EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.     

Bacterial Quorum Sensing Molecule N-3-Oxo-Dodecanoyl-L-Homoserine Lactone Causes Direct Cytotoxicity and Reduced Cell Motility in Human Pancreatic Carcinoma Cells

In spite of chemotherapeutic and surgical advances, pancreatic cancer continues to have a dismal prognosis. Metastasis due to tumor cell migration remains the most critical challenge in treating pancreatic cancer, and conventional chemotherapy is rarely curative. In the quest for more novel molecules to fight this disease, we tested the hypothesis that the Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone (O-DDHSL) would be cytotoxic to and reduce mobility of pancreatic carcinoma cells (Panc-1 and Aspc-1). Results showed a decrease in cell viability from apoptosis, diminished colony formation, and inhibition of migration of the evaluated pancreatic carcinoma cell lines. Also, cell viability decreased in the presence of O-DDHSL when cells were grown in matrigel basement membrane matrix. While messenger RNA for IQGAP-1 decreased in Panc-1 and HPDE cells upon exposure to O-DDHSL, no change was observed in Aspc-1 cells. Cofilin mRNA expression was found to be increased in both HPDE and Panc-1 cells with marginal decrease in Aspc-1 cells. RhoC, a Rho-family GTPase involved in cell motility, increased in the presence of O-DDHSL, suggesting a possible compensatory response to alteration in other migration associated genes. Our results indicate that O-DDHSL could be an effective biomolecule in eukaryotic systems with multimodal function for essential molecular targeting in pancreatic cancer.     

New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs

Poly(ADP-ribose) polymerases (PARPs) are enzymes that transfer ADP-ribose groups to target proteins and thereby affect various nuclear and cytoplasmic processes. The activity of PARP family members, such as PARP1 and PARP2, is tied to cellular signalling pathways, and through poly(ADP-ribosyl)ation (PARylation) they ultimately promote changes in gene expression, RNA and protein abundance, and the location and activity of proteins that mediate signalling responses. PARPs act in a complex response network that is driven by the cellular, molecular and chemical biology of poly(ADP-ribose) (PAR). This PAR-dependent response network is crucial for a broad array of physiological and pathological responses and thus is a good target for chemical therapeutics for several diseases.     

Expression of truncated tobacco osmotin in Escherichia coli: purification and antifungal activity

PURPOSE OF WORK: Tobacco osmotin is a functional homolog of mammalian adiponectin, and has antifungal activity. This work was undertaken to produce recombinant osmotin that has previously been unsuccessful because of its toxicity. Expression of recombinant tobacco osmotin (rOSM) in Escherichia coli inclusion bodies has been achieved. The optimal pH for rOSM expression in ZYM 505 medium is 7.0 at OD(650) of 1.5 of culture growth. The rOSM from the inclusion body was extracted with 8 M urea, and purified using CM-cellulose and cobalt-agarose bead affinity chromatography to a high purity. Approximately 80% of the rOSM remained bound to CM-cellulose and Cobalt-agarose beads after initial elution. The yield of purified rOSM was between 40 and 50 mg from 2 l of culture. Repeated elution of protein from CM-cellulose and Co-agarose increased the yield of rOSM to 200 mg from 2 l culture. The purified rOSM showed variable antifungal activities against two pathogenic yeast strains; Cryptococcus neoformans, Candida albicans, and non-pathogenic strains; Saccharomyces cerevisiae and Pichia methanolica.