Intrahyphal hyphae in corneal tissue from a case of keratitis due to Lasiodiplodia theobromae.

Lasiodiplodia theobromae, a recognized plant pathogen, was isolated in culture from a case of human mycotic keratitis. Chemotherapy with a variety of azoles was unsuccessful and the lesion was removed surgically. Electron microscopy of thin sections of the excised corneal tissue revealed several examples of intrahyphal hyphae, a unique process described previously in in vitro cultures of various zoopathogenic fungi. We believe this to be the first report of the presence of intrahyphal hyphae in parasitized animal or human tissue. The demonstration of this process in vivo is thought to be consistent with the hypothesis that intrahyphal hyphae might represent an attempt by the invading fungus to survive in an otherwise unfavourable environment.

Electron cytochemical studies of Cryptococcus neoformans grown on uric acid and related sources of nitrogen.

Cells of Cryptococcus neoformans grown on xanthine or urate as the sole sources of nitrogen produced numerous, single membrane-bound organelles, deemed to be microbodies. Electron images of these structures showed positive cytochemical staining for catalase and alpha-hydroxy acid oxidase, known marker enzyme activities for microbodies. Microbodies in xanthine and urate-grown cells were cytochemically reactive for the presence of the hydrogen peroxide-producing xanthine and urate oxidases. Molybdenum and phosphorus (elements associated with the cofactor common to nitrogen scavenging enzymes) were detected in the substrate-induced microbodies by X-ray dispersive microanalysis. The single limiting membrane of the substrate-induced microbody was stained by a modified Gomori reaction for the presence of alkaline phosphatase, thereby suggesting the participation of this enzymic activity in the events associated with microbody chemistry.

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum.

Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.

The acid phosphatases of Thermoascus crustaceus, a thermophilic fungus.

Thermoascus crustaceus, a filamentous, thermophilic ascomycete with pathogenic potential was cultured on Sabouraud's liquid medium at temperatures from 27 to 47 degrees C for periods up to 7 days. Growth rate and yield were optimal at 37 degrees C. Morphological changes were confined to the cell walls, the thickness being greatest at 47 degrees C, which were also more resistant to mechanical disruption. Significant amounts of acid phosphatase (EC 3.1.3.2) activity occurred in the spent media of all cultures but were greatest at 37 degrees C. The proportions of acid phosphatase activity which were operationally defined as soluble or bound were also documented; the optimum pH for acid phosphatase activity in all fractions was 5.0. Extracts were subjected to polyacrylamide gel electrophoresis under non-denaturing conditions and the gels were stained for acid phosphatase activity. This revealed four electrophoretically distinct acid phosphatases which had different susceptibilities to inhibition by fluoride, phosphate, or tartrate. Effects of growth temperature, or phosphate supplement in the culture medium, on the acid phosphatase isoenzyme pattern were judged to be minor. Cytochemistry at the electron microscope level indicated acid phosphatase activity on the surface, in the periplasmic space, and in the cytoplasm, but no trends with regard to growth conditions. A substantial temperature range can be tolerated by this species but it is concluded that neither the general shape of the cells nor the acid phosphatase isoenzyme pattern changes substantially; this contrasts with previously documented differences for this class of enzyme in dimorphic Sporotrix schenckii.

Mitochondrial morphology in Basidiobolus haptosporus.

Young hyphal cells of the potentially zoopathogenic fungus Basidiobolus haptosporus characteristically exhibit unusual proportions of annulate views of mitochondria in the two-dimensional perspective of thin sections. Such views exhibit a central space containing cytoplasmic ground substance and often profiles of other cytoplasmic organelles (lipid bodies, other mitochondrial forms, and especially crystalloid-containing microbodies). Three-dimensional projections are presented to suggest that these mitochondria have assumed the form of a goblet-shaped enclosure, and that the various annulate views are the consequence of plane of section viewed by electron microscopy. Their frequent occurrence and consistent morphology argues against their being random expressions of mitochondrial plasticity, but rather for close spatial associations amongst cytoplasmic organelles of young hyphae. When the fungus is grown on xanthine or its catabolites as sole sources of nitrogen, there is a proliferation of crystalloid-containing microbodies, double-membraned vesicles, and ovate to ellipsoidal mitochondria. Annulate views of mitochondria then are no longer observed, but microbodies again frequently appear in close association with mitochondria and at times in intimate contact with the mitochondrial outer membrane.

Media-induced departures from the usual, temperature-dependent cell shapes of Sporothrix schenckii and concomitant changes in the acid phosphatase isoenzyme patterns.

