High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3A resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp(108), forming an extended binding site. This is the first structural report for a bivalve lectin.     

F plasmid partition depends on interaction of SopA with non-specific DNA

Bacterial ATPases belonging to the ParA family assure partition of their replicons by forming dynamic assemblies which move replicon copies into the new cell-halves. The mechanism underlying partition is not understood for the Walker-box ATPase class, which includes most plasmid and all chromosomal ParAs. The ATPases studied both polymerize and interact with non-specific DNA in an ATP-dependent manner. Previous work showed that in vitro, polymerization of one such ATPase, SopA of plasmid F, is inhibited by DNA, suggesting that interaction of SopA with the host nucleoid could regulate partition. In an attempt to identify amino acids in SopA that are needed for interaction with non-specific DNA, we have found that mutation of codon 340 (lysine to alanine) reduces ATP-dependent DNA binding > 100-fold and correspondingly diminishes SopA activities that depend on it: inhibition of polymer formation and persistence, stimulation of basal-level ATP hydrolysis and localization over the nucleoid. The K340A mutant retained all other SopA properties tested except plasmid stabilization; substitution of the mutant SopA for wild-type nearly abolished mini-F partition. The behaviour of this mutant indicates a causal link between interaction with the cell's non-specific DNA and promotion of the dynamic behaviour that ensures F plasmid partition.     

Adjuvant potential of archaeal synthetic glycolipid mimetics critically depends on the glyco head group structure

Subunit vaccines capable of providing protective immunity against the intracellular pathogens and cancers that kill millions of people annually require an adjuvant capable of directing a sufficiently potent cytotoxic T lymphocyte response to purified antigens, without toxicity issues. Archaeosome lipid vesicles, prepared from isoprenoid lipids extracted from archaea, are one such adjuvant in development. Here, the stability of an archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) is used to advantage to synthesize a series of disaccharide archaeols and show that subtle variations in the carbohydrate head group alters the type and potency of immune responses mounted in a mammal. Critically, a glycosylarchaeol was required to elicit high cytotoxic CD8(+) T cell activity, with highest responses to the antigen entrapped in archaeosomes containing disaccharides of glucose in beta- or alpha1-6 linkage (beta-gentiobiose, beta-isomaltose), or of beta-lactose. This first study on synthetic archaeal lipid adjuvants reveals potential for this class of regulatory friendly, easily scalable, inexpensive, and potent glyco-adjuvant.     

Moduler l’action du glutamate dans le cerveau: de nouvelles pistes ouvertes grâce aux récepteurs métabotropiques

The rapid transmission of information in the central nervous system is mostly mediated by glutamate synapses. The control of this system quickly appeared as a way to modulate, and perhaps normalize, a number of brain dysfunctions. However, the central role of glutamate receptors involved in this transmission, the ionotropic AMPA and NMDA receptors, appeared as an obstacle to the development of drugs devoid of side effects. The discovery of a second family of glutamate receptors, more than fifteen years ago, offered new possibilities to act on the glutamate system. These receptors, the metabotropic glutamate (mGlu) receptors which are coupled to G-proteins, modulate excitatory synaptic transmission. Eight mGlu receptors have been identified and localized either on the post-synaptic element, where they can regulate the AMPA and NMDA receptor activity, or on the presynaptic element, where they control the release of glutamate or other neurotransmitters. Recent data highlights the therapeutic potential of drugs acting at these receptors for the treatment of a variety of pathologies including anxiety, schizophrenia and Parkinson’s disease.     

Functional magnetic resonance signal changes in neural structures to baroreceptor reflex activation.

The sequence of neural responses to exogenous arterial pressure manipulation remains unclear, especially for extramedullary sites. We used functional magnetic resonance imaging procedures to visualize neural responses during pressor (phenylephrine) and depressor (sodium nitroprusside) challenges in seven isoflurane-anesthetized adult cats. Depressor challenges produced signal-intensity declines in multiple cardiovascular-related sites in the medulla, including the nucleus tractus solitarius, and caudal and rostral ventrolateral medulla. Signal decreases also emerged in the cerebellar vermis, inferior olive, dorsolateral pons, and right insula. Rostral sites, such as the amygdala and hypothalamus, increased signal intensity as arterial pressure declined. In contrast, arterial pressure elevation elicited smaller signal increases in medullary regions, the dorsolateral pons, and the right insula and signal declines in regions of the hypothalamus, with no change in deep cerebellar areas. Responses to both pressor and depressor challenges were typically lateralized. In a subset of animals, barodenervation resulted in rises and falls of blood pressure that were comparable to these resulting from the pharmacological challenges but different regional neural responses, indicating that the regional signal intensity responses did not derive from global perfusion effects but from baroreceptor mediation of central mechanisms. The findings demonstrate widespread lateralized distribution of neural sites responsive to blood pressure manipulation. The distribution and time course of neural responses follow patterns associated with early and late compensatory reactions.     

Lipid transfer proteins from Rosaceae fruits share consensus epitopes responsible for their IgE-binding cross-reactivity

Four IgE-binding epitopes have been characterized that cover a large area (40%) of the molecular surface of lipid transfer protein allergens of Rosaceae (apple, peach, apricot, and plum). They mainly correspond to electropositively charged regions protruding on the molecular surface of the modeled apple (Mal d 3), apricot (Pru ar 3), and plum (Pru d 3) allergens. Two of these epitopes consist of consensus epitopes structurally conserved among the lipid transfer protein allergens from the Rosaceae. Their occurrence in different lipid transfer protein allergens presumably accounts for the IgE-binding cross-reactivity often observed among different Rosaceae fruits. In this respect, LTP consist of phylogenetically- and structurally-related pan allergens. However, the IgE-binding cross-reactivity due to fruit lipid transfer protein has varying degrees of clinical relevance and this cross-reactivity is not necessarily accompanied by a cross-allergenicity to the corresponding fruits.     

Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.