Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase-ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long-term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial-derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up-regulated by 4-fold 7 days after injury and gradually decreased to normal at 28 days post-lesion. In chondroitinase-treated animals, the total GAG remained at low level throughout the 28-day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.     

Characterization of eight microsatellite markers in the white sea bream, Diplodus sargus (Teleostei, Sparidae)

The white sea bream, Diplodus sargus (Teleostei, Sparidae), is a species with a high commercial importance in Mediterranean aquaculture. There is currently little information available about the genetic characteristics of cultured populations. In this survey, we have developed eight polymorphic microsatellites for the white sea bream using an enriched genome library protocol. All of them were polymorphic in the 67 individuals tested, 32 of which were wild specimens, and 35 were individuals from a captive F(1) broodstock. These markers can potentially be useful tools for use in population genetic studies.     

Comparison and Validation of Methods To Quantify Cry1Ab Toxin from Bacillus thuringiensis for Standardization of Insect Bioassays

Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC₅₀ values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.     

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.     

Increased mortality of Acanthoscelides obtectus by alkane-grown Beauveria bassiana

The effect of alkane-growth induction of theentomopathogenic fungus Beauveria bassiana(Balsamo) Vuillemin (Deuteromycotina:Hyphomycetes), on the ability to kill the beanweevil Acanthoscelides obtectus Say(Coleoptera: Bruchidae), was tested. Adultinsects were sprayed with an 0.01% Tween 20aqueous suspension of 4 × 10⁶ conidia/ml.The performance of fungi grown in complete agarmedium containing glucose as carbon source(FS₀) was compared to that of alkane-grownfungi (FS₁) with n-hexadecane as the onlycarbon source. Mortality increased (p< 0.05) from 22 ± 4.5% to 44 ±11.4% at day 7, and from 26 ± 5.5% to 60± 7.1% 14 days after treatment withFS₀ or FS₁ respectively. The insectepicuticular hydrocarbons were analysed bycapillary gas chromatography (CGC); majorcomponents were saturated hydrocarbons, 27 to29 carbons in length. A variety ofmethyl-branched isomers of C27 were theprevailing structures, and nC27 was the majorstraight chain component. Whole insecthydrocarbons were qualitatively identical tothose of the epicuticular surface. Oleic,linoleic and palmitic acids accounted foralmost 88% of the fungal fatty acids,irrespective of the carbon source used forgrowth; however, the unsaturated/saturatedratio diminished markedly from 4.32(FS₀) to 2.47 (FS₁). These resultsindicate that alkane supplementation of culturemedia might be a tool to improve the virulenceof some mycoinsecticides.     

Marine amoebae from waters of northwest Spain, with comments on a potentially pathogenic euryhaline species

Of 17 species of free-living amoebae identified in various samples of salt water, only 1, Acanthamoeba polyphaga, is known to be a potential pathogen. While no deaths occurred when laboratory animals were inoculated with A. polyphaga to test for pathogenicity, the protozoa were present in the brain, liver and lungs of some but not all of the animals.     

A spring poleward current and its influence on microplankton assemblages and harmful dinoflagellates on the western Iberian coast

Along-shore currents can propagate harmful algal blooms (HABs) over long distances in many coastal areas of the ocean. Harmful dinoflagellate blooms on the west coast of Iberia frequently occur when the Iberian poleward current (IPC) establishes on the continental slope. This has led to the suggestion that HABs could be transported northward by the IPC. To examine this possibility, the microplankton composition along the west coast of Iberia was studied in May 1993 coinciding with the presence of the IPC. The microplankton of the IPC was almost exclusively composed of small flagellates, with the notable absence of the harmful species usually associated with coastal waters. The primary influence of the IPC was to confine coastal microplankton populations to the shelf, where a downwelling convergence prevented their export from the coastal environment. Microplankton assemblages on the shelf revealed a north–south gradient related to different stages of succession. Earlier stages of succession in which diatoms were prominent were found on the northern shelf, whereas dinoflagellates were more abundant in the south. The toxic species Gymnodinium catenatum, which was only present in the southern shelf, did not show a northward transport associated with the IPC. It is suggested that the northward spreading of HABs along the west coast of Iberia must be related to the interaction between the IPC, which accumulates coastal populations on the shelf, and the latitudinal progress of microplankton succession that determines species composition. Thus, during the course of the season, HABs are likely to be observed in the south prior to their development in the north.     

Histochemical localization of NADPH-diaphorase in the rat accessory olfactory bulb

The distribution of NADPH-diaphorase activity was examined inthe accessory olfactory bulb of the rat using a direct histochemicaltechnique. Labeled fibers and somata were found in all layersof the accessory olfactory bulb. The entire vomeronasal nerveand all vomeronasal glomeruli were strongly labeled, contraryto the main olfactory bulb, where only dorsomedial olfactoryglomeruli displayed NADPH-diaphorase activity. NADPH-diapborasepositive neurons were identified as periglomerular cells inthe glomerular layer and external plexiform layer, horizontalcells in the internal plexiform layer, and granule cells anddeep short-axon cells in the granule cell layer. The labeleddendrites of the granule cells formed a dense neuropile in thegranule cell layer, internal plexiform layer and external plexiformlayer. The staining pattern in the accessory olfactory bulbwas more complex than what has been previously reported, anddemonstrated both similarities and differences with the distributionof NADPH-diaphorase in the main olfactory bulb.     

Integration of UAV Photogrammetry and SPH Modelling of Fluids to Study Runoff on Real Terrains

Roads can experience runoff problems due to the intense rain discharge associated to severe storms. Two advanced tools are combined to analyse the interaction of complex water flows with real terrains. UAV (Unmanned Aerial Vehicle) photogrammetry is employed to obtain accurate topographic information on small areas, typically on the order of a few hectares. The Smoothed Particle Hydrodynamics (SPH) technique is applied by means of the DualSPHysics model to compute the trajectory of the water flow during extreme rain events. The use of engineering solutions to palliate flood events is also analysed. The study case simulates how the collected water can flow into a close road and how precautionary measures can be effective to drain water under extreme conditions. The amount of water arriving at the road is calculated under different protection scenarios and the efficiency of a ditch is observed to decrease when sedimentation reduces its depth.