The HeLa medium showed slightly increasing levels of B-D-glucosidase activity with time. 34 35 Table l. Extracellular B-D-glucosidaie activities in spent tissue culture medium Time (hrs) 0 12“” 24 36 48fi' 60 HeLa 48 48 51 54.5 54.5 54.5 Fungus 0 0 0 0 0 0 HeLa plus Fungus 0 0 0 0 _ 0 0 HeLa Medium Control 44 44 44 44 44 44 Fungus Medium Control 0 0 0 0 0 0 * The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. With respect to N-acetyl-B-glucosaminidase activity (Table 2), the Fungus flask medium remained essentially unchanged. Enzyme activity in the media of both of the controls was also constant. The medium with the HeLa plus Fungus combination was uniform in enzyme activity, except at the end of the experiment, where this activity dropped off completely. Increasing levels of N-acetyl-B- glucosaminidase with time were seen in the HeLa flask medium. The Fungus medium and the Fungus Medium Control showed no activity in either of the galactosidase assays (Table 3). The HeLa medium and its control yielded essentially identical and consistent enzyme activities in the assay for B-D-galactosidase over the 60 hour period. The HeLa plus Fungus combination medium demonstrated a lack of 36 Table 2. Extracellular N-acetyl-B-glucosaminidase activi- ties in spent tissue culture medium* Time (hrs) 0 12 24 36 48 60 HeLa 164 164 164 171 191 191 Fungus 42 42 42 44 44 44 HeLa plus Fungus 51 51 51 51 53 0 L HeLa Medium Control 22 22 22 24 24 24 Fungus Medium Control 22 22 22 23.5 23.5 23.5 * The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. Table 3. Extracellular B-D-galactosidase activities in spent tissue culture medium Time (hrs) 0 I2 ’24 36 48 6D HeLa 29 29 29 29 29 29 Fungus 0 0 0 0 0 0 HeLa plus Fungus 0 0 0 0 0 29 HeLa Medium Control 29 29 29 27.5 29 29 Fungus Medium Control 0 0 0 0 0 0 8 The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. 37 B-D-galactosidase activity, except at the endpoint of the experiment, where enzyme activity first became measurable. The Fungal Medium Control demonstrated consistency in phosphatase activities (Tables 4 and 5) with an absence of activity in the zero acid phosphatase tube. The Fungus medium yielded essentially consistent enzyme activities in all but the high reading in the zero acid phosphatase tube. Decreasing levels of alkaline and acid phosphatase were seen in the HeLa flask medium. The HeLa Medium Con- trol flask showed a slight increase in acid phosphatase activity, with slight variability occurring in alkaline phosphatase. Phosphatase levels decreased in time in the medium with the HeLa plus Fungus. Glutamic-Oxalacetic Transaminase, Glutamic-Pyruvic Transaminase The extracellular activities of the glutamic trans- aminases in spent tissue culture medium are given in Tables 6 and 7. Standard curves represent decreasing levels of a-ketoglutaric acid, while either oxalacetic or pyruvic acid levels are increasing. Color intensity of the resulting hydrazones is proportional to transaminase activity. The Fungus and HeLa media controls exhibited identical and unchanging levels of GOT throughout the 60 hour testing period (Table 6). The media of the flasks with the Fungus and the HeLa plus Fungus also yielded con- sistent levels of GOT, with the combination flask medium 38 Table 4. Extracellular alkaline phosphatase activities in spent tissue culture medium* Time (hrs), 0 12 24 36 48 60 HeLa 28 23 23 18 l8 l4 Fungus 28 28 28 30 28 30 HeLa plus Fungus 83 32 32 32 30 9 HeLa Medium Control 98 102 106 102 102 100 Fungus Medium Control 21 21 21 23 21 21 R The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. Table 5. Extracellular acid phosphatase activities in spent tissue culture medium Time (hrs) 0 12—7 24 36 48 EU HeLa 112 94 91 72 72 72 Fungus 39 10 10 10 10 10 HeLa plus Fungus 39 28 10 10 10 10 HeLa Medium Control 6 8 8 10 10 10 Fungus Medium Control 0 10 10 10 10 10 * The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. 39 Table 6. Extracellular glutamic-oxalacetic transaminase activities in spent tissue culture medium HeLa 58 67 77 86 115 134 Fungus 48 48 48 48 48 48 HeLa plus Fungus 67 67 67 67 67 67 HeLa Medium Control 19 19 19 19 19 19 Fungus Medium Control 19 19 19 19 19 19 * The enzyme activity is expressed in umol x 10,000/ min, calculated from absorbance. Average of two assays. having a somewhat higher level of enzyme activity. A noticeable increase in GOT was seen in the HeLa flask medium. .With respect to GPT levels (Table 7), all of the media in the flasks had constant levels of enzyme activity, with the highest level being obtained by the HeLa flask medium. Alpha-Hydrgxybutyric Dehydrogenase, Lactic Dehydxggenase The extracellular activities of a-HBD in the various flasks are shown in Table 8. Both the LDH and the a-HBD standard curves illustrate enzyme activity as being inversely prOportional to color intensity. Identical and constant levels of a-HBD were found in the media of both of the controls and in the medium with the HeLa plus Fungus. 