Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)), 20637 ((157)KEKLQGQQSDSEQERRAY(173)), 20638 ((174)KEKLQEQQSDLEQERLAY(190)) and 20639 (191KEKLQEQQSDLEQERRAY(207)) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)) and 20633 ((81)DKELTMSNVKNVSQTNFKSLY(100)) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 "High Activity Binding Peptides" could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.     

The role of macroinvertebrates for conservation of freshwater systems

Freshwater ecosystems are the most threatened ecosystems worldwide. Argentinian‐protected areas have been established mainly to protect vertebrates and plants in terrestrial ecosystems. In order to create a comprehensive biodiverse conservation plan, it is crucial to integrate both aquatic and terrestrial systems and to include macroinvertebrates. Here, we address this topic by proposing priority areas of conservation including invertebrates, aquatic ecosystems, and their connectivity and land uses. Location: Northwest of Argentina. We modeled the ecological niches of different taxa of macroinvertebrates such as Coleoptera, Ephemeroptera, Hemiptera, Megaloptera, Lepidoptera, Odonata, Plecoptera, Trichoptera, Acari, and Mollusca. Based on these models, we analyzed the contribution of currently established protected areas in the conservation of the aquatic biodiversity and we propose a spatial prioritization taking into account possible conflict regarding different land uses. Our analysis units were the real watersheds, to which were added longitudinal connectivity up and down the rivers. A total of 132 species were modeled in the priority area analyses. The analysis 1 showed that only an insignificant percentage of the macroinvertebrates distribution is within the protected areas in the North West of Argentina. The analyses 2 and 3 recovered similar values of protection for the macroinvertebrate species. The upper part of Bermejo, Salí‐Dulce, San Francisco, and the Upper part of Juramento basins were identified as priority areas of conservation. The aquatic ecosystems need special protection and 10% or even as much as 17% of land conservation is insufficient for species of macroinvertebrates. In turn the protected areas need to combine the aquatic and terrestrial systems and need to include macroinvertebrates as a key group to sustain the biodiversity. In many cases, the land uses are in conflict with the conservation of biodiversity; however, it is possible to apply the connectivity of the watersheds and create multiple‐use modules.     

A comparison of HPLC pigment analyses and biovolume estimates of phytoplankton groups in an oligotrophic lake

Assessment of the contribution of distinct algal groups to phytoplanktonbiomass in oligotrophic lakes by marker pigments is comparedwith assessment by cell-counting biovolume estimates. Seasonalsamples from an oligotrophic alpine lake (Redon, Pyrenees) mostlyincluded species of chrysophytes, dinoflagellates, cryptophytesand chlorophytes. The chlorophyl a (Chl a) corresponding toeach algal group was estimated using HPLC pigment analyses andthe CHEMTAX program. Chl a estimates and biovolume showed asignificant correlation for all the groups during the ice-freeseason except for chlorophytes. However, some of the samplesfrom the initial phase of the ice cover presented a clear departurefrom the relationship during the ice-free period in most groups.On the other hand, the ratios between a specific marker pigmentand the biovolume of the marked algal group were significantlyconstant within the photic zone (>1% surface irradiance)for most of the pigments and groups, including chlorophytes.Nevertheless, the ratios increased and showed a large variabilityfor samples below the photic depth or below the ice cover. Theviolaxanthin-chrysophyte biovolume ratio presented an opposedtendency to other pigment-biovolume ratios, which increasedin inverse proportion to the depth of the sample. The resultsare discussed in terms of methodological limitations, acclimationresponses and species composition.     

Does fin damage allow discrimination among wild,escaped and farmed Sparus aurata (L.) and Dicentrarchus labrax (L.)?

The present study compares fin damages in gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) according to their wild, escaped or farmed origins. In addition, the potential applicability of fin condition indices (Fin Erosion Index ‘FEI’ and Fin Splitting Index ‘FSI’) as identification tools is discussed. Farmed seabream fins evidenced more erosion and splitting (FEI ± SD: 2.1 ± 0.3; FSI ± SD: 1.9 ± 0.6) than wild seabream fins (FEI: 0.8 ± 0.6; FSI: 1.2 ± 0.9), a result of farming conditions in open‐sea cages. Escaped seabream fin erosion was between that of farmed and wild seabream (FEI: 1.6 ± 0.4), which could indicate that fins in farmed fish recover over time from farming abrasions once they are in the wild. However, the fins of escaped seabream seem to be weaker than those of the wild fish, and therefore might be more susceptible to suffer other types of erosion such as splitting or nipping (FSI: 2.3 ± 0.7). No significant differences were found in seabass FEI according to their origins, although wild seabass presented significantly more split caudal fins (FSI: 3.3 ± 2.8) than the farmed seabass (FSI: 1.2 ± 1.1) and escapees (FSI: 2.5 ± 1.6). Therefore, FEI for seabream could be used as tools not only to distinguish between wild and farmed fish, but also to identify recent escapees, improving further assessments on the contribution of seabream escapees in fishery landings. However, the healing potential of damaged fins must be considered for the proper identification of escapees. Use of fin condition indices from both species could be helpful for aquaculture management, to assess fish welfare in fish farms stocks, and improve the knowledge of handling, stock densities and open‐sea cage environment conditions.     

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.     

Ligand-induced caveolae-mediated internalization of A1 adenosine receptors: morphological evidence of endosomal sorting and receptor recycling

The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the receptor sorting and recycling was studied in the smooth muscle cell line DDT(1)MF-2, by binding assays, by confocal microscopy, and at the structural level. The use of cholera toxin-binding subunit adsorbed to gold as a specific probe for labeling the ganglioside GM(1) and immunoelectron microscopy techniques showed that agonist stimulation produced a clustering and sequestration of adenosine receptors in caveolae. Furthermore, pull-down experiments showed there to be a direct interaction between the C-terminal domain of A(1)R and caveolin-1. Addition of exogenous adenosine deaminase (ADA), a protein that binds to A(1)R and acts as a receptor activity modifying protein (RAMP) stimulated R-PIA-induced A(1) receptor internalization. Finally, the sorting and recycling of A(1)R/ADA complexes was analyzed. Detailed electron microscopy revealed that A(1)R/ADA complexes internalize together through caveolae, are differentially sorted in endosomes, and are recycled back to the cell surface by different groups of recycling endosomes. These results give insight into the spatiotemporal regulation and traffic of A(1)R and RAMPs.     

OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.