Neurospora crassa developmental control mediated by the FLB-3 transcription factor.

Here, we report that the Neurospora crassa FLB-3 protein, the ortholog of the Aspergillus nidulans FlbC transcription factor, is required for developmental control. Deletion of flb-3 leads to changes in hyphae morphology and affects sexual and asexual development. We identified, as putative FLB-3 targets, the N. crassa aba-1, wet-1 and vos-1 genes, orthologs of the ones involved in A. nidulans asexual development and that work downstream of FlbC (abaA, wetA and vosA). In N. crassa, these three genes require FLB-3 for proper expression; however, they appear not to be required for normal development, as demonstrated by gene expression analyses during vegetative growth and asexual development. Moreover, mutant strains in the three genes conidiate well and produce viable conidia. We also determined FLB-3 DNA-binding preferences via protein-binding microarrays (PBMs) and demonstrated by chromatin immunoprecipitation (ChIP) that FLB-3 binds the aba-1, wet-1 and vos-1 promoters. Our data support an important role for FLB-3 in N. crassa development and highlight differences between the regulatory pathways controlled by this transcription factor in different fungal species.

Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways.

BACKGROUND: Glycogen and trehalose are storage carbohydrates and their levels in microorganisms vary according to environmental conditions. In Neurospora crassa, alkaline pH stress highly influences glycogen levels, and in Saccharomyces cerevisiae, the response to pH stress also involves the calcineurin signaling pathway mediated by the Crz1 transcription factor. Recently, in yeast, pH stress response genes were identified as targets of Crz1 including genes involved in glycogen and trehalose metabolism. In this work, we present evidence that in N. crassa the glycogen and trehalose metabolism is modulated by alkaline pH and calcium stresses. RESULTS: We demonstrated that the pH signaling pathway in N. crassa controls the accumulation of the reserve carbohydrates glycogen and trehalose via the PAC-3 transcription factor, which is the central regulator of the signaling pathway. The protein binds to the promoters of most of the genes encoding enzymes of glycogen and trehalose metabolism and regulates their expression. We also demonstrated that the reserve carbohydrate levels and gene expression are both modulated under calcium stress and that the response to calcium stress may involve the concerted action of PAC-3. Calcium activates growth of the Δpac-3 strain and influences its glycogen and trehalose accumulation. In addition, calcium stress differently regulates glycogen and trehalose metabolism in the mutant strain compared to the wild-type strain. While glycogen levels are decreased in both strains, the trehalose levels are significantly increased in the wild-type strain and not affected by calcium in the mutant strain when compared to mycelium not exposed to calcium. CONCLUSIONS: We previously reported the role of PAC-3 as a transcription factor involved in glycogen metabolism regulation by controlling the expression of the gsn gene, which encodes an enzyme of glycogen synthesis. In this work, we extended the investigation by studying in greater detail the effects of pH on the metabolism of the reserve carbohydrate glycogen and trehalose. We also demonstrated that calcium stress affects the reserve carbohydrate levels and the response to calcium stress may require PAC-3. Considering that the reserve carbohydrate metabolism may be subjected to different signaling pathways control, our data contribute to the understanding of the N. crassa responses under pH and calcium stresses.

Morphological, biological and molecular characterization of three strains of Trypanosoma cruzi Chagas, 1909 (Kinetoplastida, Trypanosomatidae) isolated from Triatoma sordida (Stal) 1859 (Hemiptera, Reduviidae) and a domestic cat.

A study was conducted of the biological, morphological and molecular characters of 3 strains of Trypanosoma cruzi (SI(5), SI(8) and SIGR(3)) isolated from specimens of Triatoma sordida collected in Santo Inácio and a domestic cat. In order to carry out the study, the following parameters were evaluated: pre-patent period, parasitaemia curves, morphology of the parasites, mortality rates, histopathological lesions and molecular typing. The strains presented variable pre-patent periods, low parasitaemia and no animal mortality. The morphological study of trypomastigotes showed a predominance of intermediate-width and short-length forms, as well as low nuclear index. Epimastigotes presented a low nuclear index, intermediate-width forms in strains SI(5) and SI(8), and large-width forms in SIGR(3). A shorter length could be noted in strains SI(8) and SIGR3, whereas SI(5) displayed an intermediate length. The histopathological study did not detect amastigote nests in tissues. The amplification of the divergent domain of 24Sα rRNA, HSP60 and GPI genes of strains SI(5), SI(8) and SIGR(3) classified the 3 strains into Group II. Biological parameters made it possible to classify the strains isolated in Santo Inácio (BA) into Biodeme III, Zymodeme 1 and Group II of T. cruzi.