40 Table 7. Extracellular glutamic-pyruvic transaminase activities in spent tissue culture medium Time (hrs) 0 ’12 24 36 48; 6O HeLa 211 211 211 211 211 211 Fungus 173 173 173 173 173 173 HeLa plus Fungus 154 154 154 154 154 154 HeLa Medium Control l9 19 19 19 19 19 Fungus Medium Control 154 154 154 154 154 154 * 4 The enzyme activity is expressed in umol x 10,000/ min, calculated from absorbance. Average of two assays. Table 8. Extracellular a-hydroxybutyric dehydrogenase activities in spent tissue culture medium Time (hrs) 0 12 _24 36 48 60 HeLa 250 200 50 50 0 0 Fungus 350 350 350 250 250 250 HeLa plus Fungus 450 450 450 450 450 450 HeLa Medium Control 450 450 450 450 450 450 Fungus Medium Control 450 450 450 450 450 450 * The enzyme activity is expressed in umol x 10,000/ min, calculated from absorbance. Average of two assays. 41 Decreasing levels were observed in the media of both the HeLa and the Fungus flasks. The results of the LDH activities in the various flasks are not shown, because of the nature of the standard curve. The standard curve is constructed such that the amount of pyruvate is inversely proportional to the amount of LDH activity in the sample. The values obtained were greater than 0.960 umol, which were well out of the range of sensitivity of the calibration curve recommended by Sigma (90). Hydrogen Ion Concentrations an Cell Counx§_ In the medium with the HeLa plus Fungus (Table 9), the observed fungus counts ranged from 4.8 x 106 cells/m1 at the zero hour to 7.5 x 106 cells/ml by the 60th hour. Viable HeLa cells remained attached to the plastic tissue culture flask and were not normally found floating in 5 cells/ml) were first suspension. Dead fungi (1.5 x 10 noted at the 48th hour, as opposed to dead HeLa cells floating in suspension at the 24th hour. These dead HeLa cells were not intact by the 60th hour. The hydrogen ion concentration varied little during the first 48 hours, but by the 60th hour conditions inside the flask had become more alkaline. The medium in the Fungus flask (Table 9) maintained a basic pH throughout the experiment, despite adjustment to pH 7.2-7.4 with HCl every 12 hours. The cell 42 N.H O.H O.H 0.0 N.H O.H me u: mswcnm ~.H O.H O.H O.O ~.a O.H mm o: OH»: 0.0 O.a O.“ 5.5 O.a O.a me mOH x O.m OOH x O.H O O O O He\mHHou msmeam OOOO mHmsH mOH x O.O mOH x O.H mOH x O.H O O He\mHHou OHO: OOOO . Hs\mHHou OOH x O.H OOH x O.“ OOH x A.O OOH x 0.0 OOH x 0.0 OOH x O.O weaned HOHOH OHOHH mOH x O.m OOH x O.H mOH x O.H O O He\mHHou «Hem HOHOO msmnsm\mqe: 0.0 O.O 0.0 O.O O.O O.O ma O O O O O O He\mHHou OOOO OOH x O.H OOH x O.H OOH x O.O OOH x m.m OOH x H.m OOH x O.O He\mHHou HOHOO mswcsm H.H O.A O.A ~.H 4.5 N.A :O O O O O O O Hs\nHHOu OOOO mOH x O.H mOH x O.H mOH x O.H O O O Hs\OHHou HOHOO «Ho: OO be On ON NH O OmugqloeHO sswuoe ensuaso.m=mmwu unomm cw macaumnuceocoo new cement»: one mpcsou HHoo .m oHnmh 43 6 6 concentrations ranged from 4.4 x 10 cells/ml to 7.5 x 10 cells/ml, and were 100% viable. Hydrogen ion concentrations within the HeLa flask (Table 9) ranged from pH 7.0-7.4. ’Dead cells were not seen, but viable floating HeLa cells were seen by the 36th hour. In both of the control flasks (Table 9), the hydrogen ion concentrations ranged from pH 7.0-8.0. Experiment II - Fresh Medium A_pha-D -1ucosidase,,8- D_glucosidase, . N- -acety _g%ucosam1n1dase, a nga1ac- tosidase, B __galact6§1d§3e, AIkal1ne Rhesphatase, Acid Phosphatase The extracellular activities of the above enzymes in fresh tissue culture medium with the exception of a-D- glucosidase and a-D-galactosidase are given in Tables 10 through 14. A change in absorbance from zero to two hours yielded no measurable a-D-glucosidase or a-D-galactosidase activity in any of the flasks throughout the 60 hour testing period. The HeLa flask medium and its control yielded identi- cal enzyme activities in the assay for B-D-glucosidase (Table 10), excluding the final measurement. Decreasing levels of B-D-glucosidase activity were seen in all of the media in the flasks. The HeLa medium showed increasing levels of N-acetyl- B-glucosaminidase activity (Table 11) with time. A slight increase was seen in the Fungus flask medium, and a decrease 1.1 44 Table 10. Extracellular B-D-glucosidase activities in fresh tissue culture medium* Time (hrs) 0 III ' 24 36 48 60 HeLa 76 73 73 73 69 66 Fungus 76 73 73 73 69 60 HeLa plus Fungus 76 69 68 66 66 0 General Medium Control 76 73 73 73 69 58 it The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. Table 11. Extracellular N-acetyl-B-glucosaminidase activities in fresh tissue culture medium Time (hrs) 0 12 Z47 —36 48 6D HeLa 253 257 257 277 281 283 Fungus 260 263 263 263 263 265 HeLa plus Fungus 269 257 257 257 257 0 General Medium Control 240 240 242 242 242 244 it The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. 