Endothelial Cell-Derived TGF-β Promotes Epithelial-Mesenchymal Transition via CD133 in HBx-Infected Hepatoma Cells

Background: Hepatitis B-X Protein (HBx) encoded in Hepatitis B virus (HBV) is known to play a critical role in development and progression of HBV induced hepatocellular carcinoma (HCC). HBx interacts with and activates various cells in HCC microenvironment to promote tumor initiation, progression and invasion. In this study, we investigated how surrounding stromal cells interact with HBx-infected hepatoma cells by a series of in vitro co-culture studies.Methods: Huh7 hepatoma cells were cultured and transfected with the mammalian expression vector pGFP-HBx. Co-culture assays were performed between HBx-transfected Huh7 cells and conditioned media (CM) from stromal cells [endothelial cell lines (HUVECs) and hepatic stellate cell lines (LX2 cells)]. The effect of these interactions was studied by a series of functional assays like chemotaxis, invasion, and wound healing scratch assays. Also, quantitative real time (RT)-PCRs of the mesenchymal genes was performed in the hepatoma cells with and without the co-cultures. Hep3B cells with an integrated HBV genome were taken as positive controls.Results: HBx-transfected Huh7 cells cultured in presence of CM from HUVECs illustrated enhanced migration and tube formation as compared to HBx-transfected cells cultured alone or co-cultured with LX2 cells. HBx-transfected hepatoma cells incubated with CM from HUVECs also expressed mesenchymal genes including Thy1, CDH2, TGFβR1, VIM, and CD133. ELISAs revealed increased levels of TGF-β in CM from HUVECs. In comparison to unstimulated HBx-transfected Huh7 cells, TGF-β stimulated cells displayed increased invasive properties and mesenchymal gene expression. RT-PCR and flow cytometry analysis further demonstrated that incubation with either CM from HUVECs or TGF-β significantly increased the expression of a stemness marker, CD133 in HBx-infected hepatoma cells. Gene inhibition experiments with CD133 siRNA showed a downregulation of mesenchymal gene expression and properties in TGF-β induced HBx-infected hepatoma cells as compared to that observed in control siRNA treated cells, indicating CD133 as one of the key molecules affecting epithelial to mesenchymal transition (EMT) in HBx-infected cells.Conclusion: The study indicates that secretory factors like TGF-β from neighboring endothelial cells may enhance expression of CD133 and impart an aggressive EMT phenotype to HBx-infected hepatoma cells in HBV induced HCC

Vacuolar Targeting of Cry1Ac and its Effects on Expression and Stability in Tobacco.

Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes.Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes

CPP-ZFN: A potential DNA-targeting anti-malarial drug

<p>Abstract</p> <p>Background</p> <p>Multidrug-resistant <it>Plasmodium </it>is of major concern today. Effective vaccines or successful applications of RNAi-based strategies for the treatment of malaria are currently unavailable. An unexplored area in the field of malaria research is the development of DNA-targeting drugs that can specifically interact with parasitic DNA and introduce deleterious changes, leading to loss of vital genome function and parasite death.</p> <p>Presentation of the hypothesis</p> <p>Advances in the development of zinc finger nuclease (ZFN) with engineered DNA recognition domains allow us to design and develop nuclease of high target sequence specificity with a mega recognition site that typically occurs only once in the genome. Moreover, cell-penetrating peptides (CPP) can cross the cell plasma membrane and deliver conjugated protein, nucleic acid, or any other cargo to the cytoplasm, nucleus, or mitochondria. This article proposes that a drug from the combination of the CPP and ZFN systems can effectively enter the intracellular parasite, introduce deleterious changes in its genome, and eliminate the parasite from the infected cells.</p> <p>Testing the hypothesis</p> <p>Availability of a DNA-binding motif for more than 45 triplets and its modular nature, with freedom to change number of fingers in a ZFN, makes development of customized ZFN against diverse target DNA sequence of any gene feasible. Since the <it>Plasmodium </it>genome is highly AT rich, there is considerable sequence site diversity even for the structurally and functionally conserved enzymes between <it>Plasmodium </it>and humans. CPP can be used to deliver ZFN to the intracellular nucleus of the parasite. Signal-peptide-based heterologous protein translocation to <it>Plasmodium</it>-infected RBCs (iRBCs) and different <it>Plasmodium </it>organelles have been achieved. With successful fusion of CPP with mitochondrial- and nuclear-targeting peptides, fusion of CPP with 1 more <it>Plasmodium </it>cell membrane translocation peptide seems achievable.</p> <p>Implications of the hypothesis</p> <p>Targeting of the <it>Plasmodium </it>genome using ZFN has great potential for the development of anti-malarial drugs. It allows the development of a single drug against all malarial infections, including multidrug-resistant strains. Availability of multiple ZFN target sites in a single gene will provide alternative drug target sites to combat the development of resistance in the future.</p

