3′-C-Hydroxymethylthymidine: Synthesis and Incorporation into Oligodeoxynucleotide Analogues

Abstract

The stereoselective synthesis of 3′-C-hydroxymethylthymidine (5) in five steps from thymidine has been accomplished and this nucleoside has been incorporated into oligodeoxynucleotides (ODNs) in different ways.     

8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction

Small molecule nonpeptidic mimics of α-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as α-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an α-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%.     

Evaluation of Oligonucleotides with Novel Modifications

Abstract

Oligodeoxynucleotides modified with carboxamide linked dimeric nuclcotides and an acyclic nucleoside were prepared and investigated for their hybridization properties toward DNA.     

Phosphorylation Variation during the Cell Cycle Scales with Structural Propensities of Proteins

Phosphorylation at specific residues can activate a protein, lead to its localization to particular compartments, be a trigger for protein degradation and fulfill many other biological functions. Protein phosphorylation is increasingly being studied at a large scale and in a quantitative manner that includes a temporal dimension. By contrast, structural properties of identified phosphorylation sites have so far been investigated in a static, non-quantitative way. Here we combine for the first time dynamic properties of the phosphoproteome with protein structural features. At six time points of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels, whereas regions with predominantly regular secondary structures retain more constant phosphorylation levels. The two groups show preferences for different amino acids in their kinase recognition motifs - proline and other disorder-associated residues are enriched in the former group and charged residues in the latter. Furthermore, these preferences scale with the degree of disorderedness, from regular to irregular and to disordered structures. Our results suggest that the structural organization of the region in which a phosphorylation site resides may serve as an additional control mechanism. They also imply that phosphorylation sites are associated with different time scales that serve different functional needs.     

Reconstructing an annual cycle of interaction: natural infection and antibody dynamics to avian influenza along a migratory flyway

Migratory animals may play an important role in connecting disparate ecosystems, including the introduction of various pathogens. The incidence of these pathogens may vary over time and space, such that events along the entire migratory flyway are likely to be important in the interaction between pathogens and their migratory hosts. On this premise, the annual cycle of a naturally occurring host–pathogen system was reconstructed by examining infection with and antibodies to avian influenza virus along the flyway of a long‐distance Arctic migrant, the Svalbard‐breeding pink‐footed goose Anser brachyrhynchus. A highly‐localized transmission period was identified in winter, in contrast to the north–south decline expected from dabbling ducks, indicating the dynamics of infection may differ among host species. In spring, 63% (95% CI: 57.1, 68.9) of adults had detectable antibodies to the nucleoprotein of avian influenza virus, compared to just 15% (95% CI: 8.7, 23.4) of juveniles, suggesting inter‐annual antibody maintenance. Nevertheless, adult seroprevalence declined by approximately 30% from spring to late summer, indicating significant seroreversion in the population. Integrating these findings in an epidemiological model, detectable antibodies to nucleoprotein were estimated to persist for just 343 days (95% CI: 221, 607); considerably shorter than for other wildlife diseases in long‐lived bird species. The investigation of wildlife diseases in migratory populations is an inherently complex task, yet, by integrating disease incidence and seroprevalence along a migratory flyway, our findings suggest that the ecological interactions and life history of the host, as well as the life‐history of the pathogen, can influence the dynamics of infection and host immune response.     

Acclimation responses to short‐term temperature treatments during early life stages causes long lasting changes in spontaneous activity of adult Drosophila melanogaster

Ecotherms adjust their physiology to environmental temperatures. Long‐term exposures to heat or cold typically induce acclimation responses that generate directional, but reversible shifts in thermal tolerance and performance. However, less is known about how short exposure in different life stages will affect the adult phenotype. In the present study, we compared the effects of long‐term temperature exposure to 15, 19 and 31 °C with that of brief (16 h) exposure periods at the same temperatures in Drosophila melanogaster eggs, larvae, pupae, or adults, respectively. The acclimation responses are evaluated using activity measurements at 11, 15, 19, 27, 31 and 33 °C and by measuring upper and lower thermal limits (CT_max and CT_min) in 5‐day‐old adult males. As expected, long‐term cold exposure reduces relative CT_min, whereas long‐term heat exposure increases relative CT_max. By contrast, we find little effect on thermal limits when using short‐term exposures at different life stages. Long‐term exposures to 31 and 15 °C both suppressed activity relative to the 19 °C control, suggesting that development at high and low temperatures may lead to reduced activity later in life. Short‐term cold exposure early in development reduces activity in the adult stage, whereas the effects of short‐term heat exposure on behaviour are dependent on life stage and test temperature. Together, our results highlight how the thermal sensitivity of the trait measured determines the ability to detect acclimation responses.     

Retention of the Native Epigenome in Purified Mammalian Chromatin

A protocol is presented for the isolation of native mammalian chromatin as fibers of 25–250 nucleosomes under conditions that preserve the natural epigenetic signature. The material is composed almost exclusively of histones and DNA and conforms to the structure expected by electron microscopy. All sequences probed for were retained, indicating that the material is representative of the majority of the genome. DNA methylation marks and histone marks resembled the patterns observed in vivo. Importantly, nucleosome positions also remained largely unchanged, except on CpG islands, where nucleosomes were found to be unstable. The technical challenges of reconstituting biochemical reactions with native mammalian chromatin are discussed.