Effect of ion-binding and chemical phospholipid structure on the nanomechanics of lipid bilayers studied by force spectroscopy

The nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the amount of ions present in the measuring system has a strong effect on the force needed to puncture a 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer with an atomic force microscope tip, thus highlighting the role that monovalent cations (so far underestimated, e.g., Na(+)) play upon membrane stability. The increase in the yield threshold force has been related to the increase in lateral interactions (higher phospholipid-phospholipid interaction, decrease in area per lipid) promoted by ions bound into the membrane. The same tendency has also been observed for other phosphatidylcholine bilayers, namely, 2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dioleoyl-sn-3-phosphocholine, and also for phosphatidylethanolamine bilayers such as 1-palmitoyl-2-oleoyl-sn-3-phosphoethanolamine. Finally, this effect has been also tested on a natural lipid bilayer (Escherichia coli lipid extract), showing the same overall tendency. The kinetics of the process has also been studied, together with the role of water upon membrane stability and its effect on membrane nanomechanics. Finally, the effect of the chemical structure of the phospholipid molecule on the nanomechanical response of the membrane has also been discussed.     

Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.     

Morphological Stasis of Two Species Belonging to the L-morphotype in the <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Species Complex

Detection and characterization of sibling species complexes in zooplankton are critical to understanding their ecological responses and patterns of evolution. The taxon Brachionus plicatilis is a complex of at least 14 species with three major, deeply diverged clades, which are morphologically distinct. We studied morphometric differences between two species – B. plicatilis sensu stricto and B. ‘Manjavacas’– which belong to the L-(large) morphotype and often co-occur in ponds or lakes. B. plicatilis s.s. was on average 6% longer than B. ‘Manjavacas’. They differed significantly in the measurements related to lorica spines. A significant discriminant function relating spine measurements was found, however, individuals from each species showed extensive overlap. Our morphometric data provide additional evidence for the species status of B. plicatilis s.s. and B. ‘Manjavacas’. Since these are ancient species, our results support that a morphological stasis occurs in these taxa. We identified COI restriction sites for PvuII and KpnI which are diagnostic for B. ‘Manjavacas’ and B. plicatilis s.s., respectively. We conclude that morphometry is not useful in classifying the two species. At present, this can only be done reliably using molecular methods.     

Amazing stability of the arginine-phosphate electrostatic interaction

Electrostatic interactions between a basic epitope containing adjacent arginine residues and an acidic epitope containing a phosphorylated serine are involved in receptor heteromerization. In the present study, we demonstrate that this arginine-phosphate electrostatic interaction possesses a "covalent-like" stability. Hence, these bonds can withstand fragmentation by mass spectrometric collision-induced dissociation at energies similar to those that fragment covalent bonds and they demonstrate an extremely low dissociation constant by plasmon resonance. The present work also highlights the importance of phosphorylation-dephosphorylation events in the modulation of this electrostatic attraction. Phosphorylation of the acidic epitope, a casein kinase one consensus site, makes it available to interact with the basic epitope. On the other hand, phosphorylation of serine and/or threonine residues adjacent to the basic epitope, a protein kinase A consensus site, slows down the attraction between the epitopes. Although analyzed here in the frame of receptor heteromerization, the arginine-phosphate electrostatic interaction most likely represents a general mechanism in protein-protein interactions.     

Opposite modulatory roles for adenosine A1 and A2A receptors on glutamate and dopamine release in the shell of the nucleus accumbens. Effects of chronic caffeine exposure

Previous studies have demonstrated opposing roles for adenosine A1 and A2A receptors in the modulation of extracellular levels of glutamate and dopamine in the striatum. In the present study, acute systemic administration of motor-activating doses of the A2A receptor antagonist MSX-3 significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens (NAc) and counteracted both dopamine and glutamate release induced by systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, exposure to caffeine in the drinking water (1 mg/mL, 14 days) resulted in tolerance to the effects of systemic injection of CPT or caffeine, but not MSX-3, on extracellular levels of dopamine and glutamate in the NAc shell. The present results show: first, the existence of opposite tonic effects of adenosine on extracellular levels of dopamine and glutamate in the shell of the NAc mediated by A1 and A2A receptors; second, that complete tolerance to caffeine's dopamine- and glutamate-releasing effects which develops after chronic caffeine exposure is attributable to an A1 receptor-mediated mechanism. Development of tolerance to the dopamine-releasing effects of caffeine in the shell of the NAc may explain its weak addictive properties and atypical psychostimulant profile.     

The genus Smittium (Trichomycetes, Harpellales) in the Iberian Peninsula

A study of larval Diptera (Chironomidae, Culicidae and Simuliidae) from Spain has been carried out to fulfill a catalogue of species of the genus Smittium (Harpellales: Legeriomycetaceae) present within these hosts. Among the reported taxa, eight are new species: Smittium brevisporum, S. bulbosporo-phorus, S. gracilis, S. hecatei, S. heterosporum, S. inex-pectans, S. prostratum and S. pseudodimorphum. We also report six previously described species, which are new for the Iberian Peninsula: S. alpinum, S. dipterorum, S. megazygosporum, S. pusillum, S. typhellum and S. fecundum. Three other species (S. simulii, S. culicis and S. culisetae) previously were reported from Spain. In two of them (S. fecundum and S. culicis), we describe for the first time the presence of zygospores. Some of the included species have been artificially cultured as well as ultrastructurally studied using scanning electron microscopy (SEM), with the purpose of observing the surface of both trichospores and the trichospore collar and the morphology of the trichospore appendage.     

Study of the polyol pathway in the porcine epididymis

Concentrations of D-glucose, D-fructose and D-sorbitol were quantified in porcine epididymal fluid by spectrofluorimetric assays and aldose reductase (AR) and sorbitol dehydrogenase (SDH) were located immunohistochemically in the epididymal epithelium. Glucose and fructose concentrations were low (<1 mM) and decreased in the cauda whereas sorbitol concentration (4-7 mM) was rather uniform along the duct. AR was luminally located on microvilli in the caput and corpus with less presence distally and was present in the lumen. SDH was present apically and basally in epithelial cells throughout the epididymis and in the lumen. The observations are consistent with diffusion of circulating glucose into the lumen, its conversion via AR to sorbitol which accumulates in the lumen and the action of SDH on sorbitol to produce fructose. Sperm metabolism of glucose and fructose may explain their lower concentrations in the cauda and sorbitol could be a metabolic substrate or osmolyte required for volume regulation.     

Endoplasmic reticulum-retention C-terminal sequence enhances production of an 11S seed globulin from Amaranthus hypochondriacus in Pichia pastoris

The methylotrophic yeast Pichia pastoris was used to express an 11S seed globulin from Amaranthus hypochondriacus. Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum-retention signal KDEL, were able to express the proamarantin in P. pastoris. After 48 h of induction, KDEL-proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum-retention C-terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein in P. pastoris. The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.