45 through the 48th hour in the HeLa plus Fungus flask medium. No appreciable change in enzyme activity occurred in the General Medium Control. The media of both the HeLa flask and its control yielded essentially identical B-D-galactosidase activities (Table 12). The HeLa flask medium maintained a constant level of B-D-galactosidase activity, with a slightly Table 12. Extracellular B-D-galactosidase activities in A fresh tissue culture medium Time (hrs) US 12’ 24' 36 48’ 6D HeLa ' 44 39 4o 40 4o 40 Fungus 39 39 44 44 44 44 HeLa plus Fungus 40 40 40 42 44 11 General Medium Control 39 39 39 39 39 39 * The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. increased level at the zero hour. The General Medium Con- trol did not change from the zero hour to the 60th hour in its level of B-D-galactosidase. Increasing levels of activity were seen in the Fungus flask medium, and up to the 60th hour in the medium with the HeLa plus Fungus, where the activity dropped drastically. Overall decreases in phosphatase activity (Tables 13 and 14) were observed in the media of the HeLa plus Fungus 46 Table 13. Extracellular alkaline phosphatase activities in fresh tissue culture medium Time (hrs) _0 12 24 36 48 60 HeLa 83 111 106 102 88 69 Fungus 98 120 106 116 102 83 HeLa plus Fungus 116 111 106 102 83 65 General Medium Control 153 130 106 102 88 69 * The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. Table 14. Extracellular acid phosphatase activities in fresh tissue culture medium Time (hrs) 02* 512 24’ 36 48 266 HeLa 39 54 61 54 72 58 Fungus 58 60 50 36 25 6 HeLa plus Fungus 76 61 47 47 36 10 General Medium Control 36 36 36 27 27 10 * The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. 47 flask and in the General Medium Control. In both the HeLa flask and the Fungus flask media, decreasing levels of alkaline phosphatase were seen after 12 hours. Variability with time was seen in acid phosphatase activity in the HeLa flask medium. Glutamic-Oxalagetic Transaminase, 'Glutamic4PyruVic TranSaminase The extracellular activities of the glutamic trans- aminases in fresh tissue culture medium are given in Tables 15 and 16. The HeLa, the Fungus, and the HeLa plus Fungus media exhibited increasing levels of GOT (Table 15) throughout the 60 hour period. The General Medium Control showed a constant level of enzyme activity. Table 15. Extracellular glutamic-oxalacetic transaminase activities in fresh tissue culture medium Time (hrs) *6— I2 24 36 48 60 HeLa 77 77 86 86 115 163 Fungus 67 67 86 96 96 96 HeLa plus Fungus 86 86 96 96 96 115 General Medium Control 67 67 67 67 67 67 * The enzyme activity is expressed in umol x 10,000/ min, calculated from absorbance. Average of two assays. With respect to GPT levels (Table 16), the media for the Fungus, the HeLa plus Fungus, and the General Medium 48 Table 16. Extracellular glutamic-pyruvic transaminase activities in fresh tissue culture medium Time (hrs) 0 12' 24 36 48 60 HeLa 77 96 96 96 111 111 Fungus 86 86 86 86 86 86 HeLa plus Fungus 86 86 86 86 86 86 General Medium Control 86 86 86 86 86 86 * The enzyme activity is expressed in umol x 10,000/ min, calculated from absorbance. Average of two assays. control demonstrated identical and constant levels of GPT with time. Only the HeLa flask medium showed any kind of change in activity, which was an increase over the 60 hour period. A1pha-Hydroxybutyric Dehydrogenase, Lactic DehydrggenaSe The extracellular activities of a-HBD in the various media are shown in Table 17. Increasing levels of activity were observed in both the HeLa and in the HeLa plus Fungus media. The General Medium Control demonstrated a constant level of activity, whereas the enzyme activity in the Fungus flask medium decreased with time. The difficulty in measuring LDH in Experiment I also occurred in this experiment. The values obtained were well out of the range of the standard curve. 