RESUMO
Amylomyces rouxii is a zygomycete that produces extracellular protease and tyrosinase. The tyrosinase activity is negatively regulated by the proteases and, which attempts to purify the tyrosinase (tyr) enzyme that has been hampered by the presence of a protease that co-purified with it. In this work we identified genes encoding aspartic protease II (aspII) and VI of A. rouxii. Using an RNAi strategy based on the generation of a siRNA by transcription from two opposite-orientated promoters, the expression of these two proteases was silenced, showing that this molecular tool is suitable for gene silencing in Amylomyces. The transformant strains showed a significant attenuation of the transcripts (determined by RT-qPCR), with respective inhibition of the protease activity. In the case of aspII, inhibition was in the range of 43-90 % in different transformants, which correlated well with up to a five-fold increase in tyr activity with respect to the wild type and control strains. In contrast, silencing of aspVI caused a 43-65 % decrease in protease activity but had no significant effect on the tyr activity. The results show that aspII has a negative effect on tyr activity, and that the silencing of this protease is important to obtain strains with high levels of tyr activity.
Assuntos
Ácido Aspártico Proteases , Mucorales , RNA Interferente Pequeno , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Mucorales/genéticaRESUMO
A wide variety of biological functions, including those involved in the morphogenesis process of basidiomycete fungi, have been attributed to laccase enzymes. In this work, RNA interference (RNAi) was used to evaluate the role of the laccase (lacc2) gene of Pleurotus ostreatus PoB. Previously, transformant strains of P. ostreatus were obtained and according to their level of silencing they were classified as light (T7), medium (T21) or severe (T26 and T27). The attenuation of the lacc2 gene in these transformants was determined by RT-PCR. Silencing of lacc2 resulted in a decrease in laccase activity between 30 and 55%, which depended on the level of laccase expression achieved. The silenced strains (T21, T26, and T27) displayed a delay in the development of mycelium on potato dextrose agar (PDA) medium, whereas in the cultures grown on wheat straw, we found that these strains were incapable of producing aerial mycelium, primordia, and fruiting bodies. Scanning electron microscopy (SEM) showed the presence of toxocyst-like structures. The highest abundance of these structures was observed in the wild-type (PoB) and T7 strains. However, the abundance of toxocysts decreased in the T21 and T26 strains, and in T27 they were not detected. These results suggest that the presence and abundance of toxocyst-like structures are directly related to the development of fruiting bodies. Furthermore, our data confirm that lacc2 is involved in the morphogenesis process of P. ostreatus.
Assuntos
Ascomicetos , Pleurotus , Lacase/genética , Lacase/metabolismo , Ascomicetos/metabolismoRESUMO
Methanogenesis may represent a key process in the terminal phases of anaerobic organic matter mineralization in sediments of coastal lagoons. The aim of the present work was to study the temporal and spatial dynamics of methanogenic archaea in sediments of tropical coastal lagoons and their relationship with environmental changes in order to determine how these influence methanogenic community. Sediment samples were collected during the dry (February, May, and early June) and rainy seasons (July, October, and November). Microbiological analysis included the quantification of viable methanogenic archaea (MA) with three substrates and the evaluation of kinetic activity from acetate in the presence and absence of sulfate. The environmental variables assessed were temperature, pH, Eh, salinity, sulfate, solids content, organic carbon, and carbohydrates. MA abundance was significantly higher in the rainy season (10(6)-10(7) cells/g) compared with the dry season (10(4)-10(6) cells/g), with methanol as an important substrate. At spatial level, MA were detected in the two layers analyzed, and no important variations were observed either in MA abundance or activity. Salinity, sulfate, solids, organic carbon, and Eh were the environmental variables related to methanogenic community. A conceptual model is proposed to explain the dynamics of the MA.
Assuntos
Archaea/crescimento & desenvolvimento , Archaea/metabolismo , Sedimentos Geológicos/microbiologia , Metano/metabolismo , Acetatos/metabolismo , Contagem de Células , Modelos Biológicos , Sulfatos/metabolismo , Temperatura , Clima Tropical , Água/químicaRESUMO
Road traffic injuries are a leading public health problem in Colombia. Pedestrians are the most vulnerable road users, especially in the main urban centers of Bogotá, Medellin and Cali. Data analyzed in this report include official statistics from the National Police and the National Institute of Legal Medicine and Forensic Sciences for 1996-2000, and results of a study conducted at the National University of Colombia in 2000. Methods from the Highway Capacity Manual were used for determining physical and technical variables, and a Geographical Information System tool was used for the location and spatial analysis of the road traffic crashes. Pedestrians accounted for close to 32% of injuries and 40% of the deaths from road traffic crashes. The problem of road traffic crashes existed predominately in urban areas. In the main urban centers, pedestrians constituted nearly 68% of road traffic crash victims. The high level of risky road use behaviors demonstrated by pedestrians and drivers, and inadequate infrastructure for safe mobility of pedestrians in some sections of the road network were the main contributing factors. Major improvements were achieved in Bogotá following enhancements to the municipal transport system and other policies introduced since 1995. In conclusion, policies and programs for improving road safety, in particular pedestrian safety, and strengthening urban planning are top priority.