Structural basis for α-conotoxin potency and selectivity

Parkinson’s disease is a debilitating movement disorder characterized by altered levels of α₆β₂1 (1 indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. α-Conotoxin MII (α-CTx MII) is a highly useful ligand to probe α₆β₂ nAChRs structure and function, but it does not discriminate among closely related α₆1 nAChR subtypes. Modification of the α-CTx MII primary sequence led to the identification of α-CTx MII[E11A], an analog with 500–5300-fold discrimination between α₆1 subtypes found in both human and non-human primates. α-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity α₆ nAChR, a subtype that is selectively lost in Parkinson’s disease. Here, we present the three-dimensional solution structure for α-CTx MII[E11A] as determined by two-dimensional ¹H NMR spectroscopy to 0.13 ± 0.09 ? backbone and 0.45 ± 0.08 ? heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of α-CTx MII[E11A] relative to other members of the α-CTx family may be responsible for its exceptionally high affinity for α6α4β21 nAChR as well as discrimination between α₆β₂ and α₃β₂ containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for α₆1 nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson’s disease.     

Life Expectancies of South African Adults Starting Antiretroviral Treatment: Collaborative Analysis of Cohort Studies

Background

Few estimates exist of the life expectancy of HIV-positive adults receiving antiretroviral treatment (ART) in low- and middle-income countries. We aimed to estimate the life expectancy of patients starting ART in South Africa and compare it with that of HIV-negative adults.

Methods and Findings

Data were collected from six South African ART cohorts. Analysis was restricted to 37,740 HIV-positive adults starting ART for the first time. Estimates of mortality were obtained by linking patient records to the national population register. Relative survival models were used to estimate the excess mortality attributable to HIV by age, for different baseline CD4 categories and different durations. Non-HIV mortality was estimated using a South African demographic model. The average life expectancy of men starting ART varied between 27.6 y (95% CI: 25.2–30.2) at age 20 y and 10.1 y (95% CI: 9.3–10.8) at age 60 y, while estimates for women at the same ages were substantially higher, at 36.8 y (95% CI: 34.0–39.7) and 14.4 y (95% CI: 13.3–15.3), respectively. The life expectancy of a 20-y-old woman was 43.1 y (95% CI: 40.1–46.0) if her baseline CD4 count was ≥200 cells/µl, compared to 29.5 y (95% CI: 26.2–33.0) if her baseline CD4 count was <50 cells/µl. Life expectancies of patients with baseline CD4 counts ≥200 cells/µl were between 70% and 86% of those in HIV-negative adults of the same age and sex, and life expectancies were increased by 15%–20% in patients who had survived 2 y after starting ART. However, the analysis was limited by a lack of mortality data at longer durations.

Conclusions

South African HIV-positive adults can have a near-normal life expectancy, provided that they start ART before their CD4 count drops below 200 cells/µl. These findings demonstrate that the near-normal life expectancies of HIV-positive individuals receiving ART in high-income countries can apply to low- and middle-income countries as well. Please see later in the article for the Editors'' Summary     

Predictive systems ecology

Human societies, and their well-being, depend to a significant extent on the state of the ecosystems that surround them. These ecosystems are changing rapidly usually in response to anthropogenic changes in the environment. To determine the likely impact of environmental change on ecosystems and the best ways to manage them, it would be desirable to be able to predict their future states. We present a proposal to develop the paradigm of predictive systems ecology, explicitly to understand and predict the properties and behaviour of ecological systems. We discuss the necessary and desirable features of predictive systems ecology models. There are places where predictive systems ecology is already being practised and we summarize a range of terrestrial and marine examples. Significant challenges remain but we suggest that ecology would benefit both as a scientific discipline and increase its impact in society if it were to embrace the need to become more predictive.     

The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 class ubiquitin ligase.

During development, the Notch receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. We have previously defined Su(dx) genetically as a negative regulator of Notch signaling. Here we present the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. We have identified a seven-codon deletion in a Su(dx) mutant allele and we show that expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling.     

