Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in Caenorhabditis elegans

The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in Caenorhabditis elegans have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the “eat-me” signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.     

Dietary Plasticity of Generalist and Specialist Ungulates in the Namibian Desert: A Stable Isotopes Approach

Desert ungulates live in adverse ecosystems that are particularly sensitive to degradation and global climate change. Here, we asked how two ungulate species with contrasting feeding habits, grazing gemsbok (Oryx g. gazella) and browsing springbok (Antidorcas marsupialis), respond to an increase in food availability during a pronounced rain period. We used a stable isotope approach to delineate the feeding habits of these two ungulates in the arid Kunene Region of Namibia. Our nineteen months field investigation included two time periods of drought when food availability for ungulates was lowest and an intermediate period with extreme, unusual rainfalls. We documented thirteen isotopically distinct food sources in the isotopic space of the study area. Our results indicated a relatively high dietary plasticity of gemsbok, which fed on a mixture of plants, including more than 30% of C3 plants during drought periods, but almost exclusively on C4 and CAM plant types when food was plentiful. During drought periods, the inferred gemsbok diets also consisted of up to 25% of Euphorbia damarana; an endemic CAM plant that is rich in toxic secondary plant compounds. In contrast, springbok were generalists, feeding on a higher proportion of C3 than C4/CAM plants, irrespective of environmental conditions. Our results illustrate two dietary strategies in gemsbok and springbok which enable them to survive and coexist in the hostile Kunene arid ecosystem.     

Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.     

The promise of circulating tumor cell analysis in cancer management

Enumeration and molecular characterization of circulating tumor cells isolated from peripheral blood of patients with cancer can aid selection of targeted therapy for patients, monitoring of response to therapies and optimization of drug development, while also providing valuable information about intratumoral heterogeneity.     

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.Download video file.^{(453M, mp4)}     

Interacting effects of wind direction and resource distribution on insect pest densities

Considerable effort has been made to investigate how landscape composition and spatial structures of habitats influence distribution patterns of species. In particular, specialist insect herbivores are known to be affected by spatial and temporal accessibility of their host plants. We studied three important insect pests of oilseed rape (OSR): Meligethes aeneus, Ceutorhynchus pallidactylus and Dasineura brassicae. In a landscape with northwest winds prevailing, we analysed their densities by comparing the predictive power of OSR area in differently orientated landscape sectors. Regression analyses showed that OSR area downwind from a sample site explained up to 72% of the variance in the density of M. aeneus, whereas OSR area in other directions had little effect. The correlation between downwind OSR area and M. aeneus density was negative and observable up to a distance of 1250 m. In contrast, the densities of C. pallidactylus and D. brassicae showed little response to OSR area in whatever direction. We suggest that mainly resource detection mechanisms and dispersal capabilities are responsible for the detected patterns: Odour-orientated upwind anemotaxis apparently drives directional dispersal of mobile species (M. aeneus). However, OSR area along the dispersal path seems to reduce pest density in upwind direction, because the majority of individuals detect resource patches early during the dispersal process. For less mobile species (C. pallidactylus and D. brassicae), similar effects were not detectable at the landscape scale because dispersal capabilities probably were too short.     

Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy

MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF_x structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 Å. With GDP and AlF_x, which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlF_x requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.     

Colorectal cancer metastases affect the biochemical characteristics of the human liver beta-adrenoceptor-G-protein-adenylate cyclase system

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked beta2-adrenoceptors (beta2-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of beta2-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of beta-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of beta-ARs (B(max)) was significantly reduced, compared to NA-NM liver tissues (40.09+/-2.83 vs. 23.09+/-3.24 fmol/mg protein; P<0.001). A similar decrease in the beta-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6+/-2.2 vs. 23.8+/-3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized beta-ARs were uncoupled from stimulatory G-protein (G(S)), as total density of beta-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition-concentration curves of ICI 118.551 showed the presence of a homogeneous population of the beta2-AR subtypes. Neither the binding patterns nor the inhibition constant (K(i)) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic beta-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence beta-AR-mediated functions in both organs.     

Newly resolved relationships in an early land plant lineage: Bryophyta class Sphagnopsida (peat mosses)

? Premise of the study: The Sphagnopsida, an early-diverging lineage of mosses (phylum Bryophyta), are morphologically and ecologically unique and have profound impacts on global climate. The Sphagnopsida are currently classified in two genera, Sphagnum (peat mosses) with some 350-500 species and Ambuchanania with one species. An analysis of phylogenetic relationships among species and genera in the Sphagnopsida were conducted to resolve major lineages and relationships among species within the Sphagnopsida. ? Methods: Phylogenetic analyses of nucleotide sequences from the nuclear, plastid, and mitochondrial genomes (11 704 nucleotides total) were conducted and analyzed using maximum likelihood and Bayesian inference employing seven different substitution models of varying complexity. ? Key results: Phylogenetic analyses resolved three lineages within the Sphagnopsida: (1) Sphagnum sericeum, (2) S. inretortum plus Ambuchanania leucobryoides, and (3) all remaining species of Sphagnum. Sister group relationships among these three clades could not be resolved, but the phylogenetic results indicate that the highly divergent morphology of A. leucobryoides is derived within the Sphagnopsida rather than plesiomorphic. A new classification is proposed for class Sphagnopsida, with one order (Sphagnales), three families, and four genera. ? Conclusions: The Sphagnopsida are an old lineage within the phylum Bryophyta, but the extant species of Sphagnum represent a relatively recent radiation. It is likely that additional species critical to understanding the evolution of peat mosses await discovery, especially in the southern hemisphere.     

IGERS: Inferring Gibbs Energy Changes of Biochemical Reactions from Reaction Similarities

Mathematical analysis and modeling of biochemical reaction networks requires knowledge of the permitted directionality of reactions and membrane transport processes. This information can be gathered from the standard Gibbs energy changes (ΔG⁰) of reactions and the concentration ranges of their reactants. Currently, experimental ΔG⁰ values are not available for the vast majority of cellular biochemical processes. We propose what we believe to be a novel computational method to infer the unknown ΔG⁰ value of a reaction from the known ΔG⁰ value of the chemically most similar reaction. The chemical similarity of two arbitrary reactions is measured by the relative number (T) of co-occurring changes in the chemical attributes of their reactants. Testing our method across a validated reference set of 173 biochemical reactions with experimentally determined ΔG⁰ values, we found that a minimum reaction similarity of T = 0.6 is required to infer ΔG⁰ values with an error of <10 kJ/mol. Applying this criterion, our method allows us to assign ΔG⁰ values to 458 additional reactions of the BioPath database. We believe our approach permits us to minimize the number of ΔG⁰ measurements required for a full coverage of a given reaction network with reliable ΔG⁰ values.