49 Table 17. Extracellular a-hydroxybutyric dehydrogenase activities in fresh tissue culture medium* Time (hrs) 0 12 24 36 ‘48 I60 HeLa 150 150 270 450 450 450 Fungus 250 150 150 150 150 10 HeLa plus Fungus 150 150 250 250 250 650 General Medium Control 150 150 150 150 150 150 * The enzyme activity is expressed in umol x 10,000/ min, calculated from absorbance. Average of two assays. H dro en Ion Congentrations and Cell Counts In the HeLa plus Fungus medium (Table 18), the 6 observed fungus counts ranged from 1.1 x 10 cells/ml at the zero hour to 1.4 x 106 cells/m1 by the 60th hour. Dead fungi and dead HeLa cells floating in suspension were both first noted at the 48th hour. Total lysis of HeLa cells occurred by the 60th hour. The hydrogen ion concentrations ranged from pH 7.0-8.4. The Fungus medium (Table 18) remained at pH 7.8 for the final 48 hours. The cell concentrations ranged from 6 cells/ml to 1.6 x 106 cells/ml, and were 100% 1.3 x 10 viable. Hydrogen ion concentrations within the HeLa medium (Table 18) ranged from pH 7.0-7.4. Dead cells were not seen, but viable floating cells were seen by the 48th hour. 50 {'1 O.A O.A O.H O.u O.H O.H me u: Hmpecou 0.0 O.H O.H H.H O.H O.O ma OOH x O.O OOH x O.H O O O O Hs\OHHou OOOOOO OOOO OHOOH OOH x O.H O O O O Ha\OHHou OHom OOOO HE\mHH00 OOH x O.H OOH x O.H OH x H.H OOH x O.H OOH x H.H OOH x H.H memes» HOHOH OHOOH OOH x O.H O O O O Ha\OHHou OH»: HOHOO mamcnm\mqo: O.A O.A O.a O.u O.a 0.0 ma O O O O O O He\OHHou OOOO OOH x O.H OOH x O.H OH x H.H OOH x O.H OOH x O.H OOH x O.H Ha\OHHOu HOHOH mswcam O.“ O.a H.H H.H O.A O.H an O O O O O O Ha\OHHou OOOO OOH x O.H OOH x O.H O O O O He\OHHou HOHOH «Hem OO Ame OO yea NH .1 O (OOHOO oeHe eswwes eunuazo enmmwu smehm :O Ocowuwhuceocoo cow cemopvxn ecu muasoo HHeu .wH canes 51 In the General Medium Control (Table 18), the hydrogen ion concentration remained at pH 7.6. Experiment III - Alkaline Phosphatase Alkaline Phosphatase The results of the enzyme activity after the addition of alkaline phosphatase into Spent tissue culture medium are indicated in Table 19. Throughout the 60 hour testing period, results were identical and consistent in all but the medium in the HeLa flask. In the HeLa flask medium, a notable decrease in enzyme activity occurred with time. Table 19. Extracellular alkaline phosphatase activities in spent tissue culture medium Timeg(hrs) 0* 12 24 36 48 60 HeLa 770 745 702 584 346 314 Fungus 752 752 752 752 752 752 HeLa plus Fungus 752 752 752 752 752 752 HeLa Medium Control 752 752 752 752 752 752 Fungus Medium Control 752 752 752 752 752 752 * The enzyme activity is expressed in umol/Z hr, calculated from a change in absorbance over a 2 hour period. Average of two assays. 52 Hydrogen Ion Concentrations and Cell Counts In the HeLa plus Fungus medium (Table 20), the fungus 6 cells/ml at the zero hour counts ranged from 5.25 x 10 to 7.4 x 106 cells/ml by the 60th hour. Dead HeLa cells were first noted floating in suspension at the 24th hour, and dead fungi were first noted at the 48th hour. Lysis of HeLa cells occurred by the 60th hour. The hydrogen ion concentrations ranged from pH 7.2-8.5. The medium in the Fungus flask (Table 20) remained at pH 8.0 until the 48th hour, when it dropped to pH 7.6. The cell concentrations ranged from 5.0 x 106 cells/m1 to 7.3 x 106 cells/ml, and were 100% viable. Hydrogen ion concentrations in the medium of the HeLa flask (Table 20) ranged from pH 6.9-7.1. Dead cells were not seen, but viable floating cells were seen by the 36th hour. The hydrogen ion concentrations of the HeLa Medium Control ranged from pH 7.2-8.0, and in the Fungus Medium Control ranged from pH 7.3-8.0 (Table 20). Experiment IV - Phagocytosis After combining HeLa cells with B. dermatitidis cells, coverslips were examined at l, 2, 3, 5, 9, 24, 51 and 77 hours to determine the total number of intracellular yeast bodies per 43X field. Rounded HeLa cells and yeasts were seen throughout the 77 hour period, usually in segregated clumps. By the 77th hour, poikilocytic HeLa cells were observed. The coverglass removed at the 5th hour contained 53 O.H O.a O.A 0.0 O.“ 0.0 me u: msmcam O.a O.a O.H O.A N.“ O.O :O O: «HO: O.A N.O 0.0 N.A O.“ 0.0 ma OOH x O.O OOH x O.H O O O O Ha\OHHou OOOOOO OOOO OHOOH OOH x 0.0 OOH x O.H OOH x O.H O O He\OHHou OHO: OOOO Ha\mHHeo OOH x O.A OOH x O.H OOH x 0.0 OOH x 0.0 OH x 0.0 OOH x O~.O OOOOOO HOHOO OHOHH OOH x O.O OOH x O.H OOH x O.H O O Ha\OHHou «Ho: HOHOO msmcam\eqom O.A O.“ 0.0 0.0 0.0 0.0 an O O O O O O HO\OHHou OOOO OOH x 0.0 OOH x O.H OOH x 0.0 OOH x 0.0 OH x 0.0 OOH x 0.0 Ha\OHHou HOHOO msmcsm H.H O.O O.O O.O O.O O.H me O O O O O O He\OHHou OOOO OOH x O.H OOH x O.H OOH x O.H O O O He\OHHou HOOOO OHom OO, OO Om: OH NH» O OOHHO OOHO eswvoe ousuaso memmfiu «comm :O Oceaumpuceucoo cow :owouu»: was mucsou HHou .om oHan 54 large, rounded, eosinophilic-staining HeLa cells, with 0-1 cells per 43X field which had phagocytized 1-2 yeast cells each. Phagocytized cells were not observed at any other time. .