Molecular characterization and expression analysis of hsp60 gene homologue of sheep blowfly, Lucilia cuprina.

The 60kDa heat shock protein (Hsp60) or chaperonin is one among the highly conserved families of heat shock proteins, known to be involved in variety of cellular activities, including protein folding, thermal protection, etc. In this study we sequence characterized hsp60 gene homologue of Lucilia cuprina, isolated and cloned from the genomic library as well as by genomic PCR, followed by RACE- PCR. The L. cuprina hsp60 gene/protein expression pattern was analyzed in various tissues, either at normal temperature (25±1°C) or after exposure to heat stress (42°C). The analysis of nucleotide sequence of Lchsp60 gene revealed absence of intron and the nuclear localizing signal (NLS). The deduced amino acid sequence showed presence of unique conserved sequences, such as those for mitochondrial localization, ATP binding, etc. Unlike Drosophila, Lucilia showed presence of only one isoform, i.e., hsp60A. Phylogenetic analysis of hsp60 gene homologues from different species revealed Lchsp60 to have >88.36% homology with D. melanogaster, 76.86% with L. sericata, 58.31% with mice, 57.99% with rat, and 57.72% with human. Expression analysis using Real Time PCR and fluorescence imaging showed significant enhancement in the expression level of Lchsp60 upon heat stress in a tissue specific manner, indicating its likely role in thermo-tolerance as well as in normal cellular activities.

Synthesis, anticancer, and antibacterial activities of piplartine derivatives on cell cycle regulation and growth inhibition.

A series of piplartine derivatives were synthesized via Baylis-Hillman reaction and evaluated for anticancer and antibacterial activities. The cytotoxicity of these compounds was examined in two different human tumor cell lines, IMR-32 and HeLa. The antibacterial activity was examined in Staphylococcus aureus and Pseudomonas aeruginosa. The results showed that compounds 2b, 2e, and 2j were found to be the most active compounds, which displayed line no cytotoxicity, but G2-M cell cycle arrest in tumor cells, and showed cytostatic effects in bacteria.

Anti-microbial principles of selected remedial plants from Southern India.

OBJECTIVE: To examine the anti-bacterial activity of leaf extracts of Morus alba L. (Moraceae) and Piper betel L. (Piperaceae), and seed extracts of Bombax ceiba L. (Borabacaceae). METHODS: We have partially purified plant extracts by solvent extraction method, and evaluated the effect of individual fractions on bacterial growth using Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) bacterial strains. RESULTS: Compared with Morus and Bombax fractions, Piper fractions showed significant growth inhibition on all the three types of bacteria studied. The EtOAc-hexane fractions of Piper leaves exhibited significant anti-bacterial activity with minimum inhibitory concentrations (MIC) of 50 µg/mL culture against both gram-positive and gram-negative bacteria. The EtOAc-fractions I, II, and IV inhibited bacterial colony formation on soft agar in addition to growth inhibition. A combination treatment of piper fractions with ampicillin resulted in significant growth inhibition in E. coli and P. aeruginosa, and combination with anticancer drug geldanamycin (2µg/mL) showed selective growth inhibition against P. aeruginosa and S. aureus. Three major compounds, i.e., eugenol, 3-hexene-ol and stigmasterol, were primarily identified from Piper betel leaf extractions. Among the individual compounds, eugenol treatment showed improved growth inhibition compared with stigmasterol and 3-hexene-ol. CONCLUSIONS: We are reporting potential anti-bacterial compounds from Piper betel against both gram-positive and gram-negative bacteria either alone or in combination with drug treatment.

Diferuloylmethane augments the cytotoxic effects of piplartine isolated from Piper chaba.

Natural compound based anticancer drug discovery is gaining interest against a wide variety of tumors. E-piplartine (trans-piplartine), a natural compound isolated from Piper chaba roots is examined against rat histiocytoma (BC-8), mouse embryonal carcinoma (PCC4), mouse macrophages (P388D1 and J774), and human neuroblastoma (IMR32) tumor cells. While Z-piplartine (cis-piplartine) failed to induce cytotoxicity (even at higher concentrations, 50 microM), E-piplartine induced a dose-dependent cytotoxicity (2-24 microM) in different tumor cells. The combinatorial treatment of piplartine with diferuloylmethane (curcumin), an anti-inflammatory and anticancer agent, significantly enhanced the piplartine induced cytotoxicity in tumor cells. Diferuloylmethane itself is not cytotoxic at 15 microM concentration; however, potentiated the piplartine induced cytotoxicity. The tumor cell killing with piplartine is preceded by G1 cell cycle arrest, and surpassed diferuloylmethane induced G2/M arrest when used in combination. In PCC4 cells, piplartine inhibited the cell cycle progression by inactivating cdk2 and destabilizing cyclin D1, whereas diferuloylmethane combination inhibited the ERK1/2 and Raf-1 signaling in addition to the inhibition of cell cycle progression. The over expression of heat shock protein 70, Hsp70 in rat histiocytic tumor cells interfered with piplartine induced cytotoxicity, hence, a cross talk between stress response and anticancer agents is presented. Our data demonstrates the biological and medicinal importance of piplartine isolated from the roots of P. chaba, and indicates that E-piplartine may be a promising candidate to use in combinatorial treatments to combat cancer.

Activation of stress response by ionomycin in rat hepatoma cells.

All living systems respond to a variety of stress conditions by inducing the synthesis of stress or heat shock proteins (HSPs), which transiently protect cells. HSP synthesis was preceded by an increase in intracellular free calcium concentration [(Ca(2+))i]. In this study, we show that Ca(2+) ionophore, ionomycin, induced an immediate increase in intracellular free Ca(2+) and examined how this increase affects heat shock response in rat hepatoma cell line H4II-E-C3. Results indicate that incubating H4II-E-C3 cells with 0.3 microM ionomycin at 37 degrees C for 15 min results in the induction of HSP 70 in both Ca(2+)-containing and Ca(2+)-free medium. Associated with this increase in free Ca(2+) is an in vivo change in membrane organization and activation of signaling molecules like ERKS and SAPKs/JNK. In Ca(2+) containing medium HSP 70 induction mediated by HSF-HSE interaction was faster upon ionomycin treatment as compared to heat shock. Our results show that ionomycin, at sub lethal concentration, increases intracellular free Ca(2+) concentration, activates SAPK/JNK and HSF-HSE interaction, and induces HSP 70 synthesis.

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma.

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells.

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.

Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line.

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC-8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42 degrees C for 30 min. BC-8 is a clone of AK-5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK-5. An increase in either the incubation temperature or time or both to BC-8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE-like cysteine proteases. Results presented here demonstrate that BC-8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42 degrees C.