Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis.

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 degrees C; pH optimum was 7-7.5; Mg+2 optimum was 5-10 mM. The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.

Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid-fast smear-positive specimens.

A total of 463 respiratory specimens, all smear positive for acid-fast bacteria, were inoculated onto routine solid media and into BACTEC 7H12 Middlebrook medium for detection of mycobacterial growth. Conventional drug susceptibility testing (1% proportion method) was performed on Middlebrook 7H10/7H11 medium, and radiometric susceptibility testing was performed on BACTEC 7H12 medium. The average detection times for BACTEC-positive cultures were 8.3 days for Mycobacterium tuberculosis and 5.2 days for mycobacteria other than tuberculosis; by conventional methods, they were 19.4 and 17.8 days, respectively. These detection times do not include time required for identification, which was done by the conventional method only. There was an excellent correlation in the recovery rates of mycobacteria by the two methods. Drug susceptibility test results of M. tuberculosis isolates by the two methods showed 95.1 to 100% overall agreement. The average reporting time for drug susceptibility results ranged from 4.2 to 6.9 days for the BACTEC method and 13.7 to 21 days for the conventional methods. An average of 18 days was required by the BACTEC method for complete recovery and drug susceptibility testing of M. tuberculosis, as compared with 38.5 days for the conventional methods.

Comparative activities of glycolytic enzymes in yeast and mycelial forms of Candida albicans.

Through use of a synthetic defined medium which allows for the exclusive growth of yeast or mycelial forms of Candida albicans the activity of several major glycolytic enzymes in these forms were examined and compared. The results indicate vast metabolic differences between the forms. These data are discussed in relationship to the phenomenon of morphogenesis in C. albicans which in turn relates to problems in immunology and pathogenics of this important opportunistic organism.

Production and characterization of a hybridoma-derived antibody to Blastomyces dermatitidis.

A hybridoma cell line was isolated which produced monoclonal antibody to one protein component of a yeast-phase cytoplasmic antigenic complex of Blastomyces dermatitidis. The immunoglobulin M antibody product was characterized by immunodiffusion, autoradiography of polyacrylamide gels, and cellulose acetate electrophoresis. By attaching the antibody to an affinity gel, one major protein band was identified by polyacrylamide gel electrophoresis as the antigen for which the antibody was specific.

The nutritional status of patients with chronic obstructive pulmonary disease.

The nutritional status of 38 patients with chronic obstructive pulmonary disease (COPD) was assessed by dietary intake, anthropometric measurements biochemical analysis, and immunologic testing. The mean intakes for 9 nutrients were significantly greater than the 1974 Recommended Dietary Allowances of the National Academy of Sciences. Results of the anthropometric measurements for usual weight for height, fat stores, and muscle mass were significantly less than standard. Of the 32 subjects evaluated for immunocompetence, 9 were anergic (induration, 0) on all 3 skin tests. The results of this study indicated that the marasmic type of protein calorie malnutrition is a common finding among patients with COPD, and that patients with COPD who are immunoincompetent may be more susceptible to mixed protein calorie malnutrition of the kwashiorkor-marasmus type.

Primary acute histoplasmosis in guinea pigs exposed to aerosolized Histoplasma capsulatum.

Guinea pigs were examined as a possible in vivo model for human histoplasmosis. Guinea pigs were exposed to an aerosol of viable microconidia and mycelial fragments of Histoplasma capsulatum generated in a Henderson apparatus. Colonization and infection of the lungs occurred, with subsequent involvement of the regional lymph nodes and reticuloendothelial organs. Cultural recovery of the fungus from the nasopharynx and bronchoalveoli was initially high, but decreased with time. The mean number of colonies recovered from the lungs gradually increased, reaching a peak at 2 weeks, with involvement of the regional lymph nodes. Extrathoracic dissemination to the liver and spleen occurred in only a few animals. After 4 weeks, all tissues except the cervical and tracheobronchial lymph nodes were culturally negative; all specimens were culturally negative after 8 weeks. Disappearance of H. capsulatum from tissues appeared to correlate inversely with the development of hypersensitivity as measured by skin test reactivity. Histopathological studies supported cultural results and were similar to those described for primary human and canine histoplasmosis.

