Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.     

Mass‐trapping trials for the control of pine processionary moth in a pine woodland recreational area

The pine processionary moth, Thaumetopoea pityocampa, causes serious defoliation to Cedrus, Pinus and Pseudotsuga trees, as well as health problems in humans, pets and farm animals due to their urticating hairs. Environmentally friendly strategies for the management of T. pityocampa include: removal of egg batches, removal of nests, trapping of migrant larvae, spraying microbial or Insect Growth Regulator (IGR) insecticides and biocontrol, as well as pheromone‐based adult trapping and mating‐disruption. In the present paper, results on innovative technology for the control of T. pityocampa infestation using pheromone mass‐trapping are reported. Two 1‐ha plots were identified in the study area (central‐south Italy), a pine woodland recreational site growing Pinus halepensis. In the experimental plot (MT‐plot), 10 G‐traps (funnel trap type) baited with (Z)‐13‐hexadecen‐11‐ynyl acetate sex pheromone component were placed for mass‐trapping of adults; the other plot was used as a control‐plot (C‐plot). The T. pityocampa population was monitored using the two central traps in the MT‐plot and two traps positioned in the C‐plot. In addition, the winter nests made by T. pityocampa larvae overwintering on pine trees were counted. After 2 years of mass‐trapping, the number of adults trapped by the monitoring pheromone traps decreased in the MT‐plot, but not in the C‐plot, whereas the number of nests decreased in both plots. Statistical results highlighted significant differences in trap catches between the two plots but not between years. In the case of nests, differences among plots were not significant before the mass‐trapping, but significant after 1‐year treatment. According to our results, the mass‐trapping technique is able to reduce T. pityocampa infestations. This pheromone method can be applied in combination with other control systems in the context of integrated pest management in recreational areas.     

In vivo tyrosine phosphorylation sites of activated ephrin-B1 and ephB2 from neural tissue

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.     

Dual role of sequence-dependent DNA curvature in nucleosome stability: the critical test of highly bent Crithidia fasciculata DNA tract

In spite of the knowledge of the nucleosome molecular structure, the role of DNA intrinsic curvature in determining nucleosome stabilization is still an open question. In this paper, we describe a general model that allows the prediction of the nucleosome stability, tested on 83 different DNA sequences, in surprising good agreement with the experimental data, carried out in ours as well as in many other laboratories. The model is based on the dual role of DNA curvature in nucleosome thermodynamic stabilization. A critical test is the evaluation of the nucleosome free energy relative to a Crithidia fasciculata kinetoplast DNA fragment, which represents the most curved DNA found so far in biological systems and, therefore, is generally believed to form a highly stable nucleosome.     

Eph receptors in the adult brain

The Eph receptors are a large family of receptor tyrosine kinases with important roles in the establishment of neuronal and vascular networks during embryonic development. The functions of Eph receptors in the adult brain have only recently been investigated, and the results are forcing us to amend the conventional view that these molecules function predominantly in a developmental context. This review summarizes this rapidly expanding new area of research, which has shown that the Eph receptors regulate the structure and physiological function of excitatory synapses through multiple mechanisms, and might thus play a significant role in higher brain functions.     

Exercise-induced and nitroglycerin-induced myocardial preconditioning improves hemodynamics in patients with angina

In humans, regional myocardial dysfunction during ischemia may be improved by ischemic and pharmacological preconditioning. We assessed the possibility that exercise- and nitroglycerin-induced myocardial preconditioning may improve global cardiac performance during subsequent efforts in patients with angina. Ten patients suffering from chronic stable angina and ten healthy volunteers were studied. Through impedance cardiography we assessed hemodynamics during a maximal exercise test, which was used as a baseline (Bas test) and considered as a preconditioning exercise. The Bas test was followed by a sequence of maximal efforts performed during the first (FWOP; 30 min after the Bas test) and second (SWOP; 48 h after the Bas test) windows of protection conferred by ischemic preconditioning. Hemodynamics was further evaluated during maximal exercise performed 48 h later with pharmacologically induced SWOP (PI-SWOP) obtained by transdermal administration of 10 mg of nitroglycerin. In the angina patients, FWOP, SWOP, and PI-SWOP delayed the time to ischemia and allowed them to achieve higher workloads compared with the Bas test. Furthermore, heart rate and cardiac output at peak exercise were enhanced during all the preconditioning phases with respect to the Bas test. However, only SWOP and PI-SWOP increased myocardial contractility and stroke volume. No changes in hemodynamics were detectable in the control subjects. This study demonstrates that in patients with stable angina, although hemodynamics during exercise can be positively improved during both FWOP and SWOP, differences exist between these two phases. Furthermore, the mimicking of exercise-induced SWOP by PI-SWOP with transdermal nitroglycerin may represent an important clinical aspect.