Neoxanthin prevents H2O2-induced cytotoxicity in HepG2 cells by activating endogenous antioxidant signals and suppressing apoptosis signals

Molecular Biology Reports - The liver has a solid inbuilt antioxidant defense system to regulate oxidative stress. However, exposure to an excessive level of ROS causes liver injury. This study...     

From infection to cancer: how DNA tumour viruses alter host cell central carbon and lipid metabolism

Infections cause 13% of all cancers globally, and DNA tumour viruses account for almost 60% of these cancers. All viruses are obligate intracellular parasites and hijack host cell functions to replicate and complete their life cycles to produce progeny virions. While many aspects of viral manipulation of host cells have been studied, how DNA tumour viruses manipulate host cell metabolism and whether metabolic alterations in the virus life cycle contribute to carcinogenesis are not well understood. In this review, we compare the differences in central carbon and fatty acid metabolism in host cells following infection, oncogenic transformation, and virus-driven cancer of DNA tumour viruses including: Epstein–Barr virus, hepatitis B virus, human papillomavirus, Kaposi''s sarcoma-associated herpesvirus and Merkel cell polyomavirus.     

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.     

Kinetic analysis of the coordinated interaction of SgrAI restriction endonuclease with different DNA targets

SgrAI restriction endonuclease cooperatively interacts and cleaves two target sites that include both the canonical sites, CPuCCGGPyG, and the secondary sites, CPuCCGGPy(A/T/C). It has been observed that the cleaved canonical sites stimulate SgrAI cleavage at the secondary sites. Equilibrium binding studies show that SgrAI binds to its canonical sites with a high affinity (Ka = 4-8 x 10(10) M-1) and that it has a 15-fold lower affinity for the cleaved canonical sites and a 30-fold lower affinity for the secondary sites. Steady-state kinetics reveals substrate cooperativity for SgrAI cleavage on both canonical and secondary sites. The specificity of SgrAI for the secondary site CACCGGCT, as measured by kcat/K is about 500-fold lower than that for the canonical site CACCGGCG, but this difference is reduced to 10-fold in the presence of the cleaved canonical sites. The efficiency of canonical site cleavage also increases by 3-fold when the cleaved canonical sites are present in the reaction. Furthermore, the substrate cooperativity for SgrAI cleavage is abolished for both types of sites in the presence of cleaved canonical sites. These results indicate that target site cleavage occurs via a coordinated interaction of two SgrAI protein subunits, where the subunit bound to the cleaved site stimulates the cleavage of the uncut site bound by the other subunit. The free subunits of SgrAI have the flexibility to bind different target sites and, consequently, assemble into various catalytically active complexes, which differ in their catalytic efficiencies.     

Visualization and quantification of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> intraerythrocytic merozoites

Malaria, a leading parasitic killer, is caused by Plasmodium spp. The pathology of the disease starts when Plasmodium merozoites infect erythrocytes to form rings, that matures through a large trophozoite form and develop into schizonts containing multiple merozoites. The number of intra-erythrocytic merozoites is a key-determining factor for multiplication rate of the parasite. Counting of intraerythrocytic merozoites by classical 2-D microscopy method is error prone due to insufficient representation of merozoite in one optical plane of a schizont. Here, we report an alternative 3-D microscopy based automated method for counting of intraerythrocytic merozoites in entire volume of schizont. This method offers a considerable amount of advantages in terms of both, ease and accuracy.     

Predicting stable functional peptides from the intergenic space of E. coli

Expression of synthetic proteins from intergenic regions of E. coli and their functional association was recently demonstrated (Dhar et al. in J Biol Eng 3:2, 2009. doi:10.1186/1754-1611-3-2). This gave birth to the question: if one can make ‘user-defined’ genes from non-coding genome—how big is the artificially translatable genome? (Dinger et al. in PLoS Comput Biol 4, 2008; Frith et al. in RNA Biol 3(1):40–48, 2006a; Frith et al. in PLoS Genet 2(4):e52, 2006b). To answer this question, we performed a bioinformatics study of all reported E. coli intergenic sequences, in search of novel peptides and proteins, unexpressed by nature. Overall, 2500 E. coli intergenic sequences were computationally translated into ‘protein sequence equivalents’ and matched against all known proteins. Sequences that did not show any resemblance were used for building a comprehensive profile in terms of their structure, function, localization, interactions, stability so on. A total of 362 protein sequences showed evidence of stable tertiary conformations encoded by the intergenic sequences of E. coli genome. Experimental studies are underway to confirm some of the key predictions. This study points to a vast untapped repository of functional molecules lying undiscovered in the non-expressed genome of various organisms.     

High Frequency of Transmitted HIV-1 Gag HLA Class I-Driven Immune Escape Variants but Minimal Immune Selection over the First Year of Clade C Infection

In chronic HIV infection, CD8+ T cell responses to Gag are associated with lower viral loads, but longitudinal studies of HLA-restricted CD8+ T cell-driven selection pressure in Gag from the time of acute infection are limited. In this study we examined Gag sequence evolution over the first year of infection in 22 patients identified prior to seroconversion. A total of 310 and 337 full-length Gag sequences from the earliest available samples (median = 14 days after infection [Fiebig stage I/II]) and at one-year post infection respectively were generated. Six of 22 (27%) individuals were infected with multiple variants. There was a trend towards early intra-patient viral sequence diversity correlating with viral load set point (p = 0.07, r = 0.39). At 14 days post infection, 59.7% of Gag CTL epitopes contained non-consensus polymorphisms and over half of these (35.3%) comprised of previously described CTL escape variants. Consensus and variant CTL epitope proportions were equally distributed irrespective of the selecting host HLA allele and most epitopes remained unchanged over 12 months post infection. These data suggest that intrapatient diversity during acute infection is an indicator of disease outcome. In this setting, there is a high rate of transmitted CTL escape variants and limited immune selection in Gag during the first year of infection. These data have relevance for vaccine strategies designed to elicit effective CD8+ T cell immune responses.     

Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.     

USER friendly DNA engineering and cloning method by uracil excision

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.