Recyclable markers based on site-specific recombination allow repetitive gene targeting in filamentous fungi. Here we describe for the first time functionality of the bacterial recombination system employing ␤ serine recombinase acting on six recognition sequences (␤-rec/six) in a fungal host, the human pathogen Aspergillus fumigatus, and its use in establishing a self-excising resistance marker cassette for serial gene replacement.

This study was performed to evaluate the protective efficacy of metoclopramide (MCP) against the organophosphates paraoxon (POX)- and malathion (MLT)-induced apoptosis in the murine L929 skin fibroblasts. L929 cells were exposed to either POX (10 nm) or 1.0 μm MLT in the absence and presence of increased concentrations of MCP. The protective effect of MCP on these organophosphate-stimulated apoptotic events was evaluated by flow cytometry analysis after staining with annexin-V/propidium iodide, processing and activation of the executioner caspase-3, cleavage of the poly-ADP ribose polymerase, fragmentation of the nucleosomal DNA and disruption of the mitochondrial membrane potential (Δψ). Our results showed that increased doses of MCP alone (≥10 μm) did not induce apoptosis or activation of caspase-3. Pretreatment of the cells with MCP attenuated all the apoptotic events triggered by the organophosphate compounds in a dose-dependent manner reaching ~70-80% protection when they were preincubated at 1 and 5 μm of the drug before the addition of POX and MLT, respectively. Interestingly, MCP did not offer a significant protective effect against the cytotoxicity of tumor necrosis factor-α, cisplatinum, etoposide or paclitaxel, which stimulate apoptosis by various mechanisms, suggesting that the anti-apoptotic effect of the drug is specific to organophosphates. The strong and specific anti-apoptotic activity of subclinical doses of MCP against the cytotoxicity of organophosphate compounds suggests its potential clinical application in treating their poisoning.