Sporothrix schenckii grew as mycelia at 20 degrees C or yeast at 35 degrees C on a common culture medium, YNG (yeast extract, neopeptone and glucose), in agreement with previous observations that standard media support the mycelial phase at the lower temperature and the yeast phase at the higher temperature. Special media, M-1 and M-2, were used to generate yeast at 20 degrees C and mycelia at 35 degrees C, respectively, i.e. the reverse of the typically-observed, temperature-dependent phases. In this context the M-1 induced (or forced) generation of yeast cells at 20 degrees C was judged to be abnormal yet these cells had a typical yeast ultrastructure including the definitive microfibrillar zone of the cell envelope. Electropherograms of extracts of these cells (M-1 at 20 degrees C) displayed the typical acid phosphatase isoenzyme pattern of yeast grown at 35 degrees C, and that is much more complicated than the typical pattern for extracts of mycelia grown at 20 degrees C on YNG. On the other hand the fungus was held in the mycelial phase on M-2 at 35 degrees C, at least for short term cultures, to complement the study and provide a departure from the cell shape which is expected at the higher temperature. These mycelia were judged to be ultrastructurally abnormal in that they had a thin microfibrillar zone not previously seen in S. schenckii mycelia produced on standard media at lower temperatures. The cell-free extract of these abnormal mycelia grown at 35 degrees C exhibited an isoenzyme pattern which was closer to that of yeast grown at 35 degrees C than to mycelia grown at 20 degrees C, although one isoenzyme showed intermediate electrophoretic mobility. The findings with M-2 medium at 35 degrees C were less meaningful because of the eventual conversion to yeast phase. Overall the results indicate an association between acid phosphatase isoenzyme pattern and cell shape, rather than simply an expression of the various acid phosphatases with growth temperature.

Acid phosphatases of Sporothrix schenckii.

Sporothrix schenckii cells were grown on a medium containing yeast extract, neopeptone and glucose at 20 degrees C to obtain a mixture of mycelia and conidia, and at 35 degrees C to obtain yeast-like cells. The organism was maintained in the mycelial form, and its transformation to yeast at the higher temperature proceeded via conidia and 'intermediate cells' that then gave rise to yeast by a blastic mechanism. Cell-free extracts were analysed by PAGE at pH 8.0 and acid phosphatases (EC 3.1.3.2) were revealed by a sensitive detection reagent at pH 5.0. Mycelial, conidial and yeast extracts all had some acid phosphatase activity (M-I, C-I and Y-I) at the origin, although the proportion was highest for the yeast extracts. All of the bands that penetrated the gels had different electrophoretic mobilities. Mycelial and conidial extracts each had one other isoenzyme (M-II and C-II), while the yeast extracts had a total of five electrophoretically distinct acid phosphatases. Isoenzyme Y-II was further resolved into five closely related bands (Y-IIa to Y-IIe), the relative intensities of which varied with the phosphate nutrition of the yeast cells and the history of the extracts. The acid phosphatase isoenzymes were inhibited to various extents by sodium fluoride, L(+)-tartrate and phosphate, and showed interactions with citrate as opposed to acetate as the background buffer at pH 5.0.

Virus-like particles in Basidiobolus species.

Transmission electron microscopy of ultrathin sections of hyphal cells of Basidiobolus haptosporus and B. ranarum revealed the presence of particulate inclusions consistent in morphology with that described in certain other fungi as virus-like particles. Two types of particles were observed which differed in substructure and distribution within the host cytoplasm. Relative numbers of these particles were never great, and their presence in the host was not associated with any demonstrable cytopathological changes detectable at the ultrastructural level. We believe this report to be the first description of an association of virus-like particles with fungi of the Entomophthoraceae.

Electron cytochemical demonstration of phosphatase activity with microbody membranes of Basidiobolus haptosporus.

Growth of cells of the potentially zoopathogenic fungus Basidiobolus haptosporus on a nutritionally defined medium with xanthine or urate as the nitrogen source results in greatly increased populations of microbodies. Modified Gomori procedures at the electron microscopic level suggested the single limiting membrane (and in some cases the granular matrix) of immature microbodies to be the exclusive subcellular locale(s) of alkaline phosphatase, 5'-nucleotidase and nucleoside diphosphatase activities. When grown in the presence of low inorganic phosphate, additional alkaline phosphatase activity was further identified cytochemically at and along profiles of endoplasmic reticulum and on inclusions previously described as "double-membraned vesicles". Cytochemical localization of acid phosphatase at microbody membranes was minimal if not ambiguous; Mg++-dependent adenosine triphosphatase and glucose-6-phosphatase were not identified at these locales. Quantitative biochemical estimates of alkaline phosphatase activity levels in particulate fractions initially increased with age of cells, perhaps as a function of the cultural induction and marked increase in immature microbody populations. We suggest that this enzyme may participate in some manner with protein translocation mechanisms associated with microbody biogenesis, ontogeny, and/or physiological function.

