The Mechanism of Antifungal Action of Essential Oil from Dill (Anethum graveolens L.) on Aspergillus flavus

The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 µl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus

pXmnATG: an E. coli vector for expression of unfused proteins.

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DNA-mediated transformation of Chlamydomonas reinhardi cells: use of aminoglycoside 3'-phosphotransferase as a selectable marker.

Using a modified vector, we developed a method for DNA-mediated transformation of Chlamydomonas reinhardi with increased efficiency. The vector contained the yeast 2 microns origin of replication as a heterologous replicon. The aminoglycoside 3'-phosphotransferase (APH) gene linked to the simian virus 40 early promoter was used as an antibiotic selectable marker. The C. reinhardi transformants were resistant to 12 micrograms of G418 or 150 micrograms of kanamycin per ml. A quick-blot mRNA analysis demonstrated the presence of RNase-sensitive transcripts from the APH gene in the transformants, suggesting that the acquisition of antibiotic resistance was due to the expression of the APH gene. Southern blot analysis revealed the presence of free plasmid DNA in the transformant. The transforming vector was recovered by transforming recipient bacteria with the total DNA extracted from the C. reinhardi transformant

Isolation and characterization of a species-specific DNA probe for Candida albicans.

As a model for the isolation of species-specific sequences of DNA, we isolated and characterized a species-specific DNA fragment from the Candida albicans genome. We used a series of differential colony hybridization experiments, in which a C. albicans genomic library was hybridized with genomic DNA probes from related organisms to minimize the number of potentially specific C. albicans DNA fragments to be tested. Six clones were tested by dot blot analysis, and one of them, a 1348 bp EcoRI DNA fragment, was confirmed as specific for C. albicans. This species-specific fragment could be utilized as a DNA probe for rapid, sensitive, and specific diagnosis of C. albicans DNA in clinical specimens