Curariform antagonists bind in different orientations to acetylcholine-binding protein

Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to approximately 170 degrees rotation and approximately 30 degrees degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.     

TASK-1, a two-pore domain K+ channel, is modulated by multiple neurotransmitters in motoneurons

Inhibition of "leak" potassium (K+) channels is a widespread CNS mechanism by which transmitters induce slow excitation. We show that TASK-1, a two pore domain K+ channel, provides a prominent leak K+ current and target for neurotransmitter modulation in hypoglossal motoneurons (HMs). TASK-1 mRNA is present at high levels in motoneurons, including HMs, which express a K+ current with pH- and voltage-dependent properties virtually identical to those of the cloned channel. This pH-sensitive K+ channel was fully inhibited by serotonin, norepinephrine, substance P, thyrotropin-releasing hormone, and 3,5-dihydroxyphenylglycine, a group I metabotropic glutamate receptor agonist. The neurotransmitter effect was entirely reconstituted in HEK 293 cells coexpressing TASK-1 and the TRH-R1 receptor. Given its expression patterns and the widespread prevalence of this neuromodulatory mechanism, TASK-1 also likely supports this action in other CNS neurons.     

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas^1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica ''Rubra'', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety⁴ (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances^5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass^8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential^3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.     

Virtual screening against acetylcholine binding protein

The nicotinic acetylcholine receptors (nAChRs) are a member of the ligand-gated ion channel family and play a key role in the transfer of information across neurological networks. The X-ray crystal structure of agonist-bound α(7) acetylcholine binding protein (AChBP) has been recognized as the most appropriate template to model the ligand-binding domain of nAChR for studying the molecular mechanism of the receptor-ligand interactions. Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against AChBPs revealed 51 potential candidates. In vitro radioligand competition assays using [(3)H] epibatidine against the AChBPs from the freshwater snails, Lymnaea stagnalis, and from the marine species, Aplysia californica and the mutant (AcY55W), revealed seven compounds from the list of candidates that had micromolar to nanomolar affinities for the AChBPs. Further investigation on α(7)nAChR expressing in Xenopus oocytes and on the recombinant receptors with fluorescence resonance energy transfer (FRET)-based calcium sensor expressing in HEK cells showed that seven compounds were antagonists of α(7)nAChR, only one compound (NSC34352) demonstrated partial agonistic effect at low dose (10 μM), and two compounds (NSC36369 and NSC34352) were selective antagonists on α(7)nAchR with moderate potency. These hits serve as novel templates/scaffolds for development of more potent and specific in the AChR systems.     

P. falciparum enhances HIV replication in an experimental malaria challenge system

Co-infection with HIV and P. falciparum worsens the prognosis of both infections; however, the mechanisms driving this adverse interaction are not fully delineated. To evaluate this, we studied HIV-1 and P. falciparum interactions in vitro using peripheral blood mononuclear cells (PBMCs) from human malaria na?ve volunteers experimentally infected with P. falciparum in a malaria challenge trial. PBMCs collected before the malaria challenge and at several time points post-infection were infected with HIV-1 and co-cultured with either P. falciparum infected (iRBCs) or uninfected (uRBCs) red blood cells. HIV p24Ag and TNF-α, IFN-γ, IL-4, IL-6, IL-10, IL-17, and MIP-1α were quantified in the co-culture supernatants. In general, iRBCs stimulated more HIV p24Ag production by PBMCs than did uRBCs. HIV p24Ag production by PBMCs in the presence of iRBCs (but not uRBCs) further increased during convalescence (days 35, 56, and 90 post-challenge). In parallel, iRBCs induced higher secretion of pro-inflammatory cytokines (TNF-α, IFN-γ, and MIP-1α) than uRBCs, and production increased further during convalescence. Because the increase in p24Ag production occurred after parasitemia and generalized immune activation had resolved, our results suggest that enhanced HIV production is related to the development of anti-malaria immunity and may be mediated by pro-inflammatory cytokines.     

