Quantitative analysis of the binding of simian virus 40 large T antigen to DNA

SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5'-GAGGC-3' sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5'-GAGGC-3' sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an approximately 10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.     

Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA

The interaction of simian virus 40 (SV40) T antigen (T-ag) with the viral origin has served as a model for studies of site-specific recognition of a eukaryotic replication origin and the mechanism of DNA unwinding. These studies have revealed that a motif termed the "beta-hairpin" is necessary for assembly of T-ag on the SV40 origin. Herein it is demonstrated that residues at the tip of the "beta-hairpin" are needed to melt the origin-flanking regions and that the T-ag helicase domain selectively assembles around one of the newly generated single strands in a manner that accounts for its 3'-to-5' helicase activity. Furthermore, T-ags mutated at the tip of the "beta-hairpin" are defective for oligomerization on duplex DNA; however, they can assemble on hybrid duplex DNA or single-stranded DNA (ssDNA) substrates provided the strand containing the 3' extension is present. Collectively, these experiments indicate that residues at the tip of the beta-hairpin generate ssDNA in the core origin and that the ssDNA is essential for subsequent oligomerization events.     

PDE-10A inhibitors as insulin secretagogues

Modulation of cAMP levels has been linked to insulin secretion in preclinical animal models and in humans. The high expression of PDE-10A in pancreatic islets suggested that inhibition of this enzyme may provide the necessary modulation to elicit increased insulin secretion. Using an HTS approach, we have identified quinoline-based PDE-10A inhibitors as insulin secretagogues in vitro. Optimized compounds were evaluated in vivo where improvements in glucose tolerance and increases in insulin secretion were measured.     

Seed bank dynamics govern persistence of Brassica hybrids in crop and natural habitats

Background and Aims Gene flow from crops to their wild relatives has the potential to alter population growth rates and demography of hybrid populations, especially when a new crop has been genetically modified (GM). This study introduces a comprehensive approach to assess this potential for altered population fitness, and uses a combination of demographic data in two habitat types and mathematical (matrix) models that include crop rotations and outcrossing between parental species.Methods Full life-cycle demographic rates, including seed bank survival, of non-GM Brassica rapa × B. napus F₁ hybrids and their parent species were estimated from experiments in both agricultural and semi-natural habitats. Altered fitness potential was modelled using periodic matrices including crop rotations and outcrossing between parent species.Key Results The demographic vital rates (i.e. for major stage transitions) of the hybrid population were intermediate between or lower than both parental species. The population growth rate (λ) of hybrids indicated decreases in both habitat types, and in a semi-natural habitat hybrids became extinct at two sites. Elasticity analyses indicated that seed bank survival was the greatest contributor to λ. In agricultural habitats, hybrid populations were projected to decline, but with persistence times up to 20 years. The seed bank survival rate was the main driver determining persistence. It was found that λ of the hybrids was largely determined by parental seed bank survival and subsequent replenishment of the hybrid population through outcrossing of B. rapa with B. napus.Conclusions Hybrid persistence was found to be highly dependent on the seed bank, suggesting that targeting hybrid seed survival could be an important management option in controlling hybrid persistence. For local risk mitigation, an increased focus on the wild parent is suggested. Management actions, such as control of B. rapa, could indirectly reduce hybrid populations by blocking hybrid replenishment.     

Evidence to support mitochondrial neuroprotection,in severe traumatic brain injury

Journal of Bioenergetics and Biomembranes - Traumatic brain injury (TBI) is still the leading cause of disability in young adults worldwide. The major mechanisms – diffuse axonal injury,...