Assessment of Programmed Cell Death of Aspergillus flavus by Triggered Cysteine-dependent Aspartate-directed Proteases (Meta-caspase3) Lethality Mechanism of Novel Compounds Isolated from Ethyl Acetate Extract of Spondias mombin
Biochemistry and Modern Applications, Vol. 2, No 1, p.30-34.
Spondias mombin is a plant that has been traditionally noted for its medicinal with a preliminary results report a wide range of antibacterial and antifungal properties. Meta-caspases and Caspases are essential in cells for programmed cell death, in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. A 12 hours old culture of each microorganism was re-suspended in plant extract at 1000 μg mL in a total volume of 500 μl for 0, 15, 30, 45, 60, and 180 minutes. The cells were pelleted by centrifugation at 5000 g for 5 minutes. The pellets were rinsed twice in phosphate buffer saline (PBS). Then 1/10 volume of 95% ethanol plus 5% saturated phenol were added to the pellets to stabilize cellular RNA. The cells were then re-harvested by centrifugation (8200 g, 4°C and 2 minutes). The supernatant was aspirated and pellets re-suspended in 800 μl of lysis buffer (10 mMTris, adjusted to pH 8.0 with HCl, 1 mM EDTA) and 8.3 U/ml Ready- LyseTM Lysozyme Solution. After the pellets were re-suspended, 80 μl of a 10% SDS solution was added, mixed and incubated for 2 minutes at 64 °C. Then 88 μl of 1 M NaOAc (pH 5.2) was mixed with the lysate followed by an equal volume of water and saturated phenol was added. Total RNA was quantified using Spectrophotometric absorbance at 260 nm DNA was removed with Turbo DNA-free (Ambion, Inc.). Reverse Transcription-PCR reaction was performed in a 15.0 μl final volume (kit number-DNA-PCR739288). Assessment of Polymerase Chain Reaction products (amplicons) were electrophoreses in 0.5% of agarose gel using 0.5 × TBE buffer ( 2.6 g of Tris base, 5 g of Tris boric acid and 2 ml of 0.5M EDTA and adjusted to pH 8.3 with the sodium hydroxide pellet) with 0.5 μl ethidum bromide. The mechanism of action of isolated novel compounds using Metacaspase3 to programme the death of test organism (Aspergillus flavus) between 0 and 180 minutes interval. It was observed that cell (via DNA) were completely destroyed at 180 minutes with all the isolated compounds. The purpose of this research work is to evaluate the programmed cell death (PCD) of Aspergillus flavus by triggered Cysteine-dependent Aspartate-directed proteases (meta-caspase3) lethality mechanism of novel compound isolated from ethyl acetate extract of Spondias mombin.