Testing the effects of an introduced palm on a riparian invertebrate community in southern california

Despite the iconic association of palms with semi-arid regions, most are introduced and can invade natural areas. Along the San Diego River (San Diego, California, USA), the introduced Canary Island date palm (Phoenix canariensis) forms dense patches among native riparian shrubs like arroyo willow (Salix lasiolepis). The structural differences between the palm and native shrubs are visually obvious, but little is known about palm's effects on the ecosystem. We tested for the effects of the palm on a riparian invertebrate community in June 2011 by comparing the faunal and environmental variables associated with palm and willow canopies, trunks and ground beneath each species. The palm invertebrate community had lower abundance and diversity, fewer taxa feeding on the host (e.g., specialized hemipterans), and more taxa likely using only the plant's physical structure (e.g., web-builders, oak moths, willow hemipterans). There were no observed effects on the ground-dwelling fauna. Faunal differences were due to the physical and trophic changes associated with palm presence, namely increased canopy density, unpalatable leaves, trunk rugosity, and litter accumulations. Palm presence and resulting community shifts may have further ecosystem-level effects through alteration of physical properties, food, and structural resources. These results were consistent with a recent study of invasive palm effects on desert spring arthropods, illustrating that effects may be relatively generalizable. Since spread of the palm is largely localized, but effects are dramatic where it does occur, we recommend combining our results with several further investigations in order to prioritize management decisions.     

Insights into the Establishment of the Manila Clam on a Tidal Flat at the Southern End of an Introduced Range in Southern California,USA

Coastal ecosystem modifications have contributed to the spread of introduced species through alterations of historic disturbance regimes and resource availability, and increased propagule pressure. Frequency of occurrence of the Manila clam (Venerupis phillipinarum, Veneridae) in Southern California estuaries has increased from absent or sparse to common since the mid-1990s. Potential invasion vectors include seafood sales and aquaculture, and spread from established northern populations over decades. The clam’s post-settlement habitat preferences are, however, uncertain in this region. Our project aimed to identify factors associated with established patches of the clam within a bay toward the southern end of this introduced range. During summer 2013, we sampled 10 tidal flat sites in Mission Bay, San Diego; each containing an area with and without hard structure (e.g., riprap, boulders). We measured likely environmental influences (e.g., sediment variables, distance to ocean). Manila clam densities across the bay were most strongly associated with site, where highest densities were located in the northern and/or back halves of the bay; and weakly correlated with lower porewater salinities. Within sites, Manila clam density was enhanced in the presence of hard structure in most sites. Prevailing currents and salinity regimes likely contribute to bay wide distributions, while hard structures may provide suitable microhabitats (refuge from predators and physical stress) and larval entrapment within sites. Results provide insights into decisions about future shoreline management efforts. Finally, we identify directions for future study to better understand and therefore predict patterns of establishment of the Manila clam in the southern portion of its introduced range.     

Developing hybrid approaches to predict pKa values of ionizable groups

Accurate predictions of pKa values of titratable groups require taking into account all relevant processes associated with the ionization/deionization. Frequently, however, the ionization does not involve significant structural changes and the dominating effects are purely electrostatic in origin allowing accurate predictions to be made based on the electrostatic energy difference between ionized and neutral forms alone using a static structure and the subtle structural changes be accounted by using dielectric constant larger than two. On another hand, if the change of the charge state is accompanied by a large structural reorganization of the target protein, then the relevant conformational changes have to be explicitly taken into account in the pKa calculations. Here we report a hybrid approach that first predicts the titratable groups whose ionization is expected to cause large conformational changes, termed "problematic" residues, and then applies a special protocol on them, while the rest of the pK(a)s are predicted with rigid backbone approach as implemented in multi-conformation continuum electrostatics (MCCE) method. The backbone representative conformations for "problematic" groups are generated with either molecular dynamics simulations with charged and uncharged amino acid or with ab-initio local segment modeling. The corresponding ensembles are then used to calculate the titration curves of the "problematic" residues and then the results are averaged to obtain the corresponding pKa.