Browsing by Subject "ER stress"
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Item Identification and characterization of cell-adapted mutations in West Nile Virus and replicons(2007-12-19) Shannan Lee Rossi; Peter W. Mason; Scott Weaver; Robert Tesh; Ilya Frolov; Charles RiceFlavivirus persistence in cell culture is achieved by altering the interaction between the viral genome and the host cell. To identify some of the factors that contribute to a persistent West Nile virus (WNV) infection, WNV subgenomic replicon (WNR) genomes were modified to contain neomycin phosphotransferase (antibiotic-resistance) gene and cells that persistently harbored this WNR were selected for using G418. There were many changes to the genomes harvested from WNR-bearing cell cultures compared to the parental genome, perhaps the most striking was the prevalence of NS2A mutations. WNR and WNV genomes harboring these mutations replicated more poorly than wt WNR or WNV genomes in vitro. These NS2A mutant genomes, as well as a genome with a large deletion in the 3’ UTR, were highly attenuated Swiss-Webster outbred mice. Low levels of IFN were produced from cells infected with NS2A or 3’ UTR deletion WNR genomes compared to wt WNR genomes. Since the WNR and WNV cannot efficiently replicate, little stress is placed on the cell during replication, resulting in minimal engagement of the cell’s stress and apoptotic responses and a noncytopathic infection. As a result, little cytopathic effect and apoptotic death were observed in cells infected with NS2A or 3’ UTR deletion WNR mutants compared to cells infected with wt WNR genomes. This lack of death may have been attributed to low levels of CHOP, a pro-apoptotic protein that is induced during endoplasmic reticulum stress. Taken together, these data support the hypothesis that WNR and WNV genomes that replicate poorly do not efficiently produce signs of their replication that can be recognized by the cell and place little stress upon the cell, resulting in an attenuated noncytopathic, persistent infection.Item Investigating the molecular biology of aging and extended longevity: Proteomic and genomic analysis of mouse liver(2005-11-05) William H. Boylston; John Papaconstantinou, Ph.D.; Steven G. Widen, Ph.D.; Philip T. Palade, Ph.D.; Pamela L. Larsen, Ph.D.; Jeffrey P. Rabek, Ph.D.; Giulio Taglialatela, Ph.D.Recent advances in molecular gerontology have provided important clues about the fundamental biology of the aging process including the role of oxidative stress and the genetic basis of longevity. Progressive accumulation of oxidative damage to macromolecules is thought to underlie the aging-associated decline in physiologic function characteristic of the senescent phenotype. Mitochondria are a major intracellular source of reactive oxygen species (ROS); however, other organelles are also endogenous sources of oxyradicals and oxidants that can damage macromolecules. This investigation examines the relationship between aging and oxidative damage to ER resident proteins, which exist in a strongly oxidizing environment necessary for disulfide bond formation. In these studies, young and aged mouse liver homogenates were separated into enriched sub-cellular fractions, and the ER/mitochondrial fraction was resolved by 2-dimensional gel electrophoresis and then assayed for oxidative damage as indicated by protein carbonylation. MALDI/TOF analysis and N-terminal sequencing of these proteins identified BiP/Grp78, protein disulfide isomerase (PDI), and calreticulin as exhibiting a specific age-associated increase in carbonyl content. This increase in oxidative damage to critical ER proteins in aged liver strongly indicates an impairment in protein folding, disulfide cross-linking, and glycosylation which may significantly contribute to the functional decline observed in aging liver.\r\nProviding evidence for the genetic basis of aging, several murine models demonstrate that longevity can be increased by mutations affecting endocrine signaling, particularly via the GH/IGF-1 axis. In this investigation of long-lived GH/IGF-1-deficient mice, characteristic patterns of hepatic gene expression in Pit1dw/dwJ dwarf mice were revealed. Comparative microarray analysis of young and aged male livers was utilized to identify specific genes differentially expressed in Pit1dw/dwJ mice. Further examination of both male and female livers by real-time RT-PCR demonstrated striking transcriptional differences in Pit1dw/dwJ mice comprised of genes regulating cholesterol biosynthesis, fatty acid utilization, and lipoprotein metabolism. Affecting global energy homeostasis, this programmatic shift in hepatic expression may contribute to longevity by influencing bioenergetic and oxidative reactions occurring within mitochondria, ER, and peroxisomes. Intriguingly, these long-term patterns in metabolic gene expression in Pit1dw/dwJ livers mirror many transcriptional changes induced by caloric restriction and fasting, further implicating energy metabolism in longevity.\r\n