Studies on the mode of action of tolnaftate in Microsporum gypseum.

Studies were performed on the mode of action of tolnaftate and resistance to this drug in Microsporum gypseum. Cells grown in the presence of tolnaftate (at the IC 50) showed a reduced content of total phospholipids and sterols whereas there was an increase in total RNA content. Incubation of cells with tolnaftate (at 10 x MIC), followed by addition of different macromolecule precursors revealed inhibition of the biosynthesis of all macromolecules except for RNA. The activity of membrane-bound enzymes did not change on treatment with tolnaftate (10 x MIC) whereas an increase in the leakage of intracellular 32P was observed. The content of total phospholipids was higher in tolnaftate-resistant cells, whereas the content of total sterols, DNA, RNA and protein was comparable to that of susceptible cultures. Activity of phosphodiesterase decreased and 5'-nucleotidase increased in tolnaftate-resistant cells. Our results suggest that the antifungal activity of tolnaftate is due to differential action on various targets site(s) which are modified in strains resistant to the drug.

The value of electrophoretic protein patterns for the study of Microsporum canis.

Whole-cell protein analysis of clinical isolates of Microsporum canis by polyacrylamide gel electrophoresis (PAGE) showed small but inconsistent differences in protein patterns. All clinical isolates closely resembled the (-) mating type of Arthroderma otae. A reference strain of A. otae (+) mating type from Japan gave a PAGE pattern which was distinct from those of the clinical isolates. Mating studies showed that the progeny of these reference (+) and (-) mating types had a variety of distinct protein patterns. Protein patterns can therefore distinguish between genetically different strains of M. canis (A. otae) suggesting that clinical isolates have a common clonal origin.

Ribosomal phosphoproteins in Microsporum canis.

Ribosomal phosphoproteins of Microsporum canis labelled in vivo were characterised by two-dimensional and SDS polyacrylamide gel electrophoresis. A small subunit protein, S6, was the only phosphoprotein identified in 40S and 80S in basic-acidic two-dimensional gels. Three different forms of phosphorylated S6 were also observed in 40S subunit. On SDS gels five phosphoproteins were identified in 80S; of these three were present in 40S and two in 60S. S6 was the only basic phosphoprotein, while the other four were acidic.

Separation of cytoplasmic ribosomal proteins of Microsporum canis.

The cytoplasmic ribosomal proteins of Microsporum canis were characterised in basic-acidic and basic-SDS two-dimensional polyacrylamide gel electrophoresis systems. The small subunit contained 28 proteins and the large subunit 38 proteins. The molecular weights of these proteins were in the range of 32,500 to 7600 and 48,000 to 11,000 in the small and large subunits, respectively. The 80S ribosomes showed 65 and 66 protein spots in basic-acidic and basic-SDS gel systems, respectively.

Isolation and identification of the principal siderophore of the dermatophyte Microsporum gypseum.

The dermatophyte Microsporum gypseum has been shown to produce two siderophores under conditions of low-iron stress. These compounds have been separated as Fe(III) complexes on silica gel, and the principal siderophore has been identified as ferricrocin using the methods of amino acid analysis, comparative thin-layer chromatography, partial sequencing by gas chromatography-mass spectrometry, ultraviolet spectroscopy, and proton nuclear magnetic resonance spectroscopy of the Al(III) complex.

Studies in the differentiation between Microsporum ferrugineum Ota and Trichophyton soudanense Joyeux.

