ABSTRACT
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).
Subject(s)
Gene Expression/genetics , Genetic Vectors/genetics , Plasmids , Restriction Mapping/methods , Trypanosoma cruzi/genetics , Blotting, Western , Expressed Sequence Tags/metabolism , Green Fluorescent Proteins , Life Cycle Stages/genetics , Mutagenesis, Insertional , Tetracycline/pharmacology , Trypanosoma cruzi/drug effectsABSTRACT
We studied antioxidant, antibacterial and tripanocide activities of Alvaradoa subovata extracts. The ethanolic extracts showed the greatest DPPH radical scavenging capacity, especially that of bark with an IC50 = 4.7 +/- 0.18 ug/mL. Wood dichloromethane extract displayed growth inhibition of the phytopathogenic bacteria Xanthomona axonopodis in the disk diffusion assay and showed a MIC value of 100 ug/ml. It also showed growth inhibition of Trypanosoma cruzi (IC50 = 0.063 +/- 0.003 mg/mL). A fraction of this extract, which has emodin as the main component, showed tripanocide activity (60 percent of growth inhibition at 100 ug/mL). The main compounds in wood dichloromethane extract were anthraquinones, identified as chrysophanol and emodin, and coumarins, of which scopoletin was identified. These three compound s could serve as analytical markers of the extract. The results of this study show that wood extract of A. subovata constitute a source of bioactive compounds such as antiparasitic and pesticides agents.
En el presente trabajo se estudió la actividad antioxidante, antibacteriana y tripanocida de extractos de Alvaradoa subovata. La mayor actividad depuradora de radicales libres se observó en el extracto etanólico de corteza (CI50 = 4.7 +/- 0.18 ug/mL). El extracto en diclorometano de madera inhibió el crecimiento de la bacteria fitopatógena Xanthomona axonopodis con una CIM = 100 ug/mL. El mismo extracto mostró inhibición del crecimiento de Trypanosoma cruzi (CI50 = 0.063 +/- 0.003 mg/mL). Una fracción de este extracto (100 ug/mL), cuyo componente mayoritario es emodina, inhibió en un 60 por ciento el crecimiento del parásito. Los compuestos mayoritarios detectados en el extracto de madera fueron antraquinonas, entre las cuales se identificaron emodina y crisofanol, y la cumarina escopoletina. Estos tres compuestos podrían servir como marcadores analíticos del extracto. Los resultados de este trabajo muestran que los extractos de A. subovata constituyen una fuente de compuestos bioactivos con potencial como antiparasitarios y plaguicidas.
Subject(s)
Anti-Bacterial Agents/pharmacology , Plant Extracts/pharmacology , Simaroubaceae/chemistry , Trypanocidal Agents/pharmacology , Antioxidants/pharmacology , Biphenyl Compounds/chemistry , Free Radical Scavengers , Microbial Sensitivity Tests , Picrates/chemistry , XanthomonasABSTRACT
Here we present a one-tube nested PCR test, which allows the detection of minimal quantities of Chlamydia trachomatis in human fluids. This assay includes the use of an internal control to avoid false negative results due to the presence of inhibitors. The results obtained show that this assay is robust enough to be used for clinical diagnosis