Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications

Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNAI were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNAI was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent Tm of ∼60°C. In contrast, the Apical Stem unfolds independently and with much higher apparent Tm of ∼83°C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNAI Central Domain structure essential for PKR inhibitory activity was identified

Analysis of Common and Specific Mechanisms of Liver Function Affected by Nitrotoluene Compounds

BACKGROUND: Nitrotoluenes are widely used chemical manufacturing and munitions applications. This group of chemicals has been shown to cause a range of effects from anemia and hypercholesterolemia to testicular atrophy. We have examined the molecular and functional effects of five different, but structurally related, nitrotoluenes on using an integrative systems biology approach to gain insight into common and disparate mechanisms underlying effects caused by these chemicals. METHODOLOGY/PRINCIPAL FINDINGS: Sprague-Dawley female rats were exposed via gavage to one of five concentrations of one of five nitrotoluenes [2,4,6-trinitrotoluene (TNT), 2-amino-4,6-dinitrotoluene (2ADNT) 4-amino-2,6-dinitrotoulene (4ADNT), 2,4-dinitrotoluene (2,4DNT) and 2,6-dinitrotoluene (2,6DNT)] with necropsy and tissue collection at 24 or 48 h. Gene expression profile results correlated well with clinical data and liver histopathology that lead to the concept that hematotoxicity was followed by hepatotoxicity. Overall, 2,4DNT, 2,6DNT and TNT had stronger effects than 2ADNT and 4ADNT. Common functional terms, gene expression patterns, pathways and networks were regulated across all nitrotoluenes. These pathways included NRF2-mediated oxidative stress response, aryl hydrocarbon receptor signaling, LPS/IL-1 mediated inhibition of RXR function, xenobiotic metabolism signaling and metabolism of xenobiotics by cytochrome P450. One biological process common to all compounds, lipid metabolism, was found to be impacted both at the transcriptional and lipid production level. CONCLUSIONS/SIGNIFICANCE: A systems biology strategy was used to identify biochemical pathways affected by five nitroaromatic compounds and to integrate data that tie biochemical alterations to pathological changes. An integrative graphical network model was constructed by combining genomic, gene pathway, lipidomic, and physiological endpoint results to better understand mechanisms of liver toxicity and physiological endpoints affected by these compounds

Aminomercuration of carbonyl-containing olefins

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