Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.     

Landscape dynamics and habitat selection by the alien invasive <Emphasis Type="Italic">Fallopia</Emphasis> (Polygonaceae) in Belgium

Habitat patch colonization dynamics and distribution patterns were analysed at a landscape scale in four invasive Fallopia (Polygonaceae) species. Fallopia sachalinensis and F. aubertii were uncommon and population expansion was not evident during the three consecutive years of study. The two most widespread species, F. japonica and F. × bohemica displayed similar habitat selection patterns with ruderal and natural/semi-natural forests favoured. The highest densities of F. japonica and F. × bohemica individuals were at the edge of preferred habitat patches with different patterns of edge selection. Linear network played an important role in species invasion, with 71% of all F. japonica and F. × bohemica occurring within a 10 m buffer of total linear networks (roads, railways, and rivers). However, the buffer represented only 14.5% of the total landscape surface. The rate of population increase was higher for F. japonica (75.8% and 35.2%, in 2002 and 2003, respectively) than for F. × bohemica (63.6% and 0% in 2002 and 2003, respectively) and was largely the result of intra-patch dynamics with low inter-patch colonization. The total surface area occupied by Fallopia clones in the landscape grew by 34.7% over 2 years of the study, with comparable area growth means for F. japonica and F. × bohemica (34.9% and 34.7%, respectively). The hypothesis that F. × bohemica exhibits higher invasive dynamics due to both clonal and sexual reproduction was not supported by our results.     

A novel, multi-layered methanotrophic microbial mat system growing on the sediment of the Black Sea

A novel microbially diverse type of 1- to 5-cm-thick mat performing anaerobic oxidation of methane (AOM) and covering several square metres of the seafloor was discovered in the Black Sea at 180 m water depth. Contrary to other AOM-mat systems of the Black Sea these floating mats are not associated to free gas and are not stabilized by authigenic carbonates. However, supply of methane is ensured by the horizontal orientation of the mats acting as a cover of methane enriched fluids ascending from the underlying sediments. Thorough investigation of their community composition by molecular microbiology and lipid biomarkers, metabolic activities and elemental composition showed that the mats provide a clearly structured system with extracellular polymeric substances (EPS) building the framework of the mats. The top black zone, showing high rates of AOM (15 μmol  g_dw⁻¹ day⁻¹), was dominated by ANME-2, while the following equally active pink layer was dominated by ANME-1 Archaea . The lowest AOM activity (2 μmol  g_dw⁻¹ day⁻¹) and cell numbers were found in the greyish middle part delimited towards the sediment by a second pink, ANME-1-dominated and sometimes a black outer layer (ANME-2). Our work clearly shows that the different microbial populations are established along defined chemical gradients such as methane, sulfate or sulfide.     

Structural genomics and signaling domains.

Many novel signal transduction domains are being identified in the wake of genome sequencing projects and improved sensitivity in homology-detection techniques. The functions of these domains are being discovered by hypothesis-driven experiments and structural genomics approaches. This article reviews the recent highlights of research on modular signaling domains, and the relative contributions and limitations of the various approaches being used.     

Vulpinic acids inhibit influenza (RNA) viruses but not herpes (DNA) viruses

Three synthetic vulpinic acids inhibited two influenza RNA viruses, type A (Philippine) and B (Paraha), in tissue culture with ID₅₀ values ranging from 3.9 to 15.5 g/ml. They had no activity against a third influenza virus or against two herpes viruses.

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Résumé Trois acides vulpiniques de synthèse inhibent deux virus à DNA de l'influenza, types A (Philippine) et B (Paraha), en culture tissulalre avec des valeurs d'ID₅₀ s'étalant de 3.9 à 15.5 g/ml. Ces acides vulpiniques ne présentent d'activité ni contre un trolalème virus de l'influenza, ni contre deux virus de l'herpes.

     

Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Alpha subunits, together with beta 2 and/or beta 4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3'-untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha 3- and chimeric alpha 6-containing nAChRs; BuIA blocks alpha 6/alpha 3 beta 2 nAChRs with a 40,000-fold lower IC(50) than alpha 4 beta 2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta 4 subunit have very slow off-times, compared with the corresponding beta 2 subunit-containing nAChR. In each instance, rat alpha x beta 4 may be distinguished from rat alpha x beta 2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha 3 beta 2 to mouse alpha 3 beta 4, and human alpha 3 beta2 to human alpha 3 beta 4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta 2- and beta 4-containing nAChRs.     

