Assembly,plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at “non-plastic” NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Hepatocyte growth factor/scatter factor and its interaction with heparan sulphate and dermatan sulphate

Hepatocyte growth factor (HGF)/scatter factor (SF) is a unique growth factor, in that it binds both heparan sulphate (HS) and dermatan sulphate (DS). The sequences in HS and DS that specifically interact with and modulate HGF/SF activity have not yet been fully identified. Ascidian DS, which uniquely possesses O-sulphation at C-6 (and not C-4) of its N -acetylgalactosamine unit, was analysed for HGF/SF-binding activity in the biosensor. The kinetic analysis revealed a strong, biologically relevant interaction with an equilibrium dissociation constant ( K (d)) of approx. 1 nM. An Erk activation assay also demonstrated stimulation of the MAP kinase pathway downstream of the Met receptor following addition of both HGF/SF and ascidian DS to the glycosaminoglycan-deficient CHO-745 mutant cell line. Furthermore, the activation of Met and the MAP kinase pathway by HGF/SF and ascidian DS leads to a cellular response in the form of migration.     

Spatial distribution of Culicoides species in Portugal in relation to the transmission of African horse sickness and bluetongue viruses

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midge vectors was carried out at 87 sites within a 50 x 50 km grid distributed across Portugal, using light trap collections at the time of peak midge abundance. Culicoides imicola (Kieffer) made up 66% of the 55 937 Culicoides in these summer collections. It was highly abundant in the central eastern portion of Portugal, between 37 degrees 5' N and 41 degrees 5' N, and in a band across to the Lisbon peninsula (at around 38 degrees 5' N). Of all the complexes, its distribution was most consistent with that of previous outbreaks of Culicoides-borne disease, suggesting that it may remain the major vector in Portugal. Its distribution was also broadly consistent with that predicted by a recent climate-driven model validating the use of remote sensing datasets for modelling of Culicoides distribution. Adult C. imicola were found to have overwintered at 12 of 20 sites re-surveyed in winter but it did so in very low numbers. Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) complex midges were widespread despite their low summer abundance. The observed coincidence of high abundances of C. imicola and high abundances of C. pulicaris in summer lead us to suggest that C. imicola could bring African horse sickness virus or bluetongue virus into contact with C. pulicaris and the latter complex, together with C. obsoletus, could then transmit these viruses across much wider areas of Europe. The fact that adult C. pulicaris are present in high abundances in winter may provide a mechanism by which these viruses can overwinter in these areas.     

Evolution of rDNA FISH patterns in the Fagaceae

The Fagaceae is one of the most important plant families in European forest ecosystems, and it includes several genera distributed in the Northern hemisphere. In this work we studied the genome organization and evolution within the family, by karyotyping and physically mapping rDNA in ten European and Asian species of the genera Fagus, Quercus, and Castanea. All of the species studied had a chromosome number of 2n=2x=24, except for the first report of a single individual of Quercus suber which proved to be triploid (2n=3x=36). The rDNA physical mapping revealed several patterns: the dominant one is present in European and Asian Quercus subgenus Quercus, and in Castanea sativa and Castanea crenata, consisting of two 18S–25S rDNA loci (one subterminal major and one pericentromeric minor) and one 5S rDNA pericentromeric locus. In Fagus sylvatica and in Quercus sessilifolia, different patterns were observed: four terminal 18S–25S rDNA loci and two 5S rDNA pericentromeric loci in the former, and five 18S–25S rDNA loci (three terminal and two intercalary) and one 5S rDNA pericentromeric locus in the latter. In Castanea mollissima a distinct rDNA distribution pattern with two intercalary 18S–25S rDNA loci and two 5S rDNA was found. These findings suggest rDNA loci restructuring during Castanea evolution, and variability of 18S–25S loci between Quercus and Cyclobalanopsis subgenera.     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.     

Alpha-methylated derivatives of 2-arachidonoyl glycerol: synthesis, CB1 receptor activity, and enzymatic stability

Alpha-methylated analogues of the endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), were synthesized aiming to the improved enzymatic stability of 2-AG. In addition, the CB1 activity properties of fluoro derivatives of 2-AG were studied. The CB1 receptor activity was determined by the [35S]GTPgammaS binding assay, and the enzymatic stability of alpha-methylated analogues was determined in rat cerebellar membranes. The results indicate that even if the alpha-methylated 2-AG derivatives are slightly weaker CB1 receptor agonists than 2-AG, they are clearly more stable than 2-AG. In addition, the results showed that the replacement of the hydroxyl group(s) of 2-AG by fluorine does not improve the CB1 activity of 2-AG.     

Effects of pasture implantation on the termite (Isoptera) fauna in the Central Brazilian Savanna (Cerrado)

The advance of agricultural frontier may cause the Cerrado (Brazilian savanna) to disappear before 2030. This work focuses on measuring the impact of pasture implantation on a cerrado’s termite fauna. Termites were sampled in a cerrado sensu stricto and a pasture, originally cerrado. All species were classified as their feeder group, accumulation curves were made and Shannon-Wiener indexes and β diversity were calculated for both areas. Cerrado was richer than pasture and species composition differed considerably, leading β diversity to a high value. The humivorous was the most representative species, followed by grass/litter feeders, xylophagous and, less representative, the intermediates. There were more xylophagous and intermediates species on cerrado than in pasture; the grass/litter feeders were more abundant in pasture, but didn’t differed in number or species; and humivorous didn’t differed neither in richness nor in abundance. This work shows that the simplification of the habitat is indeed causing the extinction of populations that depend on some specifics resource.     

Role of histidine for charge regulation of unstructured peptides at interfaces and in bulk

Histidine‐rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are—in contrast to the majority of proteins—optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)‐histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine‐rich antimicrobial peptides. Proteins 2014; 82:657–667. © 2013 Wiley Periodicals, Inc.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.