The interferon-alpha (IFN-alpha)-inducible protein IFI44 is associated with hepatitis C virus (HCV) infection, and its function is unknown. We show here in two human melanoma cell lines (ME15 and D10) that transcription starts 4 h after induction, and peak protein levels are reached 24 h after stimulation. We show by immunofluorescence, viral overexpression, and cellular fractionation that IFI44 is a cytoplasmic protein. Overexpression of IFI44 cDNA induces an antiproliferative state in vitro, even in cells that are not responsive to IFN-alpha. IFI44 contains a perfect GTP binding site but has no homology to known GTPases or G proteins. Based on these results, we propose a model in which IFI44 binds intracellular GTP, and this depletion abolishes extracellular signal-regulated kinase (ERK) signaling and results finally in cell cycle arrest.
Tetracycline-regulated gene expression in eukaryotic cell lines, plants, and transgenic mice has become a powerful tool for the analysis of eukaryotic gene expression and function. The system consists of two plasmids, one encoding the transactivator protein under control of a viral cytomegalovirus promoter, and the second being the tet-operator minimal promoter driving the gene of interest. Here we show that these control elements, when integrated in cis on a single plasmid, allow efficient and tight control of reporter gene expression in vitro and in vivo. Dependent on the route of administration of tetracycline, gene expression can be partially or fully repressed in transgenic mice, whereas removal of the antibiotic induces the reporter gene in various tissues to levels up to 800-fold more than the two-plasmid system. In addition, crossing and analysis of animals transgenic for the individual components of the system are unnecessary, and genetic segregation of the control elements during breeding is prevented.
The successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity. We further demonstrate that the hybridization-dependent toxic potential of LNA-ASOs is also evident in different cell types from different species, which indicates probable translatability of the in vitro results to humans. Additionally, we show that the melting temperature (Tm) of LNA-ASOs maintained below a threshold level of about 55°C greatly diminished the hepatotoxic potential. In summary, we have established a sensitive in vitro screening approach for assessing the hybridization-dependent toxic potential of LNA-ASOs, enabling prioritization of candidate molecules in drug discovery and early development.
The current spread of multidrug-resistant malaria demands rapid vaccine development against the major pathogen Plasmodium falkiparum. The high quantities of protein required for a worldwide vaccination campaign select recombinant DNA technology as a practical approach for large-scale antigen production. We describe the vaccination of Aotus monkeys with two recombinant blood-stage antigens (recombinant p41 and 190N) that were considered as vaccine candidates because parasite-derived antigen preparations could protect susceptible monkeys from an otherwise lethal malaria infection. In contrast to the natural antigen, recombinant p41 protein (P. falciparum aldolase) could not protect monkeys, although all animals seroconverted. 190N antigen, a recombinant protein containing conserved sequences of the major merozoite surface antigen p190, protected two of five monkeys from critical levels ofinfection with the highly virulent FVO isolate of P. falckarum. However, the B-and T-cell responses to 190N antigen were similar in protected and unprotected animals so that other unknown factors may contribute to protection. Higher purity or lack of protective epitopes or different structure of protective epitopes in the recombinant proteins might explain the better performance of parasite-derived antigens in vaccination trials. The partial protection obtained with 190N antigen suggests that this molecule could contribute to a vaccine mixture against P. falciparum.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.