-— ru—tw DISCUSSION The interpretation of the experimental data will be discussed by comparing the activity in the media of the HeLa plus Fungus flask to the activity in the medium of each of the separate test flasks, and by comparing the activity in the medium of the separate test flasks to the activity of their respective media in the control flasks. Increased activity of any enzyme may indicate that the enzyme is no longer needed within the cells or that during the time of greatest activity within the cells and lowest extracellular levels, there was active synthesis of these enzymes, followed by release when they were no longer required intracellularly. A decrease in enzyme activity could result from the conservation of the enzymes by the cells which synthesize them in order that they be used within the cells. Assays were performed to detect the presence of two glucosidases, but only B¥D-g1ucosidase showed any activity. In Experiment I, the only test flask medium to demonstrate any activity was the HeLa cell medium. The HeLa Medium Control, when subtracted from its test flask medium, yielded slightly increasing levels of activity with time. The medium from the combination population flask, like the 55 56 Fungus flask medium, demonstrated no activity. Any enzyme activity present in the HeLa flask medium was quickly reduced. A factor involved in this reduction may have been the time element, since no enzyme activity was seen in the 11 day old control medium, as opposed to that which was seen in the 3 day old control medium. It is also possible that the medium in the HeLa flask may have been completely devoid of enzyme, since the 3 day old medium control demonstrated a level of enzyme production similar to that of the HeLa flask medium. The fetal calf serum may have been the only source of enzyme, which was degraded before the eleventh day. The media in all flasks had B-D-glucosidase activity in fresh tissue culture medium, except medium in the combination flask, which was at the zero hour equal to the activity in the medium of each of the separate flasks, but then gradually decreased to an undetectable level of enzyme activity. This suggests destruction or inhibition of the enzyme activity by metabolites of one or both of the two cell types. Again, the actual production of exo- enzyme by one of the two cell types is questionable, as the Medium Control showed similar activity to the tests. The overall significance here, and with the other enzymes assayed, lies not in the production of the enzyme but rather in its change in activity. At the zero hour in Experiment 11, the combining of HeLa cells with fungal cells produced no change in B-D-glucosidase levels. At the 60th hour, enzyme activity was totally absent, 57 possibly destroyed or inhibited by the lytic HeLa cell products. The highest level of N-acetyl-B-glucosaminidase in the first experiment was produced in the medium of the HeLa flask, which was approximately three times greater than that produced in the media by either the Fungus or the HeLa plus Fungus. A major portion of the enzyme produced by the HeLa cells was not preserved at any point in time up to 60 hours when the two cell types were com- bined. Whether all of the HeLa enzyme was destroyed and the fungus stimulated to produce slightly more enzyme in the presence of HeLa cells is speculative. The level of enzyme in the medium of the combination flask was lost at 60 hours, possibly because products of dead HeLa cells destroyed or inhibited the enzyme. The question of whether endogenous serum enzyme is the only source of enzyme is answered by the lower level of enzyme activity observed in the media controls as opposed to the media in the test flasks. The consistent and almost identical enzyme activity in both the 11 day and the 3 day controls might also lend support to the stability of the enzyme in the experimental system. In the second experiment, the medium in the HeLa plus Fungus flask demonstrated variable enzyme activity for N-acetyl-B-glucosaminidase relative to the activity in the medium of the separate flasks. Yem and wu (107) reported that this exoenzyme from bacteria does not vary significantly with respect to growth media, carbon source, 58 or phase of growth. If this is true, the variation in enzyme activity for the HeLa plus Fungus flask medium may be