A study, conducted with 20 isolates of Microsporum ferrugineum and 12 isolates of Trichophyton soudanense, revealed that some of the discrepancies in the literature regarding their characteristics and differentiation were due to methodology, strain variation and the use of an insufficient number of isolates. We found all isolates of T. soudanense to be urease negative and gelatinase positive (usually by the first week); to produce brown to black colonies on Lowenstein-Jensen medium; to rapidly decompose casein and more slowly tyrosine; to grow well or better at 37 degrees C as compared to room temperature; to produce reflexive branching on cornmeal Tween agar and abundant microconidia on casero medium and to exhibit no sexual reaction with either mating type of arthroderma simii. All but one isolate demonstrated restricted growth on lactose agar and only three isolates perforated hair. In contrast, we found 18 of 20 isolates of M. ferrugineum to be urease positive in urea broth (most isolates were negative on urea agar); all produced light-colored colonies on Lowenstein-Jensen medium; spreading colonies on lactose agar and failed to perforate hair in vitro or to produce reflexive branching. Most isolates manifested poorer to no growth at 37 degrees C compared to room temperature and all but one failed to decompose casein and tyrosine. A few strains produced macroconidia and/or microconidia on casero medium and some reacted sexually with A. simii (a) (-) mating type. Gelatin hydrolysis was variable.(ABSTRACT TRUNCATED AT 250 WORDS)

Analytical isoelectric focusing of secreted dermatophyte proteins applied to taxonomic differentiation of Microsporum and Trichophyton species (preliminary studies).

Culture filtrates were prepared from dermatophytes under standard conditions and adapted for analytical isoelectric focusing in thin layer polyacrylamide gels over the pH range 3.5-9.5. Dermatophytes grown in trypticase soy broth secreted a large number of proteins displaying a wide range of isoelectric points (pIs). Trichophyton megninii extracts contained a triplet of proteins focusing in the pH 8.0-8.5 range that were absent in taxonomically related T. kuryangei isolates. Single ascospore isolates and standard tester strains of Nannizzia otae (+) mating type were differentiated from the (-) mating type by proteins focusing at pH 6.5 and 8.4. These were markedly reduced in the (+) type. The isofocused pattern of Microsporum canis conformed closely to the (-) mating type of N. otae. The protein patterns of T. megninii and T. kuryangei were distinct from those obtained with M. canis and M. equinum because of an intense-staining broad protein band, pI 7.2, and three periodic acid-Schiff-positive glycoproteins focusing in the acidic range which were absent in the Microsporum species. A characteristic protein or doublet (pI 8.7) was present in the Microsporum species and absent in the Trichophyton species. Analytical isoelectric focusing is a potentially useful method to distinguish inter- and intra-species differences in the pattern of secreted dermatophyte proteins present in culture filtrates and in trichophytins. The information derived may be useful in the classification of species.

Lipid composition of Microsporum gypseum.

Lipid composition of Microsporum gypseum grown in Sabouraud's liquid media on a rotary shaker were analyzed. The organism contains 6.07% lipid (dry weight basis), of which 75.88% is neutral lipids, 0.9% phospholipids and the rest are glycolipids and pigments. Phospholipids of M. gypseum contain phosphatidyl choline (44.21%), phosphatidyl ethanolamine (17.8%), polyphospho inositide (12.13%), phosphatidyl inositol (8.24%), phosphatidic acid (4.61%) and cardiolipin. Phosphatidyl serine is absent. The neutral lipid composition (expressed as mg/g dry weight of mycelia) of M. gypseum is monoglycerides (1.35), diglyceride (2.87), triglycerides (35.79), free fatty acids (1.00), sterols (3.95) and sterol esters (1.13).

Fatty acids of dermatophytes.

The cellular fatty acids (C11-C20) from 18 species and strains within the genera Microsporum, Trichophyton, and Epidermophyton were determined by gas liquid chromatography. In addition, the effects of incubation time and temperature on fatty acid composition were investigated in selected species. The dermatophytes investigated represented anthropophilic, geophilic, and zoophilic species. Linoleic (18:2), oleic (18:1), and palmitic acid (16:0), accounted for 83.6-94.5% of the fatty acids of dermatophytes. Fatty acid composition and degree of unsaturation did not show any correlations with taxonomic status or ecological group. Incubation time influenced fatty acid composition slightly, but tendencies towards unsaturation and chain elongation were not observed. Elevated incubation temperature (37 degrees C) tended to increase the degree of total fatty acid unsaturation.

