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The present study deals with membrane-bound efflux pumps, MexAB-OprM and MexXY and their respective regulatory genes mexR, nalC, nalD and mexZ in multidrug resistant (MDR) Pseudomonas aeruginosa. Following antibiotic sensitivity testing and detection of various beta-lactamases, hyperexpression of efflux pump genes, mexB and mexY in the isolates was investigated using semi-quantitative and real-time reverse transcription-PCR. Amplicons from regulatory genes were sequenced and subjected to mutational and phylogenetic analysis. Twenty-nine clinical isolates of P. aeruginosa were obtained from a total of 144 MDR gram-negative bacteria collected from Kerala State, South India. All strains were found to be resistant to ampicillin and nalidixic acid with 13.8, 44.8 and 31% testing positive for extended-spectrum beta-lactamases, metallo-beta-lactamases and AmpC producers respectively. Increased mexB and mexY transcription was detected respectively in 10.3 and 20.7% of the isolates in comparison with P. aeruginosa reference strain, PAO (MTCC). Co-expression of MexY was also observed in MexB overproducers. Various synonymous/and non-synonymous mutations in regulatory gene sequences of efflux pump operons were detected. In the strain designate Pa16, mexR was found to harbour four novel point mutations with one transversion and three transitions which included a substitution of an ochre codon with that for serine. The gene also displayed a novel mutation involving insertion of a cysteine at the 444th base position, followed by an opal codon. The genetic divergence and homogeneity of the concatenated (mexR, nalC and nalD) regulatory gene sequences of mexAB-oprM operon was apparent in the phylogram generated with similar sequences retrieved from public database.

Plants have proved to be an important source of anti-cancer drugs. Wrightia arborea, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. This study evaluates the antiproliferative/apoptotic potential of Wrightia arborea leaf extracts, prepared in different organic solvents, on cancer cell lines. MTT assay, light and fluorescence microscopy, flow cytometry, DNA laddering, alkaline comet assay, and western blotting were some of the techniques used for evaluation. Combinations of camptothecin, either with CHK1 inhibitor-PD407824 or with W. arborea leaf extract, were deployed to determine the G2 abrogating potential of the extract. The chloroform extract (WAC) selectively killed K562 cells, without affecting cancerous MCF-7, Hep G2 cells, and normal human peripheral blood lymphocytes. Cell death was characterized by observation of apoptotic bodies, increased Ca2+ and ROS, phosphatidyl serine externalization, mitochondrial membrane depolarization, DNA laddering, increased sub-G1 population, and altered expression of caspase 3, -9, and PARP. WAC also induced DNA damage, alterations in key G2/M phase protein expression, cell cycle perturbation, and potent G2 abrogation. The present study showed that W. arborea leaf extract, WAC, is capable of selectively killing leukemic cancer cells leaving normal lymphocytes unaffected. Our results indicate that this is effectuated through DNA damage and G2 abrogation leading to mitochondrial apoptosis. Taken together, this report contributes toward a better understanding of the anticancer properties of this traditional medicinal plant extract possessing valuable bioactive constituents which can serve as a bioresource for promising complimentary/alternative/chemopreventive therapeutics.

Zerumbone, a natural cyclic sesquiterpene, has been the focus of recent research as it has been found to exhibit selective toxicity towards cancer cells compared to normal cells. Studies on the cell cycle phase-specific effects of this interesting compound, however, remain sparse. Hence, concentration and time-dependent effects of zerumbone were evaluated employing a suitable model system, the naturally synchronous surface cultures of Physarum polycephalum. Zerumbone treatment in S, early, and late G2 phases resulted in G2 arrest. Early G2 phase exhibited the highest sensitivity (P < 0.001) to the compound. Protein profiles showed a complete inhibition of cyclin B1 expression following zerumbone treatment. Furthermore, FACS and comet analysis revealed that zerumbone inhibited DNA synthesis (P < 0.001) without being genotoxic at the concentrations tested. Differential display of mRNA showed distinct zerumbone-induced variations in transcript profiles, an analysis of which suggested a likely link between cellular networks involving stress-related gene expression and G2 arrest in P. polycephalum.