accounted for by metabolic products from the cell types themselves. The HeLa plus Fungus flask medium had the greatest enzyme activity of the three test flasks' media at the zero hour, probably due to the combining of the two cell types. For the next 24 hours, all three test media were at about equal levels of activity, indi- cating that some of the enzyme present in the HeLa plus Fungus flask medium at the zero hour was inhibited or destroyed by the 12th hour. At 60 hours, HeLa cell death occurred. Lysis of these cells at this time was accom- panied by a total loss of the enzyme activity in the com- bination flask medium, probably due to the HeLa lytic products. The Medium Control demonstrated that endogenous enzyme was already present in the fetal calf serum in fresh tissue culture medium. The first experiment demon- strated a significant decrease in enzyme activity upon incubation at 37 C for at least 3 days. The controls were stable for each experimental condition. Assays were performed to detect the presence of two galactosidases, but only B-D-galactosidase activity in the HeLa flask medium was inhibited or destroyed when the fungus was added to the HeLa cells. The enzyme itself may have been present in the fetal calf serum, since the enzyme was present in the HeLa Medium Control flask. Apparently, a loss of enzyme activity occurred at some time before 11 days of incubation at 37 C. At 60 hours, 59 when HeLa cell death occurred, intracellular products of lysis or removal of inhibitor may have accounted for the sudden appearance of this enzyme in the HeLa plus Fungus medium. In fresh tissue culture medium, the B-D-galactosidase activities in the media of the respective flasks showed only slight variations. The values for this enzyme in the medium of the HeLa plus Fungus flask were similar to those observed both in the media of the separate test flasks and in the control through 48 hours. Enzyme activity in the medium of the combination flask was par- tially inhibited or destroyed in 60 hours at the time of HeLa cell lysis, probably because of intracellular HeLa cell metabolites causing a change. The presence of enzyme in the control again suggests exogenous B-D-galactosidase residing in the serum of both experiments. Intracellular B-galactosidase activity has been reported to be affected by the ionic composition of the medium (4). Salts containing SO43, Cl', Na+ and Mg++ stimulate enzyme activity. Intracellular B-galactosidase activity in yeasts has also been reported to increase near the end of the cell cycle (106). Beta-galactosidase is an inducible enzyme. The experimental design does not clarify the questionable presence of the enzyme. In the absence of lactose in the medium as the major carbon source, B-galactosidase is expected to be present at very low levels inside the cells, and not to be secreted to the external environment. No significant increase in 60 extracellular activity was seen at any time during the experiments. Neither one of the organisms together or separately showed any extracellular activity toward PNP derivatives of a-D-glucoside or a-D-galactoside. These enzymes may be bound to the cell and do not escape into the medium without cell disintegration. It is also possible that cell bound enzymes are located outside an impermeable cytoplasmic membrane. Only certain enzymes, called "partially cell-bound enzymes" by Pollock (56). may .1’ diffuse through pores of the cell wall. According to Mitchell and Moyle (57), a small amount of potentially extracellular enzyme is expected to be located on the outer layers of the cell. If this is the case, then enzymatic secretion would be dependent upon the cell wall. Enzyme release from the cell would be partially or completely prevented. Mitchell and MOyle concluded that the amount of enzyme produced depends on the properties both of the enzyme and of the cell wall. The results of the alkaline phosphatase assays of the HeLa plus Fungus activity were greater than in the separate flasks through 48 hours, particularly at the zero hour. This suggests an additive increase in activity due to combining the two cell types. The increased acti- vity of the enzyme from the medium in the combination flask dropped to the level of activity in the media of the separate flasks after the zero hour, indicating enzyme inhibition or degradation. Intracellular products of 61 lysed HeLa cells may have accounted for a sudden disap— pearance of the activity of the combination flask at 60 hours. Activity in the medium of the Fungus flask and its control were relatively consistent, whereas the enzyme in