Ombrohydrochory in Thismia panamensis (Standley) Jonk: a mycoheterotrophic species in Brazilian Cerrado forests.

Species of the genus Thismia Griff. are small herbs, considered mycoheterotrophic due to an intimate relationship with fungi. They are achlorophyllous, with complex floral structure but little information on reproductive strategies. This study evaluated structural and ecological aspects associated with the dispersal of seeds of Thismia panamensis (Standley) Jonk. The study was carried out in a forest fragment in the Brazilian Cerrado. During the reproductive period, 36 individuals were monitored for spatial distribution of the population and their fruits were collected. Samples were subjected to light microscopy and microtomography techniques, in addition to an experiment to evaluate seed dispersal by water droplets. Thismia panamensis is up to 8-cm tall, with a tuberous root and stem, without leaves. Its fruit is dehiscent, cup-shaped, 5 ± 1 mm in diameter, containing 219.33 ± 106.70 seeds, with an average length of 0.55 ± 0.07 mm. The seeds are exposed, and their coat has a thin and lignified wall. Accumulation of secretions was observed inside the fruits. The innermost cell layer of the ovary showed typical characteristics of aquiferous parenchyma. Water splash experiments showed that the seeds reached an average distance of 44.04 ± 26.58 cm. Each splash contained, on average, 1.50 ± 1.23 seeds, with 75% of the splashes containing a single seed. A total of 239 seeds were counted in the 163 splashes evaluated. The data show potential seed dispersal by ombrohydrochory in T. panamensis, favouring its maintenance in the study area and reflecting its clumped spatial distribution.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei.

AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.

Molecular cloning and characterization of a glucan synthase gene from the human pathogenic fungus Paracoccidioides brasiliensis.

1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.

Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance.

Germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved using lithium acetate treatment or electroporation. The efficiency of transformation was up to 32 and 25 transformants per microgram of plasmid DNA with the two methods, respectively. Transformants obtained by the lithium acetate method were more stable and showed a high copy number of the hph gene integrated into their genome. The other transformants, from the electroporation procedure, were stable, but unable to grow in the presence of high levels of hygromycin, and detection of the hph gene was only possible by polymerase chain reaction analysis.

Expression of glycoprotein gp43 in stage-specific forms and during dimorphic differentiation of Paracoccidioides brasiliensis.

Expression of the 43 kDa glycoprotein (gp43) was analysed in several Paracoccidioides brasiliensis isolates. Using one- and two-dimensional analysis of crude cellular extracts, it was shown that protein expression in yeast and mycelium was dependent on the isolate analysed. In two strains, in both yeast and mycelium cells. gp43 was present, whereas expression was restricted to the yeast phase of two other strains. The clinical implications of this phase-specific gp43 expression are uncertain.

Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody.

Formas tripomastigotas de Trypanosoma cruzi derivadas de cultura de tecido foram encubadas com soros humanos imunes e nao-imunes.Todos os soros humanos usados tinham titulos elevados de anticorpos das classes IgM ou IgG.Aglutinacao e entumescimento dos parasitos eram observados apos encubacao a 37oC mas muitos tripomastigotas permaneceram circulando livremente nos soros por dois a tres dias. A quantidade de soro imune capaz de lisar um maximo de 10 x 10 elevado a 6 hemacias sensibilizadas nao foi capaz de lisar 4 x 10 elevado a 3 tripomastigotas. Tipicamente, os parasitos apresentavam alteracoes ciclicas com formacao de aglomerados de amastigotas e de epimastigotas. Observou-se multiplicacao dos parasitos nos soros imunes, e a infectividade destes parasitos para camundongos nao foi perdida. Todos os soros usados produziram efeitos similares no crescimento do parasito.O anticorpo ligado ao T. cruzi parece ser endocitado pela medicao de receptor antigenico.O anticorpo conjugado a ferritina era internalizado e descarregado em fagolisossomas onde eles podem ser completamente degradados ate amino-acidos livres. Este parece ser um processo de acoplamento pelo qual a imunoglobulina e primeiramente ligada especificamente ao receptor da superficie do parasito e entao e rapidamente endocitada