Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands

Background  

G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro systems. Caenorhabditis elegans is a soil-dwelling, bacteria-feeding nematode that uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds, making this an ideal system for studying in vivo GPCR-ligand interactions. We sought to test this by functionally expressing two medically important mammalian GPCRs, somatostatin receptor 2 (Sstr2) and chemokine receptor 5 (CCR5) in the gustatory neurons of C. elegans.     

Improved analyses of human mtDNA sequences support a recent African origin for Homo sapiens

New quantitative methods are applied to the 135 human mitochondrial sequences from the Vigilant et al. data set. General problems in analyzing large numbers of short sequences are discussed, and an improved strategy is suggested. A key feature is to focus not on individual trees but on the general "landscape" of trees. Over 1,000 searches were made from random starting trees with only one tree (a local optimum) being retained each time, thereby ensuring optima were found independently. A new tree comparison metric was developed that is unaffected by rearrangements of trees around many very short internal edges. Use of this metric showed that downweighting hypervariable sites revealed more evolutionary structure than studies that weighted all sites equally. Our results are consistent with convergence toward a global optimum. Crucial features are that the best optima show very strong regional differentiation, a common group of 49 African sequences is found in all the best optima, and the best optima contain the 16 !Kung sequences in a separate group of San people. The other 86 sequences form a heterogeneous mixture of Africans, Europeans, Australopapuans, and Asians. Thus all major human lineages occur in Africa, but only a subset occurs in the rest of the world. The existence of these African-only groups strongly contradicts multiregional theories for the origin of Homo sapiens that require widespread migration and interbreeding over the entire range of H. erectus. Only when the multiregional model is rejected is it appropriate to consider the root, based on a single locus, to be the center of origin of a population (otherwise different loci could give alternative geographic positions for the root). For this data, several methods locate the root within the group of 49 African sequences and are thus consistent with the recent African origin of H. sapiens. We demonstrate that the time of the last common ancestor cannot be the time of major expansion in human numbers, and our results are thus also consistent with recent models that differentiate between the last common ancestor, expansion out of Africa, and the major expansion in human populations. Such a two-phase model is consistent with a wide range of molecular and archeological evidence.      

Trophic ecology and persistence of invasive silver carp Hypophthalmichthys molitrix in an oligotrophic South African impoundment

The alien invasive silver carp Hypophthalmichthys molitrix established a self-sustaining feral population in an oligotrophic impoundment, Flag Boshielo Dam, in South Africa. The ability of this population to persist in a dam with low algal biomass (median annual suspended chlorophyll a = 0.08 µg l^?1), and limited access to rivers considered large enough for successful spawning, has implications for their invasive potential in other systems. Stomach content and stable isotope analysis were used to assess the trophic ecology of H. molitrix, which was then compared with indigenous Mozambique tilapia Oreochromis mossambicus, on a seasonal basis during 2011. Hypophthalmichthys molitrix are generalist filter feeders, with a diet consisting primarily of sediment, vegetative detritus, dinoflagellates and diatoms. The dominance of sediments in their stomachs suggests occasional benthic scavenging. However, H. molitrix occupied a higher trophic level (TL = 2.8) than expected, suggesting that this population subsidised their diet with an unidentified dietary constituent, characterised by enriched nitrogen values. Although the stomach contents indicated dietary overlap between H. molitrix and O. mossambicus, stable isotopes revealed fine-scale resource partitioning, despite both species occupying the same trophic level. Nonetheless, the persistence of this feral H. molitrix population in an oligotrophic impoundment highlights their phenotypic plasticity.     

Widespread and ancient distribution of a noncanonical genetic code in diplomonads

Recently, a group of diplomonads has been found to use a genetic code in which TAA and TAG encode glutamine rather than termination. To survey the distribution of this characteristic in diplomonads, we sought to identify TAA and TAG codons at positions where glutamine is expected in genes for alpha-tubulin, elongation factor-1 alpha, and the gamma subunit of eukaryotic translation initiation factor-2. These sequences show that the variant genetic code is utilized by almost all diplomonads, with the genus Giardia alone using the universal genetic code. Comparative phylogenetic analysis reveals that the switch to this genetic code took place very early in the evolution of diplomonads and was likely a single event. Termination signals and downstream untranslated regions were also cloned from three Hexamita genes. In all three of these genes, the predicted TGA termination codon was found at the expected position. Interestingly, the untranslated regions of these genes are high in AT. This is incongruent with the coding regions, which are comparatively GC-rich.      

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Crystal structure of hyaluronidase, a major allergen of bee venom

BACKGROUND: Hyaluronic acid (HA) is the most abundant glycosaminoglycan of vertebrate extracellular spaces and is specifically degraded by a beta-1,4 glycosidase. Bee venom hyaluronidase (Hya) shares 30% sequence identity with human hyaluronidases, which are involved in fertilization and the turnover of HA. On the basis of sequence similarity, mammalian enzymes and Hya are assigned to glycosidase family 56 for which no structure has been reported yet. RESULTS: The crystal structure of recombinant (Baculovirus) Hya was determined at 1.6 A resolution. The overall topology resembles a classical (beta/alpha)(8) TIM barrel except that the barrel is composed of only seven strands. A long substrate binding groove extends across the C-terminal end of the barrel. Cocrystallization with a substrate analog revealed the presence of a HA tetramer bound to subsites -4 to -1 and distortion of the -1 sugar. CONCLUSIONS: The structure of the complex strongly suggest an acid-base catalytic mechanism, in which Glu113 acts as the proton donor and the N-acetyl group of the substrate is the nucleophile. The location of the catalytic residues shows striking similarity to bacterial chitinase which also operates via a substrate-assisted mechanism.     

The paradox of power-sharing: stability and fragility in postwar Lebanon

ABSTRACT

This article examines the consequences of civil war and power-sharing settlements for the development of sectarian networks of mobilization. While power-sharing presents a viable mechanism for ending civil war, it allows the participating militias-turned-parties access to state resources and leaves their population networks and organizations intact. This continuity reduces the militias-turned-parties’ start-up costs for violent mobilization in the future, enabling them to mobilize more effectively than new parties with no combat experience. I exploit rich variation in the wartime legacies and settlement status of the major postwar parties in Lebanon to explain whether and how parties mobilized during the clashes of May 2008, the most serious internal violence to plague Lebanon since the end of its civil war in 1990.     

T-cell Receptor Specificity Maintained by Altered Thermodynamics

The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (K_D = 100 nm to 270 μm). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (K_D = 0.6 nm) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.