the medium of the HeLa flask appeared to be decreasing with time, despite the presence of exogenous alkaline phosphatase. The appearance of extracellular alkaline phosphatase is associated with the nutritional level of the organism with respect to phosphorus. As long as orthophosphate is available, no enzyme is released into the surrounding medium. In the second experiment, the alkaline phosphatase activity in the medium of the combination HeLa plus Fungus flask was again greater than the media in either of the separate flasks at the zero hour. Later on, a decrease in enzyme activity caused the level in the medium of the HeLa plus Fungus flask to approximate that within the HeLa flask. Intracellular products of lysed HeLa cells may have been responsible for some destruction or inhibition of enzyme in the HeLa plus Fungus medium at 60 hours. Another interpretation is suggested by looking at the General Medium Control. The presence of exogenous alkaline phosphatase may have been due to inhibition or degradation by metabolic products of either organism. This seems unlikely as the subtracting of the levels of activities in the media of the controls from both the HeLa and the Fungus flasks demonstrate increasing activities or leveling off with time. 62 A third experiment was done to observe the effects on a specific amount of alkaline phosphatase solution placed into the spent medium system. As in Experiment I, the enzyme in the medium of the HeLa flask was degraded. All other flasks were consistent and identical, thus demonstrating no increased activity. The degradation or inhibition of pure alkaline phosphatase in the HeLa flask in this third experiment also ruled out the occurrence of artifactual decrease of enzyme in the first experiment. The level of acid phosphatase in the first experi- ment was found to decrease with time in both media of the HeLa and the Fungus flasks, despite a slight increase in the controls.‘ The relatively high levels of enzyme present initially in the tests seem to indicate removal of inhibitor or production of enzyme by the organism as opposed to only exogenous enzyme being present. Combining the two cell types caused a decrease in enzyme activity which continued through 60 hours. Acid phosphatase was not detected by 60 hours in the medium of the HeLa plus Fungus flask. In fresh tissue culture medium, the HeLa flask medium, after the control values were subtracted, showed an increase in acid phosphatase activity with time. This enzyme in fungi is either strictly or partially repressed (34,62,71, 88,103). In other words, acid phosphatase is produced in small quantities in the presence of phosphate, but markedly in its absence. Nonspecific acid phosphatase was reported to be localized extracellularly by Linko and Schmidt (96) 63 and to be derepressed by lowering the concentration of inorganic phosphate in the medium. Combining the two cell types, HeLa and fungus, yielded variable results for acid phosphatase. Only at the zero hour was the HeLa plus Fungus enzyme activity greater than that in the medium of the separate flasks. FolloWing this point in time, enzyme produced by the fungus and/or the HeLa cells was degraded or inhibited, possibly by cellular metabolites, when the cell types were combined. This decrease in acti- vity was expected, as the enzyme in the medium of the Fungus flask was gradually being destroyed. In the first experiment, glutamic-oxalacetic trans- aminase (GOT) stability with time and incubation at 37 C was observed, except in the medium of the HeLa flask, which showed increasing activity. The HeLa plus Fungus medium exhibited greater GOT activity than either the Fungus or the HeLa medium alone, but after 12 hours the combination was less than the HeLa medium. This seems to indicate an immediate partial loss of activity in the HeLa plus Fungus medium, possibly increasing with time. The increased secretion of enzyme by the HeLa cells may account for the consistent level in the medium of the combination flask, such that a decrease in activity was masked by HeLa cell enzyme production. Again, in this experimental system, it is impossible to identify which cell type is producing and which is destroying or inhibit- ing any enzyme. Electrophoretic studies would assist in answering such questions. IE1.