Identification and Functional Expression of a Glutamate- and Avermectin-Gated Chloride Channel from Caligus rogercresseyi,a Southern Hemisphere Sea Louse Affecting Farmed Fish

Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC₅₀ value of around 200 nM, being cooperative (n_H = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy.     

Temporal temperature gradient gel electrophoresis (TTGE) as a tool for the characterization of arbuscular mycorrhizal fungi

The aim of this study was to assess the feasibility of using temporal temperature gradient electrophoresis (TTGE) of PCR-amplified 18S rDNA fragments of different Glomus species for their detection and characterization. Screening of Glomus clarum, Glomus constrictum, Glomus coronatum, Glomus intraradices, Glomus mosseae and Glomus viscosum by PCR-TGGE revealed that the NS31-AM1 region of the 18S rRNA gene contained insufficient variation to discriminate between them. In contrast, TTGE analysis of the NS31-Glo1 region, which was obtained by nested PCR of the NS31-AM1 amplicon, showed that each species was characterized by a specific TTGE fingerprint. However, isolates of the same species could not be distinguished. The nested PCR-TTGE approach developed allowed identification of the Glomus species colonising the roots of different plant species.     

Inflammatory cytokines,goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice

Background

Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O₂ toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury.

Methods

An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1^+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation.

Results

Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1^+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of T_H2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics.

Conclusion

Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1^+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.     

Frugivory and Seed Dispersal by the Lowland Tapir Tapirus terrestris in the Peruvian Amazon

The lowland tapir Tapirus terrestris is the largest herbivore in the Neotropics and feeds on a large quantity of fruits, often ingesting the seeds and defecating them intact. Seed dispersal by the lowland tapir in the southwestern Amazon was studied by examining seeds from 135 dung samples collected between 2005 and 2007. Seeds of a total of 122 plant species were identified, representing 68 genera and 33 families. The species accumulation curve showed that more species can be expected with further sampling. Many species (45%) were only encountered once, and only 10 percent of all species were found in >10 samples, indicating that the lowland tapir is an opportunistic forager. Seed diversity showed a clear seasonal pattern and was highly correlated with fruit availability. Seed diameter ranged from <1 to 25 mm with 81 percent <10 mm diam. The size distribution of seeds found in lowland tapir dung generally followed that of seeds found in the forest, but had a lower proportion of seeds in the smallest size class (<2.5 mm) and a larger proportion found in the largest size class (20–25 mm). The diversity of seeds encountered in dung of the lowland tapir in this study was much higher than in previous studies. We conclude that the lowland tapir is a potential disperser for a large number of plant species, including many that previously have been thought to be dispersed only by large primates.     

Forestiera ecuadorensis: Una nueva especie endémica de Oleaceae y un nuevo registro genérico para Ecuador

Se describeForestiera ecuadorensis, una nueva especie de árbol, endémica del bosque seco tropical al occidente de Ecuador, en la provincia del Guayas, la misma que también es un nuevo registro genérico para el país.Forestiera ecuadorensis es similar aForestiera rhamnifolia distribuida en Mesoamérica y el Caribe, y aForestiera eggersiana de las Islas Vírgenes, Haití y Puerto Rico, de las que difiere por tener por presentar hojas que alcanzan mayor dimensión, con distintivo ápice largoacuminado, mayor número de nervios laterales, estambres con filamentos más cortos y distinto patrón fitogeográfico. También se presenta una clave para todas las especies de Oleaceae reportadas hasta la fecha para Ecuador.     

Phytogeography of the trees of the Osa Peninsula,Costa Rica

A phytogeographic analysis of the distributions of 454 species of trees native to the Osa Peninsula in 22 families revealed that 4.8% of the species are endemic to the Osa Peninsula and the adjacent mainland of Costa Rica. However, nearly one-fourth of the species might be regionally endemic to Central-South Mesoamerica (Costa Rica, Nicaragua, and Panama). Our sample suggests that 53.6% of the species occur in some part of Mesoamerica and sometimes range into northwestern South America, and that 44.5% of the species have wide distributions throughout tropical America. There is a strong affinity with the flora of northwestern South America, with 46.2% of the species on the Osa also found there. In addition, 50.6% of the tree species on the Osa occur on both the Atlantic and Pacific slopes of Central America or, if they reach South America, are sometimes found on both sides of the Andes. Major contributions to the tree flora of the Osa have been made by species arriving in the Osa by 1) dispersal from South and North America to islands in proto Central America before the formation of a dry-land connection between the two continents, and 2) migration from South America and North America after the closure of the Panamanian isthmus was made. This analysis demonstrates the importance of the Osa as a regional refuge for protecting species with distributions limited to the Osa and parts of Panama, Costa Rica, or Nicaragua. The Osa is also important because it harbors the last expanse of tropical wet forest on the Pacific slope of Central America large enough to ensure the survival of the Central American populations of widely distributed plants and animals.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.