Pyrolysis gas-liquid chromatography applied to a study of variation in Arthroderma tuberculatum.

Replicates of whole colonies of four species of closely related dermatophytes were analyzed by pyrolysis gas-liquid chromatography (PGLC). The four species included fifteen strains of Arthroderma tuberculatum, and two strains each of A. benhamiae, Nannizzia gypsea and N. incurvata. Individual peaks on different pyrograms were identified as homologous with the aid of internal markers by the superimposition of pyrograms. The peak height data extracted from the pyrograms of the fungal samples were analyzed to compute average similarities between pairs of pyograms. The average was calculated with each peak weighted equally, and log weighted for its information content. The results of the cluster analyses of proximities were generally similar. Most, but not all, replicates of each strain were similar enough to be clustered together. Some strains belonging to the same species were also similar enough to be grouped in one cluster. Other strains of a single species varied sufficiently to be put in separate clusters. The nearest neighbour to each OTU (pyrogram) was always a replicate of the same strain.

Lipid composition of Microsporum gypseum.

The lipid composition of Microsporum gypseum has been studied. The lipids amounted to 10.1% and phospholipids to 1.1% of the mycelial dry weight. Phosphatidyl choline, phosphatidyl serine and phosphatidyl ethanolamine were the major components, while lysophosphatidyl choline, and phosphatidyl inositol were present in smaller quantities. Neutral lipids consisted of monoglycerides, diglycerides, triglycerides, free and esterified cholesterol.

Contribution to the study on fatty acids of Epidermophyton floccosum.

The total lipid content of a dermatophyte, Epidermophyton floccosum, grown under controlled environmental conditions, varies with growth stage and represents 7.0 to 19.8% of the dry weight. The main lipid classes, determined by quantitative thin-layer chromatography, were: triglycerides, free fatty acids, sterols and phospholipids. The fatty acid composition was established with the aid of combined gas chromatography-mass spectrometry. The major components, linoleic, palmitic and stearic acids represent 42.8, 18.9 and 9.6 per cent respectively. The fatty acid biosynthesis, their taxonomic value and their role in the disease mechanism are discussed.

A study of the kinetics of microsporogenesis in Campelia zanonia (L.) H.B.K.

During maturation, microspores pass through a series of morphologically distinguishable stages or compartments. A study has been made of the systematic fluctuations in the frequency of microspores in these compartments, when plants are grown under rigidly controlled conditions. A new approach to the construction of cumulative flux rate curves is described; these give the number of cells passing the compartment boundaries per unit time. The curves obtained indicate that simple models, which assume constant flux rates and compartment transit times, will not explain the observations. It is evident that not only do microspores mature at different rates, but that the maturation rate of individual microspores varies during the developmental sequence. The overall process may be controlled by the intimate relationship which exists between the microspores and the tapetal periplasmodium in the Tradescantiae.

Carbohydrate composition of the isolated cell walls of dermatophytes.

In an attempt to evaluate taxonomic character of sugar composition of dermatophytes, the purified cell walls from 13 species are analyzed on neutral sugar composition by gas liquid chromatography. The results were principally compatible with those obtained by conventional morphological examination. Neutral sugar components of dermatophytes cell walls were mannose and glucose in the ratio of 1:2.7 for Epidermophyton and 1:1.4 for Microsporum. There were two types in Trichophyton, in which the ratios of mannose to glucose were 1:1.6 and 1:3.8. The cases of Trichophyton ferrugineum and Trichophyton mentagrophytes were exceptional. The ratio of the former was 1:1.4, which implied the relation to Microsporum group, and the ratio of the latter was 1:2.3, which was supposed to be the intermediate of two types of Trichophyton group. Albino type cell wall of Epidermophyton floccosum was more rich in glucose than pigmented type one.