Alloreactive T cell responses and acute rejection of single class II MHC-disparate heart allografts are under strict regulation by CD4+ CD25+ T cells

Skin but not vascularized cardiac allografts from B6.H-2bm12 mice are acutely rejected by C57BL/6 recipients in response to the single class II MHC disparity. The underlying mechanisms preventing acute rejection of B6.H-2bm12 heart allografts by C57BL/6 recipients were investigated. B6.H-2bm12 heart allografts induced low levels of alloreactive effector T cell priming in C57BL/6 recipients, and this priming was accompanied by low-level cellular infiltration into the allograft that quickly resolved. Recipients with long-term-surviving heart allografts were unable to reject B6.H-2bm12 skin allografts, suggesting potential down-regulatory mechanisms induced by the cardiac allografts. Depletion of CD25+ cells from C57BL/6 recipients resulted in 15-fold increases in alloreactive T cell priming and in acute rejection of B6.H-2bm12 heart grafts. Similarly, reconstitution of B6.Rag(-/-) recipients with wild-type C57BL/6 splenocytes resulted in acute rejection of B6.H-2bm12 heart grafts only if CD25+ cells were depleted. These results indicate that acute rejection of single class II MHC-disparate B6.H-2bm12 heart allografts by C57BL/6 recipients is inhibited by the emergence of CD25+ regulatory cells that restrict the clonal expansion of alloreactive T cells.     

Metabolic reprogramming enables hepatocarcinoma cells to efficiently adapt and survive to a nutrient-restricted microenvironment

Hepatocellular carcinoma (HCC) is a metabolically heterogeneous cancer and the use of glucose by HCC cells could impact their tumorigenicity. Dt81Hepa1-6 cells display enhanced tumorigenicity compared to parental Hepa1-6 cells. This increased tumorigenicity could be explained by a metabolic adaptation to more restrictive microenvironments. When cultured at high glucose concentrations, Dt81Hepa1-6 displayed an increased ability to uptake glucose (P<0.001), increased expression of 9 glycolytic genes, greater GTP and ATP (P<0.001), increased expression of 7 fatty acid synthesis-related genes (P<0.01) and higher levels of Acetyl-CoA, Citrate and Malonyl-CoA (P<0.05). Under glucose-restricted conditions, Dt81Hepa1-6 used their stored fatty acids with increased expression of fatty acid oxidation-related genes (P<0.01), decreased triglyceride content (P<0.05) and higher levels of GTP and ATP (P<0.01) leading to improved proliferation (P<0.05). Inhibition of lactate dehydrogenase and aerobic glycolysis with sodium oxamate led to decreased expression of glycolytic genes, reduced lactate, GTP and ATP levels (P<0.01), increased cell doubling time (P<0.001) and reduced fatty acid synthesis. When combined with cisplatin, this inhibition led to lower cell viability and proliferation (P<0.05). This metabolic-induced tumorigenicity was also reflected in human Huh7 cells by a higher glucose uptake and proliferative capacity compared to HepG2 cells (P<0.05). In HCC patients, increased tumoral expression of Glut-1, Hexokinase II and Lactate dehydrogenase correlated with poor survival (P = 2.47E^?5, P = 0.016 and P = 6.58E^?5). In conclusion, HCC tumorigenicity can stem from a metabolic plasticity allowing them to thrive in a broader range of glucose concentrations. In HCC, combining glycolytic inhibitors with conventional chemotherapy could lead to improved treatment efficacy.     

Cooccurrence of Aerobic and Anaerobic Methane Oxidation in the Water Column of Lake Plußsee

Dissolved methane was investigated in the water column of eutrophic Lake Plußsee and compared to temperature, oxygen, and sulfide profiles. Methane concentrations and δ-¹³C signatures indicated a zone of aerobic methane oxidation and additionally a zone of anaerobic methane oxidation in the anoxic water body. The latter coincided with a peak in hydrogen sulfide concentration. High cell numbers of aerobic and anaerobic methane-oxidizing microorganisms were detected by fluorescence in situ hybridization (FISH) or the more sensitive catalyst-amplified reporter deposition-FISH, respectively, in these layers.