Identification of the active precipitin components in a purified preparation of the A antigen of Blastomyces dermatitidis.

A purified A-antigen preparation of Blastomyces dermatitidis was determined to be composed of five major glycoprotein bands, visible with Coomassie blue and periodic acid-Schiff staining of polyacrylamide gels. At least 20 additional protein bands were detected by using a silver stain, which was 100 times more sensitive than the Coomassie method. Two components of this mixture were determined to be associated with the A-antigenic activity of B. dermatitidis. Of several antigen preparations examined in Ouchterlony precipitation tests, those reactive with a reference anti-A antiserum contained the slowest moving of the Coomassie blue bands. The antigen preparations without precipitin reactivity lacked this protein band. Two protein bands were shown to disappear from an antigen preparation after incubation with an affinity gel linked to the reference anti-A serum. One of the bands was the slowest Coomassie blue band, and the other was a fast-migrating protein detectable only with the silver stain. Characterization of the components responsible for the A-antigenic activity has important applications in the production and standardization of serological reagents for the diagnosis of blastomycosis.

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum.

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5% of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 micrograms/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.

An effective medium for the selective growth of yeast or mycelial forms of Candida albicans: biochemical aspects of the two forms.

A new synthetic medium, based on a modification of a commercially available tissue culture medium, allows Candida albicans to be grown in the yeast or mycelial form. Salient features of the system are described and comparisons with previous physiological investigations are discussed. A concise biochemical profile of these two forms of C. albicans is also presented. The results indicate vast metabolic differences between the two forms.

Crossed immunoelectrophoretic analysis of two antigen extracts of Thermoactinomyces candidus.

A pyridine extract antigen and a double-dialysis antigen (DDA) obtained from Thermoactinomyces candidus were analyzed by crossed immunoelectrophoresis. In addition, the heat lability, pronase sensitivity, and isolectric points of the components of the DDA were determined. By using antisera raised against crude pyridine extract antigen, two immunogenic components were resolved by crossed immunoelectrophoresis. A similar analysis of DDA using antisera raised against crude DDA revealed 15 immunogens. All but six components were heat labile, whereas pronase had little effect on the number of resolvable components. Intermediate gel crossed immunoelectrophoresis using antiserum raised to whole spores detected six immunogenic components, four of which were also detected by the anti-DDA serum. A total of 19 bands were obtained when the DDA was subjected to flatbed isoelectric focusing on polyacrylamide gels. The isoelectric points for the various components were found to range from 3.5 to 5.7. Crossed immunoelectrophoresis using isoelectric focusing in the first dimension yielded at least 16 immunogenic components. Six components with isoelectric points falling in the range of 4.5 to 6.4 were found to be resistant to heat. A comparison with antigens obtained from other thermophilic actinomycetes is presented.

The efficacies of common dyes in primary isolation media for recovery of pathogenic fungi.

Dyes incorporated into a basal medium of brain heart infusion, Sabhi, tryptic soy, or yeast extract--pepton--glucose (YxPG) agar for selective isolation of fungi were investigated. Dilutions of 1:500, 1:750, 1:1,000, 1:5,000, and 1:10,000 of 33 common dyes were tested against 11 gram-positive and 16 gram-negative bacteria. In addition, these dyes were tested against Cryptococcus neoformans, Candida albicans, and the dimorphic phases of Histoplasma capsulatum and Blastomyces dermatitidis. Twenty-one of the dyes did not inhibit any of the organisms tested. Brilliant green, gentian violet, and malachite green (at three dilutions) inhibited all the organisms tested. Methyl red was found to be the best dye in selecting for fungi. Several dyes were also found to inhibit selectively C. neoformans or C. albicans and the dimorphic fungi H. capsulatum or B. dermatitidis.

