METHODS AND RESULTS: Transmission electron microscopy revealed phage pPM_01 to be a siphovirus (the first reported virus to infect P. mirabilis), with its complete genome sequence successfully determined. The genome was sequenced using Illumina technology and the reads obtained were assembled using CLC Genomic Workbench v.7.0.3. The whole genome contains a total of 58,546 bp of linear double-stranded DNA with a G+C content of 46.9%. Seventy putative genes were identified and annotated using various bioinformatics tools including RAST, Geneious v.R7, National Center for Biotechnology Information (NCBI) BLAST, and tRNAscan-SE-v1.3 Search. Functional clusters of related potential genes were defined (structural, lytic, packaging, replication, modification, and modulatory). The whole genome sequence showed a low similarity to known phages (i.e., Enterobacter phage Enc34 and Enterobacteria phage Chi). Host range determination and SDS-PAGE analysis were also performed.
CONCLUSIONS: The inability to lysogenize a host, the absence of endotoxin genes in the annotated genome, and the lytic behavior suggest phage pPM_01 as a possible safe biological candidate to control P. mirabilis infection.
AIM OF THE STUDY: To determine the mechanism of action of pure clausenidin crystals in the induction of hepatocellular carcinoma (hepG2) cells apoptosis.
MATERIALS AND METHODS: Pure clausenidin was isolated from Clausena excavata Burm.f. and characterized using (1)H and (13)C NMR spectra. Clausenidin-induced cytotoxicity was determined by MTT assay. The morphology of hepG2 after treatment with clausenidin was determined by fluorescence and Scanning Electron Microscopy. The effect of clausenidin on the apoptotic genes and proteins were determined by real-time qPCR and protein array profiling, respectively. The involvement of the mitochondria in clausenidin-induced apoptosis was investigated using MMP, caspase 3 and 9 assays.
RESULTS: Clausenidin induced significant (p<0.05) and dose-dependent apoptosis of hepG2 cells. Cell cycle assay showed that clausenidin induced a G2/M phase arrest, caused mitochondrial membrane depolarization and significantly (p<0.05) increased expression of caspases 3 and 9, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, clausenidin caused decreased expression of the anti-apoptotic protein, Bcl 2 and increased expression of the pro-apoptotic protein, Bax. This finding was confirmed by the downregulation of Bcl-2 gene and upregulation of the Bax gene in the treated hepG2 cells.
CONCLUSION: Clausenidin extracted from Clausena excavata Burm.f. is an anti-hepG2 cell compound as shown by its ability to induce apoptosis through the mitochondrial pathway of apoptosis. Clausenidin can potentially be developed into an anticancer compound.