New genus of trichomatous coelomycete on Myrcia fenzliana from the Brazilian Cerrado.

Megacoelomyces (type species: Megacoelomyces sanchezii), an ascomycete asexual morph infecting Myrcia fenzliana (Myrtaceae) from the Brazilian Cerrado, is described as a new genus in the Phaeosphaeriaceae (Pleosporales, Dothideomycetes, Ascomycota), based on multilocus phylogeny (three nuclear ribosomal DNA and two protein-coding genes) in addition to morphological (light and scanning electron microscopy) and ecological data.

Especies oportunistas de importancia clínica de los géneros bipolaris shoemaker y curvularia boedijn: su caracterización bajo los nuevos criterios taxonómicos / Opportunistic species of clinical relevance in the bipolaris shoemaker & curvularia boedijn genera: its characterization under newest taxonomic criteria

Los géneros fúngicos Bipolaris y Curvularia,incluyen numerosas especies fitopatógenas,saprotrofas y algunas oportunistas emergentes enel ser humano como en otros animales. La distribuciónecológica de ambos taxas es cosmopolita enla gran mayoría de sus integrantes, encontrándosefrecuentemente en hojas, tallos y raíces de diferentespastos, en especial Poaceae, pero tambiénen una gran variedad de dicotiledoneas, aire y sueloen todos los continentes. Cochliobolus, Bipolarisy Curvularia integran un complejo de especiestaxonómicamente confuso, debido a los constantescambios en la nomenclatura de algunos de sus integrantesasexuales (Bipolaris y Curvularia), loscuales se han diferenciado principalmente en basea la morfología de sus conidios, situación a vecesmuy dificultosa debido a que en ambos génerosalgunas especies presentan similares característicasconidiales. Una cuidadosa identificación y unanominación precisa de las especies es crucial parael acceso a la información referente en la literaturamoderna, que gracias a la biología molecularha resuelto muchas de las dudas de la taxonomíabasada solo en el fenotipo.Las especies de ambos géneros (principalmenteCurvularia) pueden afectar a pacientesinmunocomprometidos e inmunocompetentes,en especial B. cynodontis, C. australiensis C.hawaiensis y C. spicifera, causando cuadros clínicosdiversos ya sea superficiales o profundos enmuchas áreas geográficas...

The fungal genera Bipolaris and Curvularia,include numerous plant pathogenic species,saprophitic and some emerging opportunistic inman and other animals. The ecological distributionof both taxa is cosmopolitan in the vast majority ofits members, often being in leaves, stems and rootsof different grasses, especially Poaceae, but also ina variety of dicotyledonous, air and soil on all continents. Cochliobolus, Bipolaris and Curvulariaintegrate a complex of species taxonomically confusingdue to the constant changes in the nomenclatureof some of its asexual members (Bipolarisand Curvularia), which are differentiated mainlybased on the morphology of its conidia, a situationsometimes very difficult because in both generaconidial of some species have similar characteristics. Careful identification and a precise nominationof species is crucial for access to informationconcerning in modern literature, actually thanks tomolecular biology has solved many of the doubtsof taxonomy based only on the phenotype.The species of both genera (mainly Curvularia)can affect immunocompetent or debilitatepatients, especially B. cynodontis, C. australiensis,C. hawaiensis and C.spicifera, causing varioussuperficial or systemic clinical cases in manygeographic areas...

Coelomycetes in Dominican and Mexican amber.

Three species of fossil epiphyllous coelomycetes are described from Dominican and Mexican amber in the new genera, Asteromites gen. nov., Leptostromites gen. nov. and Leptothyrites gen. nov. Characters of the pycnidia and spores most closely resemble members of their extant respective genera, Leptostroma, Asteromella, and Leptothyrium, respectively. A. mexicanus sp. nov. occurs on a petal (possibly from a caesalpinoid legume such as Peltogyne) in Mexican amber. Leptostromites ellipticus sp. nov. occurs on a dicot leaf in Dominican amber, and Leptothyrites dominicanus sp. nov. on a monocot (grass?) leaf in Dominican amber.

Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae.

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS-PAGE.

Comparison of Fonsecaea pedrosoi sclerotic cells obtained in vivo and in vitro: ultrastructure and antigenicity.

The parasitic form of Fonsecaea pedrosoi from the hyperkeratotic layer of the skin was obtained from four patients with chromoblastomycosis. Primary cultures containing hyphae and conidia were successfully converted into sclerotic cells in the presence of 800 microM propranolol and low pH as described before. The morphology of sclerotic cells of F. pedrosoi obtained in vivo and in vitro was analyzed by light and electron microscopy. Their antigenicity was also compared by immunofluorescence microscopy and ELISA assays, using serum samples from untreated patients infected with F. pedrosoi. Due to the similarity of the sclerotic cells obtained in vivo and in vitro, the latter can be more adequately in studies of host-parasite interactions in chromoblastomycosis.

Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola.

Colletotrichum graminicola, a pathogen of sorghum and corn, was investigated prior and during germination as to certain aspects of acid phosphatase activity and lipid mobilization. Ungerminated conidia cytoplasm was filled with lipid deposits, which were mobilized during the germination process. Cytochemical ultrastructural examination showed that conidia vacuoles exhibit acid phosphatase activity, which is suggestive of lytic activity. Lipid bodies, stored in the ungerminated conidia cytoplasm, were internalized by vacuoles in a process analogous to microautophagy and were apparently digested inside them. The lipid bodies disappeared and vacuoles became enlarged in conidial cells during germination. Appressoria also showed acid phosphatase activity in multiple heterogeneous vesicles which were, in most cases, juxtaposed with lipid bodies. These results suggest that the vacuolar system plays an important role during C. graminicola germination and that the initial stages of lipid metabolization are taking place inside the vacuoles.

Fine structure and cytochemical study of the interaction between Fonsecaea pedrosoi and rat polymorphonuclear leukocyte.

The induction of a granulomatous reaction is frequently observed in subcutaneous mycoses. Our previous studies demonstrated that Fonsecaea pedrosoi was able to survive and proliferate in tissue macrophages and that activated macrophages were fungistatic but not fungicidal. By contrast, our present studies revealed that neutrophils were able to kill F. pedrosoi cells in periods shorter than 20 min. Several phases of the interaction process were analysed by light and electron microscopy. The kinetic analysis demonstrated no significant difference during the first hour of F. pedrosoi-neutrophil interaction. Electron microscopy images showed that neutrophils readily associated with and killed extracellular fungi; however, few fungi were ingested. During this process the activation of respiratory burst took place as evaluated by light and electron microscopy. Cytochemical activity of acid and alkaline phosphatase was detected in low levels during the host cell parasite interaction.

Effect of environmental factors on Fonsecaea pedrosoi morphogenesis with emphasis on sclerotic cells induced by propranolol.

The influence of growth conditions, as well as of propranolol on Fonsecaea pedrosoi morphogenesis was established using the chemically defined media of Czapeck-Dox (CD) and Butterfield (BF). Mycelial growth of F. pedrosoi in both media was obtained at room temperature (25 degrees C) for 14 days, without shaking, whereas conidia formed at 37 degrees C, for 4 days, in shaken cultures and could be isolated free from the mycelium by filtration in gauze. At low pH (2.5-3.0), there appeared sclerotic cells attached to normal hyphae. When propranolol ws added to the CD medium moniliform hyphae were observed, whereas this drug in the BF medium induced formation of sclerotic cells. Ultrastructural examination revealed that the propranolol-induced sclerotic cells were very similar to those observed in infected tissues.

Characterization of Fonsecaea pedrosoi melanin.

The constituents of the melanin complex from mycelial forms of Fonsecaea pedrosoi were partially characterized. The pigment was mainly accumulated on large alkali-extractable, electron-dense cytoplasmic bodies (melanosomes) and, apparently, on the outer layer of the cell wall as external deposits within verrucose outgrowths. Using electron microscopy and Thiéry's periodate/thiosemicarbazide/silver proteinate staining method, glycogen-like particles were also detected at the periphery of the cells. Melanin constituents comprised aromatic and aliphatic/glycosidic structures with a predominance of the latter. Infrared spectra showed the presence of hydroxyl, carbonyl and carboxyl groups. The aliphatic/glycosidic moiety consisted of fatty acids and polysaccharides with protein, in a ratio protein/polysaccharide 1:15. Rhamnose, mannose, galactose and glucose (in the ratio 1:2:4:3.5) were the constituents of the polysaccharide. Lipid components included even-numbered, saturated and unsaturated fatty acids (in the ratio 2:1) ranging from C16 to C18. Palmitic and oleic acids were the prominent fatty acids. Aspartic and glutamic acids, leucine, glycine and alanine were the major amino acids. Non-pigmented cells of F. pedrosoi were studied for comparison with the pigmented forms: they did not accumulate acid-insoluble precursors of melanin.

Subcutaneous phaeohyphomycosis caused by Bipolaris hawaiiensis: a case report

Os autores descrevem um caso de feohifomicose com lesao verrucosa no halux esquerdo. O paciente nao apresentava sinais clinicos de deficiencia imunologica. Os exames direto e histopatologico mostraram celulas leveduriformes e poucas hifas septadas, demacioides, essenciais ao diagnostico desta micose. O cultivo em lamina permitiu identificar o hifomiceto como Biopolaris hawaiiensis.