KasI vs. SspDI: Comparative analysis of enzyme activity for restriction digestion

945-948Type II restriction enzymes are routinely used by molecular biologists in designing and implementation of cloning experiments without referring to the literature on enzymes in use, and at times, face some unforeseeable problems. In our laboratory too, we encountered one such problem while working with KasI restriction enzyme which recognizes GGCGCC sequence, and we further analyzed the issue. Our observations corroborate the fact that KasI acts as monomer and cleaves double stranded DNA through nicking mechanism. It introduces breaks in two strands of DNA after substantial time gap which can be owed to two independent nickase activities in the opposite strands. Moreover, this time gap between two nickase activities results in formation of different topological forms of DNA. Since molecular biologists working with common restriction enzymes are not familiar with such nickase activity, they may misinterpret their restriction digestion results. However, no such problem was observed with the use of SspDI restriction enzyme which also recognizes the same sequence (GGCGCC) and produces the identical overhangs as by KasI. Hence, SspDI suits better for routine cloning and genetic modification purposes over KasI while using GGCGCC as cloning site

A fusion gene encoding two different insecticidal proteins of Bacillus thuringiensis

204-209A chimeric fusion gene was constructed with the coding regions of insecticidal crystal protein (Cry1Ac) and vegetative insecticidal protein (Vip3Aa14) of Bacillus thuringiensis (Bt). Overexpression of the fusion gene in Escherichia coli resulted in the synthesis of a protein of ~140 kDa size, as revealed by SDS-PAGE analysis. Western hybridization analysis using polyclonal antisera raised against Cry1Ac showed the presence of a band corresponding to ~140 kDa. Stability of the fusion protein was studied by trypsin digestion. Insect bioassays of the fusion protein on three insect species viz., Helicoverpa armigera, Spodoptera litura and Plutella xylostella showed that the fusion protein retained the toxicity of Cry1Ac, but partially lost that of Vip3Aa14

Isolation of pigeon pea (<i style="">Cajanus cajan </i>L.) legumin gene promoter and identification of conserved regulatory elements using tools of bioinformatics

495-503A seed specific legumin gene promoter from pigeon pea was isolated by PCR amplification. Database assisted sequence analysis of this promoter revealed several putative cis-acting regulatory elements. Comparative analysis of 15 seed-specific legumin gene promoters from six species, viz. Cajanus cajan, Cicer arietinum, Pisum sativum, Glycine max, Vicia faba and Arachis hypogaea, revealed several conserved motifs in promoter sequences; maximum conservation was observed upstream to transcription start site. Most of the conserved motifs have known transcription factor binding sites. One unknown conserved motif of seven base pair (AG/TGTGTA) was found 19 bp upstream to legumin box, putatively named as L-19. Study of nucleosome formation potential showed that putative linker DNA is more prone to mutations as compared to DNA involved in nucleosome formation. A chimeric construct was made with pigeonpea legumin promoter and β-glucuronidase (GUS) gene. Analysis of GUS expression at different developmental stages of transgenic tobacco plant’s parts revealed that the reporter gene was expressed at a high level only in mature seeds, specifically in embryo, endosperm and in cotyledonary leaves of developing seedling. These data showed that GUS gene transcription was regulated in a tissue specific and temporally regulated manner