Localization of alkaline phosphatase activity at microbody membranes of Neurospora crassa and Aspergillus nidulans.

Hyphal cells of Neurospora crassa and Aspergillus nidulans, grown in Sabouraud glucose broth or in a defined medium with xanthine or its catabolites as the nitrogen source, contained single membrane-bound organelles cytochemically identified as microbodies. Modified Gomori procedures at the ultrastructural level revealed putative alkaline phosphatase activity sites in thin sections of cells of both species of fungi. Microbody membranes displayed electron opaque deposits (lead phosphate) which were absent in control preparations either lacking the substrate (beta-glycerophosphate) or the lead capture ion. Inhibition of this enzymic activity was achieved in parallel incubations fortified with the inhibitor levamisole. Cells grown in media containing limiting levels of inorganic phosphate revealed additional alkaline phosphatase activity at and along the nuclear membrane and endoplasmic cisternae. Hexagonal inclusions found in the cytoplasm of N. crassa (but not A. nidulans) and believed to arise from microbody precursors were without demonstrable cytochemical staining for microbody marker oxidases.

Double-membraned vesicles and their possible role in the ontogeny of microbodies of Basidiobolus haptosporus.

A concomitant but transient occurrence of large numbers of double-membraned vesicles with immature, culturally induced microbodies was demonstrated in thin sections of Basidiobolus haptosporus soon after transfer of the fungus to a defined medium containing xanthine or its catabolites as the sole source of nitrogen. Double-membraned vesicles were rapidly formed as derivatives of endoplasmic cisternae later to occur free in the cytoplasm, often as the most predominant cytoplasmic inclusion. Densitometric measurements revealed that heavy metal-binding cytoplasmic constituents were concentrated within the vesicular lumen. Most of the double-membraned vesicles appeared to undergo degeneration; they were rare to absent in thin sections of older cells. At times, double-membraned vesicles were seen connected to newly formed microbodies in a manner suggesting ontogenic relationships. The outer vesicular membrane was continuous with the single limiting membrane of the microbody via a short tubule. We interpret these observations as empirical ultrastructural evidence to suggest that some double-membraned vesicles become specialized and function as precursor inclusions in the biogenesis of new microbody populations committed to purine salvage.

Ultrastructure of Candida ingens: a yeast that can assimilate volatile fatty acids.

A defined liquid medium was developed for culture of Candida ingens on either volatile fatty acids or glucose as carbon source. Buffering capacity at pH 5.4 was achieved by inclusion of 50 mM phthalic acid which is not assimilated. The yeast grew on C2-C5 acids, with lags in some cases that were dependent on the cultural history of the inoculum. The cells were notably large for Candida species, or yeasts in general, and were significantly more elongated on glucose substrate compared with butyrate. Traditional aldehyde fixatives and heavy metal staining yielded bland micrographs. However, the periodic acid-thiocarbohydrazide-silver proteinate stain of Thiéry resulted in informative, high contrast electron images for this species. The ultrastructure of the cell envelope was not greatly affected by carbon source or by growth habit except that a slime layer was more prevalent in pellicular cells compared with those grown in submerged culture. The slime layer was apparently stabilized by colloidal iron staining. The other feature of the cell envelope was a microfibrillar zone containing periodic acid reactive constituents. Subcellular organelles were typical of yeast species although there was a propensity for large vacuoles in pellicular cells. When cells were grown on fatty acids there was an increase in the number of microbodies compared with that observed on glucose. Microbodies were best demonstrated by diaminobenzidine-hydrogen peroxide staining of protoplasts, which were generated by dissolution of cell walls with snail digestive enzymes.

Ultrastructural cytology of Basidiobolus haptosporus: morphology and electron cytochemistry of microbodies.

Vegetative and reproductive cells of Basidiobolus haptosporus possess naturally occurring organelles identified as microbodies. Cells of four other species of the genus contained morphologically indistinguishable organelles. Microbodies were invariably present in cells of the fungi grown on routine mycological media. The constitutive microbody was characterized by a single, intensely electron-opaque crystalloid body which rapidly enlarged to fill the organellar compartment. The microbody then underwent degeneration by an autolytic-like process. Growth of the fungi on xanthine and its catabolites as sole nitrogen sources (but not urea) greatly enhanced the production of new microbodies in which protein was initially accumulated as paracrystalline arrays. These inclusions then underwent reorganization and compaction to form crystalloid bodies. Key enzymes of the purine degradation pathway are believed to be core proteins of the crystalloid. D-amino acid oxidase, alpha-hydroxy acid oxidase, xanthine oxidase and urate oxidase (but not catalase) were detected cytochemically in mature microbodies. Significant levels of phosphorus and molybdenum were present in the microbody crystalloid by X-ray dispersive microanalysis; iron and copper were not detected. The ability of Basidiobolus species to assimilate xanthine and its catabolites might explain their ecological association with the gut and cloacal contents of various amphibia, reptiles and fish.