- 64 The second experiment revealed stability of the COT enzyme with respect to the control. In this case, both the Fungus medium and especially the HeLa medium showed increasing levels of enzyme activity with respect to the control medium. The combination medium was greater in activity than either of the separate media flasks through 48 hours, possibly due to the additive effect of the two cell types or the stimulation of one cell type by another to produce enzyme. Natural inhibitors of the enzyme, namely L-aspartate, L-glutamate and succinate, were not known to be present in this particular tissue culture medium. Glutamic-pyruvate transaminase (GPT) activity in the first experiment was produced in large amounts by the HeLa cells and lesser by the fungal cells. Although consistent levels of enzyme were seen in the media controls, the level appeared to be greater in incubated material. Unlike many of the other enzymes, in spent material both of the separate media flasks demonstrated higher levels of enzyme activity than the combined flask medium. This loss of activity in the HeLa plus Fungus flask medium seems to have been due to the cells or to their metabolic products, as enzyme inhibitors such as L-glutamate, L-alanine, and L-aspartate were not present in MEM. Although a loss of enzyme was observed upon combining the two cell types, no further decrease was seen over the 60 hour period. Glutamic-pyruvate transaminase activity in the second experiment varied with time only in the medium of the HeLa 65 flask, where an increase was seen. Apparently, combining HeLa cells with fungal cells in fresh tissue culture medium produced an enzyme level equal to that produced by the fungal cells alone. This activity stayed constant and equal to that of the control. The combination of the two cell types seemed to effect a degradation or inhibi- tion of enzyme. Whether or not the HeLa cells produced an enzyme that was being degraded cannot be certain with- out electrophoretic studies. The combined level of activity, although equal to that of the medium of the Fungus flask, was also equal to the control medium activity. The results indicate enzyme production by only HeLa cells or that an enzyme inhibitor was present in the other flask. Alpha-hydroxybutyric dehydrogenase (a-HBD) activity was stable with respect to the controls. An overall change in enzyme activity came about as a decrease, par- ticularly in the medium of the HeLa flask. Despite the occurrence of enzyme inhibition or degradation in media of both the HeLa and Fungus flasks, the medium of the HeLa plus Fungus activity remained quite high and con- sistent. These results suggest at least two possibilities. The two cell types together may have produced an increased level of'a~HBD. A more plausible explanation is suggested by observing the results of the controls. Similar activi- ties in the HeLa plus Fungus medium and in the media controls suggest only a lack of enzyme decrease. The immediate degradation or inhibition of enzyme occurring 66 in media of the separate flasks is not seen in the HeLa plus Fungus medium. Results of the second experiment also indicate a-HBD stability with respect to the control, but considerably lower levels of activity were observed than those in spent MEM. In this experiment, the HeLa flask medium increased in enzyme activity, while the Fungus flask medium decreased. A rapid lowering of a-HBD was observed at 60 hours in the Fungus flask medium. In the combina- tion flask at 60 hours, it is possible that the HeLa cells may have been contributing a great amount of detectable enzyme, but the source was probably intracellular, due to lysis. The number of variables that can affect enzyme levels is virtually endless. In cells that are in culture, these include nutritional, developmental, stage of the cell cycle, and viral infection (85). Whether an observed difference in measurable activity results from a differ- ence in catalytic activity of pre-existing enzyme or an altered content of enzyme can be elucidated by studying the mechanism for an altered enzyme level. Once it is established that the difference in enzyme activity results from a difference in the content of enzyme protein, the next logical question is whether the increase or decrease is due to an alteration in the rate of synthesis, the rate of degradation, or both. Inhibitors from yeasts and/or HeLa cells probably influence the activities of these enzymes. Some of these questions could be answered 67 by the incorporation of isotope into specific protein and combining these results with electrOphoretic studies. This is necessary for developing a better understanding of the extracellular enzyme activities of the host cells and of the pathogenic fungal cell. _______- LITERATURE CITED 10. LITERATURE CITED Almin, K. E., K.-E. Eriksson, and B. Pettersson. 1975. 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