Merthiolate treatment of pathogenic fungi.

The action of Merthiolate on the pathogenic yeasts Blastomyces, dermatitidis, Histoplasma capsulatum, and Sporothrix schenckii was compared to the effect of treatment with formaldehyde. Concentrations of 1:10,000 and 1:5,000 Merthiolate for three exposure times (24, 48, and 72 h) at 4 and 25 degrees C were tested on three media (brain heart infusion with and without blood, and modified Sabouraud agar). The effect of Merthiolate on these three yeasts was primarily fungistatic, with maximum effect using 1:5,000 Merthiolate at 25 degrees C for at least 48 h. Mycelial suspensions of B. dermatitidis, H. capsulatum, S. shenckii, and the yeast phase of Cryptococcus neoformans were susceptible to the 1:5,000 Merthiolate concentration after 24 h of treatment. The antifungal effect of Methiolate varies with species and growth phase of the fungus. Concentration, time of exposure, and temperature of incubation are important variables.

In Vitro and In Vivo Activity of a Synthetic Halogenated Quinoline Against Cryptococcus neoformans.

The minimum inhibitory concentrations of a halogenated quinoline, 3-amino-7-chloro-3,4-dihydro-1-hydroxycarbostyril (CBS), against nine clinical strains of Cryptococcus neoformans were determined by in vitro testing. The CBS was fungistatic at a minimum concentration of 0.2 mug/ml at 48 h for several strains. In vivo toxicity studies were carried out in mice. Mice were also infected with C. neoformans strain Price and injected with various concentrations of CBS. Mean life expectancy of treated groups of animals was increased over infected untreated controls.

Use of an alkali-soluble water-soluble extract of Blastomyces dermatitidis yeast-phase cell walls and isoelectrically focused components in peripheral lymphocyte transformations.

An alkali-soluble water-soluble extract of Blastomyces dermatitidis yeast-phase cell walls was tested for its ability to elicit a response in lymphocytes isolated from the peripheral blood of Blastomyces-infected guinea pigs. Sequential preparations of the antigen were reproducible and specific in the in vitro lymphocyte transformation assay. Cross-reactivity of the antigen was not evident in lymphocyte transformation assays on lymphocytes obtained from Histoplasma-infected guinea pigs or from animals sensitized with complete Freund adjuvant. Fractionation of the antigen was accomplished on an isoelectric-focusing column, using a sucrose density gradient support. Components were assayed for activity in skin testing and lymphocyte transformation. Comparison of column fractions to the whole antigen showed greater response to the whole antigen in in vivo and in vitro assays.

Comparative study of trichothecin, amphotericin B, and 5-fluorocytosine against Cryptococcus neoformans in vitro and in vivo.

Trichothecin (T-cin), amphotericin B (AB), and 5-fluorocytosine (FC) were compared singly and in combination for capacities to inhibit growth of Cryptococcus neoformans in culture and to protect mice bearing infections with this yeast. The minimum inhibitory concentrations for T-cin, AB, and FC were found to be 0.5, 0.2, and 5.0 mug/ml, respectively. In vitro viability studies demonstrated a marked reduction in colony counts with the AB-FC combination and additive effects with the AB-T-cin and FC-T-cin combinations for a 3-day period. In mice infected intravenously with C. neoformans, the mean effective dose for AB was 0.38 mg/kg, and for FC it was 100 mg/kg for a 30-day treatment period. No mean effective dose could be ascertained when T-cin was tested at doses of 0.1 to 50 mg/kg. Despite this, marked beneficial effects were noted in vivo with the AB-T-cin combination, whereas additive effects and indifference were observed for AB-FC and FC-T-cin combinations, respectively. High-dose T-cin controls survived despite having received a cumulative dosage of more than twice the reported (LD(50)) mean lethal dose value.