Ultrastructural aspects of cytoplasmic ribosomes from Histoplasma capsulatum and Blastomyces dermatitidis as revealed by heavy metal staining.

Cytoplasmic ribosomes were isolated and purified from sonicates of the mycelial and yeastlike growth forms of the pathogenic dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Similar ribosomal fractions were prepared from Neurospora crassa and Saccharomyces cerevisiae. These latter organisms were selected as typical filamentous and yeastlike monophasic fungi, and their ribosomes were used as reference standards. High resolution electron microscopy permitted a comparison of both positively and negatively-stained ribosomes to those dehydrated without heavy metal salt. Such studies revealed statistically significant differences in physical dimensions. Cautious interpretations of substructural detail of the various ribosomal preparations suggested both interphasic and interspecies differences.

Electron cytochemical demonstration of the capsule of yeast-like Sporothrix schenckii.

Staining of yeast-like cells of Sporothrix schenckii by the cationic dye Alcian blue 8GX and lanthanum nitrate permitted the demonstration in thin sections of an electron opaque material localized at and along the outermost wall surface. This material was associated with the characteristic microfibrillar layer of the external cell wall. It was absent or largely removed when cells were grown in shaken liquid culture or after washing of cells from static cultures. It is believed that this loosely-bound material represents a capsular substance comprised, at least in part, of a mucopolysaccharide or a mucopolysaccharide-protein complex.

Isolation and characterization of a bacteriophage factor that confers competence for genetic transformation to an exfoliative toxin-producing strain of Staphylococcus aureus.

Competence for genetic transformation in an exfoliative toxin-producing strain of Staphylococcus aureus was shown to be dependent on a virion factor that was isolated from a crude bacteriophage 80 alpha lysate. This competence-conferring factor was completely separated from infectious virus particles after either centrifugation through a neutral sucrose velocity gradient or fractionation on a Sepharose 2B gel. Since the competence-conferring factor tends to aggregate, optimal separation was obtained after treatment of the phage factor with the detergent Nonidet P-40. The competence-conferring factor had a molecular weight between 3 X 10(6) and 20 X 10(6) and an approximate sedimentation coefficient of 252. The factor was neutralized after interaction with antiserum prepared against isolated infectious 80 alpha virions. Electron microscopy of transforming cells that were exposed to isolated competence-conferring factor revealed a significant number of abnormally long and aggregated phage tail-like structures attached to the surface of recipient cells. This phenomenon was only observed in the presence of donor DNA, indicating that a phage tail-DNA-surface receptor complex might be one of the early steps in DNA-mediated transformation of S. aureus.

Fine structural studies of the Chrysosporium state of Renispora flavissima.

Aspects of the fine structural characteristics of conidial and hyphal cells of the imperfect Chrysosporium state of the newly reported gymnoascaceous fungus, Renispora flavissima, are described and illustrated by electron micrographs of ultrathin sections. Preservation of the substructure of the thick-walled, smooth to tuberculate conidia was best achieved after fixation in permanganate. The conidial wall consisted of two conspicuous zones (or layers) of varying thickness, texture, and electron opacity. The tuberculate ornamatations arose as extensions of the electron opaque outer zone. The outer layer(s) of the conidial wall proper was continuous with an outer wall layer of the conidiogenous hypha. The conidial cytoplasm was cytologically complex containing numerous but typical fungal inclusions and organelles scattered throughout. This ultrastructural feature of the tuberculate conidia of the Chrysosporium sp. differs from that observed for the morphologically similar large tuberculate conidia of the zoopathogenic Histoplasma capsulatum.

Fine structural studies of Rhodococcus species.

Fine structural aspects of Rhodococcus rhodochrous and R. equi are described and illustrated by electron micrographs after staining of cells by a variety of electron cytochemical procedures. The cell contents of these actinomycetous bacteria were those of a typical prokaryotic cell and consistent with that observed for other species of the Actinomycetales. Fixation with either osmium tetroxide or permanganate indicated the presence of an electron opaque substance at the wall exterior of R. rhodochrous which is thought to be composed of protein. Ruthenium red and Alcian blue-lanthanum stains for mucosubstances revealed that both species possess a capsular substance thought to be composed of a mucopolysaccharide or mucopolysaccharide-protein complex. This substance was non-reactive toward the PATAg stain for polysaccharide macromolecules containing vicinal glycol groups.