The NMR solution structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The 3-dimensional structure of the pheromone Er-1 isolated from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution by 1H NMR spectroscopy. The structure of this 40-residue protein was calculated with the distance geometry program DIANA on the basis of 503 upper distance constraints derived from nuclear Overhauser effects and 77 dihedral angle constraints derived from spin-spin coupling constants, and refined by restrained energy minimization with the program OPAL. The Er-1 solution structure is represented by a group of 20 conformers with an average RMS deviation relative to the mean structure of 0.55 A for the backbone atoms N, C alpha, and C', and 0.93 A for all heavy atoms of the complete polypeptide chain, residues 1-40. The molecular architecture is dominated by an up-down-up bundle of 3 alpha-helices formed by residues 2-9, 12-19, and 24-33. Although this core part coincides closely with the previously determined structure of the homologous pheromone Er-10, the C-terminal peptide segment adopts a novel conformation. This is of interest in view of previous suggestions, based on sequence comparisons, that this molecular region may be important for the different specificity of receptor recognition by different pheromones.     

Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of ¹²⁵ I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10^?9 M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10⁶ per cell of 5–7 fissions of age, to about 16 × 10⁶ at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from “immature” to “adult,” that is competent to respond as well to pheromones of conspecific, genetically different cells. © 1992 Wiley-Liss, Inc.     

Global asymptotic stability for a vector disease model with spatial spread

Summary We analyze the global behaviour of a vector disease model which involves spatial spread and hereditary effects. This model can be applied to investigate growth and spread of malaria. No immunization is considered. We prove that, if the recovery rate is less than or equal to a threshold value, the disease dies out, otherwise the infectious people density tends to a homogeneous distribution. Our results follow using contracting convexes techniques and agree with the results given by K. L. Cooke for the model without diffusion.Work supported by C.N.R., Grant No. 79.00696.01.     

Glucose Accelerates Copper- and Ceruloplasmin-induced Oxidation of Low-density Lipoprotein and Whole Serum

Glucose at pathophysiological concentrations was able to accelerate copper-induced oxidation of isolated low-density lipoprotein (LDL) and whole serum. The efficiency of glucose was favored under the following circumstances: (a) when LDL oxidation was induced by low copper concentration, (b) when LDL was partly oxidized, i.e. enriched with lipid peroxides. The glucose derivative methyl- &#102 - d -glucoside was ineffective on Cu 2+ -induced LDL oxidation, pointing out the essential role of the reactivity of the aldehydic carbon for the pro-oxidative effect. When LDL oxidation was induced by a peroxyl radical generator, as a model of transition metal independent oxidation, glucose was ineffective. Glucose was found to stimulate oxidation of LDL induced by ceruloplasmin, the major copper-containing protein of human plasma. Thus, glucose accelerated oxidation of LDL induced by both free and protein bound copper. Considering the requirement for catalytically active copper and for the aldehydic carbon, the pro-oxidative effect of glucose is likely to depend on the increased availability of Cu + ; this is more efficient in decomposing lipid peroxide than Cu 2+ , accounting for acceleration of LDL oxidation. The possible biological relevance of our work is supported by the finding that glucose was able to accelerate oxidation of whole serum, which was assessed by monitoring low-level chemiluminescence associated with lipid peroxidation.     

Different Permeability of Potassium Salts across the Blood-Brain Barrier Follows the Hofmeister Series

The passage of ions across biological membranes is regulated by passive and active mechanisms. Passive ion diffusion into organs depends on the ion-pairing properties of salts present in the serum. Potassium ions could affect brain activity by crossing the blood-brain barrier (BBB) and its accumulation in the extracellular cerebral space could precipitate seizures. In the present study, we analyze passive diffusion of a series of potassium salts in the in vitro isolated guinea pig brain preparation. Different potassium counter-anions confer ion-pairing and lipophilicity properties that modulate membrane diffusion of the salt. Extracellular recordings in different cortical areas demonstrated the presence of epileptiform activities that strongly relate to anion identity, following the qualitative order of the Hofmeister series. Indeed, highly lipophilic salts that easily cross the BBB enhanced extracellular potassium concentration measured by ion-selective electrodes and were the most effective pro-epileptic species. This study constitutes a novel contribution for the understanding of the potential epileptogenicity of potassium salts and, more generally, of the role of counter-anions in the passive passage of salts through biological membranes.     

Phylogeographical pattern of Euplotes nobilii,a protist ciliate with a bipolar biogeographical distribution

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild‐type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool.     

Biomechanical modeling and optimal control of human posture

The present work describes the biomechanical modeling of human postural mechanics in the saggital plane and the use of optimal control to generate open-loop raising-up movements from a squatting position. The biomechanical model comprises 10 equivalent musculotendon actuators, based on a 40 muscles model, and three links (shank, thigh and HAT-Head, Arms and Trunk). Optimal control solutions are achieved through algorithms based on the Consistent Approximations Theory (Schwartz and Polak, 1996), where the continuous non-linear dynamics is represented in a discrete space by means of a Runge-Kutta integration and the control signals in a spline-coefficient functional space. This leads to non-linear programming problems solved by a sequential quadratic programming (SQP) method. Due to the highly non-linear and unstable nature of the posture dynamics, numerical convergence is difficult, and specific strategies must be implemented in order to allow convergence. Results for control (muscular excitations) and angular trajectories are shown using two final simulation times, as well as specific control strategies are discussed.     

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.     

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.