Antifungal activity of altenusin isolated from the endophytic fungus Alternaria sp. against the pathogenic fungus Paracoccidioides brasiliensis

BackgroundAltenusin is a biphenyl derivative isolated from different species of fungi, which presents several biological activities.AimsWe report the antifungal activity of the altenusin isolated from the endophytic fungus Alternaria sp., against clinical isolates of Paracoccidioides brasiliensis, and its action on cell walls of P. brasiliensis and the nonpathogenic yeast Schizosaccharomyces pombe.MethodsIn vitro antifungal activity of altenusin was evaluated using the broth microdilution method against 11 strains of P. brasiliensis and one strain of S. pombe. The effects of the altenusin on the cell wall were estimated using the sorbitol protection assay.ResultsThe altenusin presented strong activity against P. brasiliensis with MIC values ranging between 1.9 and 31.2 μg/ml, and 62.5 μg/ml for S. pombe. Our results demonstrated that the MIC values for altenusin were increased for P. brasiliensis Pb18 and for S. pombe when the medium was supplemented with sorbitol. Additionally, S. pombe cells treated with altenusin were more rounded in shape than untreated cells.ConclusionsAltenusin showed activity against clinical strains of P. brasiliensis at the concentration tested, and this compound probably affects fungal cell walls. These findings suggest that altenusin could act through the inhibition of cell wall synthesis or assembly in P. brasiliensis and S. pombe, and could be considered as a lead compound for the design of new antifungals.     

N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation

Sepsis is a common cause of acute kidney injury (AKI) and acute lung injury. Oxidative stress plays as important role in such injury. The aim of this study was to evaluate the effects that the potent antioxidant N-acetylcysteine (NAC) has on renal and pulmonary function in rats with sepsis. Rats, treated or not with NAC (4.8 g/l in drinking water), underwent cecal ligation and puncture (CLP) 2 days after the initiation of NAC treatment, which was maintained throughout the study. At 24 h post-CLP, renal and pulmonary function were studied in four groups: control, control + NAC, CLP, and CLP + NAC. All animals were submitted to low-tidal-volume mechanical ventilation. We evaluated respiratory mechanics, the sodium cotransporters Na-K-2Cl (NKCC1) and the α-subunit of the epithelial sodium channel (α-ENaC), polymorphonuclear neutrophils, the edema index, oxidative stress (plasma thiobarbituric acid reactive substances and lung tissue 8-isoprostane), and glomerular filtration rate. The CLP rats developed AKI, which was ameliorated in the CLP + NAC rats. Sepsis-induced alterations in respiratory mechanics were also ameliorated by NAC. Edema indexes were lower in the CLP + NAC group, as was the wet-to-dry lung weight ratio. In CLP + NAC rats, α-ENaC expression was upregulated, whereas that of NKCC1 was downregulated, although the difference was not significant. In the CLP + NAC group, oxidative stress was significantly lower and survival rates were significantly higher than in the CLP group. The protective effects of NAC (against kidney and lung injury) are likely attributable to the decrease in oxidative stress, suggesting that NAC can be useful in the treatment of sepsis.     

Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes

Nitric oxide (NO) is a highly reactive free radical involved in intra- and intercellular signaling in various stages of reproduction. The objective of the present study was to evaluate the effect of the addition of sodium nitroprusside (SNP), a NO donor, on nuclear and cytoplasmic in vitro maturation of bovine oocytes. Analysis of variance was conducted and the means were compared by t test at a level of 5%. Low (10(-7) and 10(-9)M) and intermediate (10(-5)M) concentrations of SNP had no significant effect on nuclear maturation, however, when a greater concentration of SNP (10(-3)M) was added, oocytes remained in metaphase I (MI) after 24 h culture (P<0.05) and did not show cumulus expansion. To evaluate if this effect was reversible and if a retardation or inhibition had occurred in the progression from MI to MII, oocytes were cultured in presence of 10(-3)M of SNP for 24 h followed by culture for an additional 24 h in medium with or without SNP. After 48 h, the oocytes remained in MI even when the medium was changed at 24 h with or without SNP. The kinetics of nuclear maturation was assessed to evaluate if there had been or not a retardation in the progression of meiosis with the concentration of 10(-3)M SNP. This concentration delayed germinal vesicle breakdown (VGBD) at 8 h of culture (P<0.05), and at 12 h there was no significant difference between the control and the treated group. The concentrations that did not induce alterations in nuclear maturation were evaluated for cytoplasmic maturation. The concentration of 10(-5)M improved the percentage of peripheral cortical granules (P<0.05), and significantly increased the percentage of blastocysts. These results demonstrate that SNP at greater concentrations (10(-3)M) has a cytotoxic effect, but at intermediate (10(-5)M) concentrations it increases blastocyst rates. NO exhibits a dual effect on bovine oocytes, inhibits (10(-3)M of SNP) nuclear and cytoplasmic maturation or stimulates (10(-5)M of SNP) cytoplasmic maturation, depending on concentration in the culture medium.     

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 microg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 microM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 micro M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 microM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.     

Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [(3)H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[(3)H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17beta into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [(3)H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [(3)H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [(3)H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15 degrees C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by alpha-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17beta in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17beta injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [(3)H]uridine uptake into RNA are not precursor-pool-dependent.     

Antifungal and other Biological Activities from Oudemansiella Canarii(Basidiomycota)

Summary The ethyl acetate extract from the fungus Oudemansiella canarii grown in malt extract medium was evaluated against (a) the recombinant enzyme trypanothione reductase from Trypanosoma cruzi, (b) lymphocyte proliferation in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemaglutinin, (c) the human tumour cell lineages MCF-7, TK-10 and UACC-62, and (d) the phytopathogenic fungus Cladosporium sphaerospermum. At 10 μg/ml, the crude extract was inactive against PBMC but inhibited the growth of UACC-62 cells by 47% and the enzyme trypanothione reductase (TryR). It also presented strong inhibition in the bioautographic assay with C. sphaerospermum. Chromatographic fractionation guided by this assay allowed the isolation of oudemansin A (1), a known fungitoxic compound that showed a minimum inhibitory concentration (MIC) of 1.25 μg/spot in the bioautographic assay. As oudemansin A was not active in the other assays, other components in the extract may be responsible for the observed activities by the crude extract against the UACC-62 cells or the TryR enzyme.     

Biomphalaria spp. (Preston,1910) snails in the municipality of Juiz de Fora, Zona da Mata Mineira mesoregion, state of Minas Gerais, Brazil

This study focuses on the geographic distribution of the snail of the genus Biomphalaria and evaluates its infectivity by Schistosoma mansoni in 5264 specimens collected in the municipality of Juiz de Fora, Minas Gerais, Brazil. Of the 31 locations studied, 6 were reservoirs, 11 rudimentary holding ponds, 7 irrigation ditches, 5 lakes, 1 ornamental pond, and 1 waterfall. Intermediate hosts were found only in the rudimentary ponds and ditches, which were 100% positive. Using morphological and molecular analysis techniques, B. tenagophila, B. peregrina, and B. straminea were identified. This is the first report of B. stramínea in the municipality, and evaluation of its infective potential revealed susceptibility of 25.4%. Although we did not find specimens of Biomphalaria infected by S. mansoni, the data obtained indicate the presence of intermediate hosts, especially in the irrigation ditches in Juiz de Fora, and their proximity to contaminated areas.     

Leishmanicidal Metabolites from Cochliobolus sp., an Endophytic Fungus Isolated from Piptadenia adiantoides (Fabaceae)

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellman''s reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 µg mL⁻¹. UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC₅₀ values (effective concentrations required to kill 50% of the parasite) of 1.7 µM (95% confidence interval = 1.6 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.