Galactosylated Fucose Epitopes in Nematodes: INCREASED EXPRESSION IN A CAENORHABDITIS MUTANT ASSOCIATED WITH ALTERED LECTIN SENSITIVITY AND OCCURRENCE IN PARASITIC SPECIES

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.     

Functions and origin of plasmids in Erwinia species that are pathogenic to or epiphytically associated with pome fruit trees

The genus Erwinia includes plant-associated pathogenic and non-pathogenic species. Among them, all species pathogenic to pome fruit trees (E. amylovora, E. pyrifoliae, E. piriflorinigrans, Erwinia sp. from Japan) cause similar symptoms, but differ in their degrees of aggressiveness, i.e. in symptoms, host range or both. The presence of plasmids of similar size, in the range of 30 kb, is a common characteristic that they possess. Besides, they share some genetic content with high homology in several genes associated with exopolysaccharide production and hence, with virulence, as well as in some other genes. Knowledge of the content of these plasmids and comparative genetic analyses may provide interesting new clues to understanding the origin and evolution of these pathogens and the level of symptoms they produce. Furthermore, genetic similarities observed among some of the plasmids (and genomes) from the above indicated pathogenic species and E. tasmaniensis or E. billingiae, which are epiphytic on the same hosts, may reveal associations that could expose the mechanisms of origin of pathogens. A summary of the current information on their plasmids and the relationships among them is presented here.     

Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015-1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5-30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.     

Biology of Tupiocoris cucurbitaceus (Hemiptera: Miridae), a predator of the greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) in tomato crops in Argentina

The predator Tupiocoris cucurbitaceus is frequently found attacking Trialeurodes vaporariorum in greenhouses without pesticide applications in Argentina. The objective of these studies was to evaluate some biological characteristics of this species fed on three types of diet (whitefly nymphs, Sitotroga cerealella eggs and a mix of both) and on two host plants (tomato and tobacco), under controlled experimental conditions. Preimaginal developmental time for female and male bugs was shorter in the presence of whiteflies than with only moth eggs. Females lived longer when they ate only whitefly nymphs compared to a mixed diet or only moth eggs. The amount of adult descendants was greater when bugs could eat whiteflies, regardless of the presence of S. cerealella. Embryonic development time, male longevity and sex proportion were not affected by the diet or the host plant. Prey consumption was evaluated for three T. cucurbitaceus life history stages (fourth/fifth instar nymphs, female and male adults) on two types of prey (whitefly nymphs and S. cerealella eggs). On tomato, females were more voracious than males and nymphs. On tobacco, adults and nymphs consumed more S. cerealella than T.vaporariorum nymphs, but again, bug females preyed more than males and nymphs. Results demonstrate that T. cucurbitaceus can survive, develop and reproduce normally using both T. vaporariorum and S. cerealella eggs as prey on tobacco or tomato plants. This information can be useful for managing this predator against T. vaporariorum through conservative or augmentative biological control strategies.     

Communicating old-growth forest through an educational trail

This paper describes the experience of developing an educational trail inside an old-growth beech forest in central Italy. In the last 150 years local people exploited this stand almost exclusively for recreational purposes and now the beechwood has acquired new ecological value after having reached old-growth status. Despite the cultural-historical importance of the site, there is a substantial lack of knowledge about this magnificent stand among members of the public, who have no tools to understand its dynamics nor to accept those structural changes related to its natural evolution. Tourist facilities were restored at the site, according to non-restricting criteria, and an educational program was developed in order to promote a shared comprehension of old-growth forests, their ecological processes and value, the services they provide, and to increase tourist awareness toward this fragile ecosystem. We discuss the educational role ecotourism might potentially hold in western developed countries to link the public with the scientific world, educating people about nature, while preventing negative impacts on natural environments. Even if the importance of local communities in conservation of natural resources has been widely recognized by a number of conservationists, public involvement, particularly in forest conservation and management, still represents a difficult challenge, since it requires the implementation of a common understanding about forest ecosystems, forest goods and forest services. The educational trail inside the beech forest of Monte Cimino may represent a low impact strategy for preserving forest aesthetic functionality, limiting potential damages to the forest while increasing its importance for nature conservation.     

Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences

Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (~1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2·0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.     

Water availability modifies tolerance to photo-oxidative pollutants in transplants of the lichen Flavoparmelia caperata

The hypothesis that a daily water supply allows a lichen to endure the negative effects of environmental concentrations of NO_x and O₃ was tested with a transplant experiment. Five groups (0, A–D) of Flavoparmelia caperata samples derived from the same thalli were used for destructive, pre-exposure measurements (0), or exposed for 5 weeks in the rural collection site (A), and in a urban site with high levels of NO_x and O₃ (B–D). Two groups (C, D) were daily watered half an hour before the daily peak of NO_x (C), and O₃ (D). The comparison between pre- and post-exposure measurements of stress biomarkers revealed that the different thallus hydration regime modified the pollution tolerance as well as the physiology of the exposed samples. The non-watered group B suffered an evident decrease in F_v/F_m and reduced glutathione, but increased ion leakage, whereas the watered groups C and D showed only decreased non-photosynthetic-quenching, possibly derived from NO_x exposure. Ozone, which was higher in the rural than in the urban site, did not significantly affect the lichen metabolism. Our results re-open the discussion on the so-called “drought hypothesis”, which suggests that the lichen desert observed in urban areas of central and eastern Europe is more a matter of dry microclimate than of air pollution.     

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.