Subcutaneous phaeohyphomycosis caused by Bipolaris hawaiiensis. A case report.

A case of phaeohyphomycosis caused by Bipolaris hawaiiensis is reported. The patient, an immunocompetent host, presented a verrucous lesion on the first finger of the left foot. Dematiaceous septate hyphae and yeast-like elements were seen in direct and histological examination. The isolated strain was identified on the basis of micro and macromorphological aspects. Treated with electrocoagulation, the lesions healed and presented no relapse after two years follow-up.

Ultrastructure of phagocytosed Paracoccidioides brasiliensis in nonactivated or activated macrophages.

Transmission electron microscopy was used to study ultrastructures in Paracoccidioides brasiliensis yeast cells after ingestion by nonactivated or cytokine-activated murine peritoneal macrophages. Yeast cells ingested by nonactivated macrophages had typical bi- and trilayered cell walls, plasma membranes, mitochondria, nuclei, vacuoles, etc., which remained intact for 24 h of coculture. In contrast, yeast cells ingested by activated macrophages exhibited abnormal mitochondrial ultrastructures within 4 h of interaction. Subsequent events that occurred were the formation of several clear vacuoles per cell, disintegration of the cytoplasm, and development of empty cells with intact walls. These findings provide, for the first time, insights into stepwise damage to fungal cells by activated macrophages (of particular interest in this instance because of prior evidence that the damage is due to nonoxidative mechanisms) and give possible clues regarding fungicidal mechanisms.

Fine structure and cytochemistry of the interaction between Fonsecaea pedrosoi and mouse resident macrophages.

The interaction between conidia of Fonsecaea pedrosoi and mouse resident peritoneal macrophages was observed by light microscopy and by scanning (SEM) and transmission (TEM) electron microscopy. The conidia first attached to the surface of the macrophage and were then ingested. Prolonged incubation of the macrophage cultures showed proliferation of intracellular fungi as well as those which remained attached to the macrophage surface. The conidia were ingested by a typical phagocytic process, with formation of a phagosome. Macrophage lysosomes were observed to fuse with the phagosomes by immunofluorescence microscopy of macrophages previously labeled with acridine orange, by TEM of thin sections of macrophages labeled with albumin-gold, and by ultrastructural localization of acid phosphatase within the phagosomes.

Interaction between macrophage and parasite cells in lobomycosis. The thickened cell wall of Paracoccidioides loboi exhibits apertures to the extracellular milieu.

Lesioned, cutaneous, tissue fragments from five indians of the Caiabi tribe with lobomycosis, living in the Xingu National Park (Central Brazil), are analyzed by light and electron microscopy. Clusters of macrophages filled with parasite and/or cell wall debris, separated by collagen fiber bundles, characterize the morphological pattern seen in thick and thin sections. Paracoccidioides loboi within the phagocytic cells are multinucleate organisms whose cytoplasm contains mitochondria with few cristae, ribosomes and vacuoles; a large, dense, globular body and peculiarly curved mitochondrial profiles are described. From the outer portion of the double layered parasite cell wall, radial projections commonly emerge, rendering the structure conspicuously thicker and more irregularly surfaced than that seen in many other phagocytized yeast cell species. The cell wall layers from fungi possessing distinct subcellular organization show a weak or no reaction for acid phosphatase. Most of the foamy cells commonly seen by light microscopy are macrophages filled with fungal cell wall remnants which exhibit marked acid phosphatase activity. Occasionally, microchannels extending from the outer layer of the parasite cell wall to the macrophage surface and exocytic-like openings, possibly derived from the fusion of the macrophage membrane covering the parasite cell wall and the macrophage plasmalemma can be seen. Through such routes, material of texture and density similar to that of the outermost cell wall layer appears to be deposited extracellularly.

Nuclear staining of Paracoccidioides brasiliensis conidia.

More than 80% of the conidia produced by two different isolates of Paracoccidioides brasiliensis, were found to be uninucleate; however, when they were incubated at 37 degrees C and began to transform into yeast cells, they became bi- or multi-nucleated, so that when most of the conidia had already transformed into yeast cells (72-96 h), there were at least four or five nuclei per cell in approximately 80% of the conidia examined.

Scanning electron microscopic studies of Paracoccidioides brasiliensis in the yeast phase.

Scanning electron microscopy of four different Paracoccidioides brasiliensis isolates in the yeast phase revealed that mother cells generating multiple, spherically shaped buds may be firmly or tenuously associated with their progeny whereas elongated buds remain attached to the mother cell through stem-like structures and may represent early stages of hypha formation. The yeast cell surfaces were covered with a delicate network of microfibrillar components.