Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.     

Inactivation and oxidative modification of Cu,Zn superoxide dismutase by stimulated neutrophils: the appearance of new catalytically active structures.

Bovine superoxide dismutase (SOD) was inactivated during incubation with phorbol myristate acetate-stimulated neutrophils. In addition, stimulated neutrophils were able to disrupt the SOD structure. Inactivation and structural damage were dependent on the action of hypochlorous acid, an oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils. Incubation of SOD with stimulated neutrophils lead to long-wavelength fluorescence (ex, 350 nm; em, 450 nm) and the appearance of new structural forms with other isoelectric points. These additional forms possess catalytic activity. Generation of catalytically active new forms of SOD demonstrates the inaccessibility of the active centre of SOD to hypochlorite and may be a reason for the successful application of SOD during anti-inflammatory therapy.     

Lipid microdomains and membrane trafficking in mammalian cells

This overview summarizes the data for how epithelial cells sort and deliver proteins and lipids to the apical and basolateral cell surface domains. The basolateral pathway uses a Rab-SNARE mechanism for docking and fusion, while the apical route employs a different machinery. This latter mechanism is based on lipid microdomains, composed of clusters of sphingolipids and cholesterol, which function as rafts for apical delivery. The sphingolipid-cholesterol raft mechanism seems to be employed generally by mammalian cells to transport raft-associated proteins to their post-Golgi destinations.     

Disease resistance, stress response and effects of triploidy in growth hormone transgenic coho salmon

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non‐transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non‐transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and non‐transgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth‐accelerated coho salmon.     

Species loss leads to community closure

Global extinction of a species is sadly irreversible. At a local scale, however, extinctions may be followed by re-invasion. We here show that this is not necessarily the case and that an ecological community may close its doors for re-invasion of species lost from it. Previous studies of how communities are assembled have shown that there may be rules for that process and that limitations are set to the order by which species are introduced and put together. Instead of focusing on the assembly process we randomly generated simple competitive model communities that were stable and allowed for two to 10 coexisting species. When a randomly selected single species was removed from the community, the cascading species loss was recorded and frequently the resulting community was more than halved. Cascading extinctions have previously been recorded, but we here show that the relative magnitude of the cascade is dependent on community size (and not only trophic structure) and that the reintroduction of the original species lost often is impossible. Hence, species loss does not simply leave a void potentially refilled, but permanently alters the entire community structure and consequently the adaptive landscape for potential re-invaders.     

Phytochrome A regulates red-light induction of phototropic enhancement in Arabidopsis.

Phytochrome A (phyA) and phytochrome B photoreceptors have distinct roles in the regulation of plant growth and development. Studies using specific photomorphogenic mutants and transgenic plants overexpressing phytochrome have supported an evolving picture in which phyA and phytochrome B are responsive to continuous far-red and red light, respectively. Photomorphogenic mutants of Arabidopsis thaliana that had been selected for their inability to respond to continuous irradiance conditions were tested for their ability to carry out red-light-induced enhancement of phototropism, which is an inductive phytochrome response. We conclude that phyA is the primary photoreceptor regulating this response and provide evidence suggesting that a common regulatory domain in the phyA polypeptide functions for both high-irradiance and inductive phytochrome responses.     

Socioeconomic, demographic and environmental determinants of infant mortality in Nepal

The Nepal Fertility and Family Planning Survey of 1986 demonstrated that demographic variables, previous birth interval and survival of preceding child, still predominated as determinants of infant mortality, particularly in rural areas of Nepal. However, in urban Nepal, where the level of socioeconomic development is higher, an environmental variable, along with previous birth interval and survival of preceding child emerges as important in determining infant mortality. Separate policy measures for child survival prospects in rural and urban Nepal are suggested.