G-protein-coupled receptor dynamics: dimerization and activation models compared with experiment

Our previously derived models of the active state of the β2-adrenergic receptor are compared with recently published X-ray crystallographic structures of activated GPCRs (G-protein-coupled receptors). These molecular dynamics-based models using experimental data derived from biophysical experiments on activation were used to restrain the receptor to an active state that gave high enrichment for agonists in virtual screening. The β2-adrenergic receptor active model and X-ray structures are in good agreement over both the transmembrane region and the orthosteric binding site, although in some regions the active model is more similar to the active rhodopsin X-ray structures. The general features of the microswitches were well reproduced, but with minor differences, partly because of the unexpected X-ray results for the rotamer toggle switch. In addition, most of the interacting residues between the receptor and the G-protein were identified. This analysis of the modelling has also given important additional insight into GPCR dimerization: re-analysis of results on photoaffinity analogues of rhodopsin provided additional evidence that TM4 (transmembrane helix 4) resides at the dimer interface and that ligands such as bivalent ligands may pass between the mobile helices. A comparison, and discussion, is also carried out between the use of implicit and explicit solvent for active-state modelling.     

De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane

Despite the understanding of the importance of mitogen-activated protein (MAP) kinase activation in the stimulation of growth, little is known about the role of MAP kinase regulation during contact inhibited growth control. To investigate the role of the MAP kinase extracellular signal-regulated kinase (ERK) during the transition to a contact inhibited state, cultures of normal fibroblasts (BJ) were grown to different stages of confluency. The levels of MAP kinase phosphatase (MKP) expression and the amount of active ERK and MAP ERK kinase (MEK) in these cultures were assessed through western blot analysis and were compared to fibrosarcoma cell cultures (HT-1080), which lack contact inhibition. In normal fibroblasts, the amounts of active MEK and ERK decline at contact inhibition, concurrently with a rise in MKP-1, MKP-2, and MKP-3 protein levels. In contrast, fibrosarcoma cells appear to lack density-dependent regulation of the ERK pathway. Additionally, altering the redox environment of fibrosarcoma cells to a less reducing state, as seen during contact inhibition, results in increased MKP-1 expression. Taken together, these results suggest that the altered redox environment upon contact inhibition may contribute to the regulation of ERK inactivation by MKPs.     

Risk factors and outcomes among children admitted to hospital with pandemic H1N1 influenza

Background

Limited data are available on disease characteristics and outcomes of children with 2009 pandemic influenza A(H1N1) virus infection (pandemic H1N1 influenza) who have required hospital admission.

Methods

We reviewed the charts of 58 children with pandemic H1N1 influenza admitted to a large pediatric hospital in Ontario, Canada, between May 8 and July 22, 2009. We compared risk factors, severity indicators and outcomes of these children with those of 200 children admitted with seasonal influenza A during the previous 5 years (2004/05 to 2008/09).

Results

Children with pandemic H1N1 influenza were significantly older than those with seasonal influenza (median age 6.4 years v. 3.3 years). Forty-six (79%) of the children with pandemic H1N1 influenza had underlying medical conditions; of the other 12 who were previously healthy, 42% were under 2 years of age. Children admitted with pandemic H1N1 influenza were significantly more likely to have asthma than those with seasonal influenza (22% v. 6%). Two children had poorly controlled asthma, and 6 used inhaled medications only intermittently. The median length of stay in hospital was 4 days in both groups of children. Similar proportions of children required admission to the intensive care unit (21% of those with pandemic H1N1 influenza and 14% of those with seasonal influenza) and mechanical ventilation (12% and 10% respectively). None of the children admitted with pandemic H1N1 influenza died, as compared with 1 (0.4%) of those admitted with seasonal influenza.

Interpretation

Pandemic H1N1 influenza did not appear to cause more severe disease than seasonal influenza A. Asthma appears to be a significant risk factor for severe disease, with no clear relation to severity of asthma. This finding should influence strategies for vaccination and pre-emptive antiviral therapy.Influenza causes significant morbidity and mortality in childhood.¹ Infants, young children and people 65 years of age and older account for the highest rates of influenza-related hospital admission.² Earlier case series of 2009 pandemic influenza A(H1N1) virus infection (pandemic H1N1 influenza) reported small numbers of children^3,4 or did not present data on children separately.⁵ A recently published series that included 122 children confirmed typical influenza-like presentation, reported a high prevalence of underlying medical conditions (60%, including asthma in 29%) and described the need for intensive care in 20% and mechanical ventilation in 10%.⁶ A previous comparison of children with pandemic H1N1 influenza and those in previous years with seasonal influenza included only children considered to have died of influenza.⁷In this article, we present our experience with children admitted to hospital with pandemic H1N1 influenza. Our primary goal was to describe the demographic characteristics, clinical features and markers of severity of illness of these children. Our secondary goal was to identify risk factors for severe disease or poor outcome by comparing these children with those who had been admitted in previous years with seasonal influenza.     