In vitro activity of compounds isolated from Piper crassinervium against Trypanosoma cruzi.

This study describes the antichagasic potential of five compounds isolated from leaves of Piper crassinervium (Piperaceae). Two prenylated benzoic acid derivatives, one prenylated hydroquinone and two flavanones, were evaluated. The in vitro trypanocidal activity was determined against epimastigote forms of Trypanosoma cruzi (Y strain), the etiologic agent of Chagas disease. The most active compound was the prenylated hydroquinone [1,4-dihydroxy-2-(3(0),7(0)-dimethyl-1(0)-oxo-2(0)-E,6(0)-octadienyl)benzene] with an IC(50) value of 6.10 microg mL(-1), which was in the same order of activity if compared with the positive control benznidazole (IC(50) = 1.60 microg mL(-1)). This is the first report of trypanocidal activity for prenylated hydroquinone and benzoic acid derivatives.

Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m3G antibodies.

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m3G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m3G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.

Natural chromenes and chromene derivatives as potential anti-trypanosomal agents.

The aim of the study was to investigate the anti-trypanocidal activities of natural chromene and chromene derivatives. Five chromenes were isolated from Piper gaudichaudianum and P. aduncum, and a further seven derivatives were prepared using standard reduction, methylation and acetylation procedures. These compounds were assayed in vitro against epimastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. The results showed that the most of the compounds, especially those possessing electron-donating groups as substituents on the aromatic ring, showed potent trypanocidal activity. The most active compound, [(2S)-methyl-2-methyl-8-(3''-methylbut-2''-enyl)-2-(4'-methylpent-3'-enyl)-2H-chromene-6-carboxylate], was almost four times more potent than benznidazole (the positive control) and showed an IC50 of 2.82 microM. The results reveal that chromenes exhibit significant anti-trypanocidal activities and indicate that this class of natural product should be considered further in the development of new and more potent drugs for use in the treatment of Chagas disease.

Mapeamento de bandas de Trans-splicing in vitro com extratos nucleares de Tryopanosoma cruzi / Band mapping of in vitro trans-splicing reaction using Trypanosoma cruzi nuclear extracts

A maturaçäo dos pré-mRNAs em tripanosomatídeos ocorre por meio de um mecanismo denominado trans-splicing, que envolve a excisäo dos íntrons e a uniäo dos éxonsde dois transcritos independentes. Um transcrito curto , splice leader (SL) RNA, é trans-spliceado a pré-mRNAs aceptores, originando o RNA maduro. O objetivo deste trabalho foi padronizar as condiçöes necessárias que favoreçam essa reaçäo in vitro, empregando, para este fim, extratos nucleares de formas epimastigotas de T. cruzi e pré-mRNA de alfa-tubulina de tripanosoma africano. Nas condiçöes experimentais para a reaçäo de trans-splicing, em que se utilizou extrato nuclear de cepa Bolíviae o pré-mRNA a-tubulina (aceptor) marcado radioativamente, na ausência do pré-mRNA SL exógeno, foi obtida uma banda acima do pré-mRNA de alfa-tubulina (possível produto), sugerindo que esta reagiu com o SL presente no extrato nuclear. A banda foi processada, mas näo se detectou a sequência do produto esperado(SL + éxon alfa-tubulina). Entretanto, o tamanho da banda na reaçäo de trans-splicing (acima do pré-mRNA) pode trtar-se do lariat, cuja estrutura molecular näo possibilitaria seu sequenciamento pela metodologia empregada. Interessante salientar que a banda somente aparece na reaçäo de trans-splicing contendo ATP, o que sugere tratar-se de uma reaçäo de transesterificaçäo.