Papaya latex enzymes capable of detoxification of gliadin

Assay of fractions obtained from ion exchange chromatography of papaya latex on CM Sephadex-C50, size exclusion chromatography on Sephacryl S-300 and size exclusion HPLC have provided an insight into the relative contributions of the gluten-detoxifying enzymes present. This outcome has been achieved by the use of the above chromatographic techniques, coupled with assays of lysosomal activity, protease activity using benzylarginine ethyl ester (BAEE) as substrate, prolyl endopeptidase (PEP) using glycylprolylnitroanilide and a prolidase assay using acetylprolylglycine. These procedures have shown that the activity in papaya latex is due largely to caricain and to a lesser extent, chymopapain and glutamine cyclotransferase. The presence of caricain and these other enzymes was confirmed by mass spectrometry of trypsin digests of the most active fraction obtained by CM Sephadex-C50 chromatography and size exclusion HPLC. Fractions rich in caricain would be suitable for enzyme therapy in gluten intolerance and appear to have synergistic action with porcine intestinal extracts.     

Facultative Control of Matrix Production Optimizes Competitive Fitness in Pseudomonas aeruginosa PA14 Biofilm Models

As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O₂. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O₂-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities.     

Interrelated lipid alterations and their influence on the proliferation and fusion of cultured myogenic cells

We have cultured myogenic cells derived from primary explants and a cell line (L6) in a lipid-depleted medium (LDM) and produced large alterations of the fatty acyl and polar headgroup composition and of the cellular sterol levels. These alterations were produced by altering the composition of the media as follows: removing biotin and providing exogenous fatty acid; removing choline and providing exogenous ethanolamine or choline analogues; and by adding 25-OH cholesterol, an inhibitor of 3-hydroxy-3-methylglutarate (HMG)-CoA reductase. Relatively small, secondary alterations of other lipid classes accompany the large primary alteration. In general, they are not obviously compensatory for the primary alteration by retaining some physical property. We have explored the influence of these lipid alterations on myoblast proliferation and fusion into myotubes. In general, considerable variability appears tolerated, but there also appear to be limits. Long-term cultures grown in media containing a single fatty acid do not proliferate indefinitely, and the fatty acid does not become the sole fatty acyl component of the phospholipids. This phenomenon is also observed for cultures enriched in phosphatidylethanolamine (PE) or phosphatidyldimethylethanolamine (PDME). The influence of the lipid alterations on fusion is particularly interesting. The inclusion of 25-OH cholesterol inhibits fusion. Enrichment of the fatty acyl chains with elaidate or the polar headgroups with PE also inhibits fusion, but in contrast to that by 25-OH cholesterol, a significant fraction of the myoblasts are aligned and interacting with each other. Oleate enrichment enhances the rate of fusion.     

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.     

Genetic divergence and migration patterns in a North American passerine bird: implications for evolution and conservation

Like many other migratory birds, the black-throated blue warbler (Dendroica caerulescens) shows pronounced differences in migratory behaviour and other traits between populations: birds in the southern part of the breeding range have darker plumage and migrate to the eastern Caribbean during the winter, whereas those in the north have lighter plumage and migrate to the western Caribbean. We examined the phylogeography of this species, using samples collected from northern and southern populations, to determine whether differentiation between these populations dates to the Pleistocene or earlier, or whether differences in plumage and migratory behaviour have arisen more recently. We analysed variation at 369 bp of the mitochondrial control region domain I and also at seven nuclear microsatellites. Analyses revealed considerable genetic variation, but the vast majority of this variation was found within rather than between populations, and there was little differentiation between northern and southern populations. Phylogeographic analyses revealed a very shallow phylogenetic tree, a star-like haplotype network, and a unimodal mismatch distribution, all indicative of a recent range expansion from a single refugium. Coalescent modelling approaches also indicated a recent common ancestor for the entire group of birds analysed, no split between northern and southern populations, and high levels of gene flow. These results show that Pleistocene or earlier events have played little role in creating differences between northern and southern populations, suggesting that migratory and other differences between populations have arisen very recently. The implications of these results for the evolution of migration and defining taxonomic groups for conservation efforts are discussed.     

A selective inducible NOS dimerization inhibitor prevents systemic,cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.