Vaccinia Virus–Encoded Ribonucleotide Reductase Subunits Are Differentially Required for Replication and Pathogenesis

Ribonucleotide reductases (RRs) are evolutionarily-conserved enzymes that catalyze the rate-limiting step during dNTP synthesis in mammals. RR consists of both large (R1) and small (R2) subunits, which are both required for catalysis by the R1₂R2₂ heterotetrameric complex. Poxviruses also encode RR proteins, but while the Orthopoxviruses infecting humans [e.g. vaccinia (VACV), variola, cowpox, and monkeypox viruses] encode both R1 and R2 subunits, the vast majority of Chordopoxviruses encode only R2 subunits. Using plaque morphology, growth curve, and mouse model studies, we investigated the requirement of VACV R1 (I4) and R2 (F4) subunits for replication and pathogenesis using a panel of mutant viruses in which one or more viral RR genes had been inactivated. Surprisingly, VACV F4, but not I4, was required for efficient replication in culture and virulence in mice. The growth defects of VACV strains lacking F4 could be complemented by genes encoding other Chordopoxvirus R2 subunits, suggesting conservation of function between poxvirus R2 proteins. Expression of F4 proteins encoding a point mutation predicted to inactivate RR activity but still allow for interaction with R1 subunits, caused a dominant negative phenotype in growth experiments in the presence or absence of I4. Co-immunoprecipitation studies showed that F4 (as well as other Chordopoxvirus R2 subunits) form hybrid complexes with cellular R1 subunits. Mutant F4 proteins that are unable to interact with host R1 subunits failed to rescue the replication defect of strains lacking F4, suggesting that F4-host R1 complex formation is critical for VACV replication. Our results suggest that poxvirus R2 subunits form functional complexes with host R1 subunits to provide sufficient dNTPs for viral replication. Our results also suggest that R2-deficient poxviruses may be selective oncolytic agents and our bioinformatic analyses provide insights into how poxvirus nucleotide metabolism proteins may have influenced the base composition of these pathogens.     

Java GUI for InterProScan (JIPS): A tool to help process multiple InterProScans and perform ortholog analysis

Background  

Recent, rapid growth in the quantity of available genomic data has generated many protein sequences that are not yet biochemically classified. Thus, the prediction of biochemical function based on structural motifs is an important task in post-genomic analysis. The InterPro databases are a major resource for protein function information. For optimal results, these databases should be searched at regular intervals, since they are frequently updated.     

Solution, solid phase and computational structures of apicidin and its backbone-reduced analogs.

The recently isolated broad-spectrum antiparasitic apicidin (1) is one of the few naturally occurring cyclic tetrapeptides (CTP). Depending on the solvent, the backbone of 1 exhibits two gamma-turns (in CH(2)Cl(2)) or a beta-turn (in DMSO), differing solely in the rotation of the plane of one of the amide bonds. In the X-ray crystal structure, the peptidic C==Os and NHs are on opposite sides of the backbone plane, giving rise to infinite stacks of cyclotetrapeptides connected by three intermolecular hydrogen bonds between the backbones. Conformational searches (Amber force field) on a truncated model system of 1 confirm all three backbone conformations to be low-energy states. The previously synthesized analogs of 1 containing a reduced amide bond exhibit the same backbone conformation as 1 in DMSO, which is confirmed further by the X-ray crystal structure of a model system of the desoxy analogs of 1. This similarity helps in explaining why the desoxy analogs retain some of the antiprotozoal activities of apicidin. The backbone-reduction approach designed to facilitate the cyclization step of the acyclic precursors of the CTPs seems to retain the conformational preferences of the parent peptide backbone.     

Evolution of biological dispersal corridors through a tectonically active mountain range in New Zealand

Aim To assess the geological evolution and biogeographical implications of low mountain passes. In particular, we question the common biogeographical belief that major mountain belts form impervious physical barriers to biological dispersal, and that related taxa found on opposites sides of mountains are necessarily a result of vicariant tectonic processes. Location The Southern Alps of New Zealand form a long (500 km) narrow mountain belt at the oblique collisional Pacific–Australian tectonic plate boundary. High mountains were uplifted during the Pliocene (2–5 Ma) and uplift has continued to the present day. Methods We integrate previous work from several disciplines to obtain an overview of inter‐relationships between plate tectonic processes, geomorphology and biogeography along the main mountain barrier in New Zealand, and then extend this approach to other major mountain belts. Results The Southern Alps initially formed a barrier to at least some biological dispersal, including vicariant formation of separate species of freshwater non‐migratory galaxiid fish on either side. However, the high mountain barrier was breached in several places when passive transport of topography occurred, from the low‐erosion rain shadow on the eastern side towards the high‐erosion, high‐rainfall western side. This tectonic transport resulted in the capture of eastern rivers by west‐draining rivers, leaving low passes at the topographic divide. These low‐elevation corridors permitted biological dispersal across the mountains, although continued uplift raises these passes. A new set of passes has formed in the northern part of the mountains where younger faults are cutting across the older mountain topography. These potential dispersal corridors are becoming lower with continued erosion, and more common as the defining structures migrate southwards. Main conclusions Biological dispersal across the Southern Alps may be facilitated by numerous mountain passes, especially via the new passes formed by cross‐cutting faults. More low‐lying corridors existed than is readily apparent now, as old river capture‐related passes have been blocked by ongoing uplift. The dynamic mountain‐building and erosional environment typified by the Southern Alps occurs in all the world’s collisional mountain belts, such as the Andes, Himalayas, European Alps and North American Cordillera. Sister taxa occurring across mountain belts are not necessarily a result of vicariance driven by the rise of the mountains, as numerous passes may have permitted intermittent dispersal. The evolution of low passes may have been more prevalent than is currently appreciated, suggesting that topographically complex mountain ranges might be more effectively viewed as dynamic filters within a probability landscape rather than as static and impervious high‐altitude barriers to all but the rarest of biological dispersal events. In some cases, the biological disjunctions observed across mountains may more directly reflect habitat differentiation driven by orographic mountain development that has limited the probability of trans‐alpine dispersal success.