Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein

Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria

Cycloheximide and Actinomycin D Block the Toxic Effect of Glutamic Acid on PC12 Cells.

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Lower hybrid dynamics by ECE spectra in Tore Supra

The suprathermal electron cyclotron radiation is used to investigate fundamental phenomena governing electron tail formation during lower hybrid current drive (LHCD). To this end, an approximate analytical solution of the 2-D time-dependent Fokker-Planck equation is developed. Electron cyclotron emission (ECE) is analysed with this model. The pitch-angle scattering time, the characteristic time of the quasi-linear diffusion process, the perpendicular temperature and the quasi-linear diffusion coefficient are measured by fitting the temporal evolution of the ECE si,signal. (C) 2001 Elsevier Science B.V. All rights reserved

Multigene Family Encoding 3′,5′-Cyclic-GMP-Dependent Protein Kinases in Paramecium tetraurelia Cells

In the ciliate Paramecium tetraurelia, 3′,5′-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca(2+) stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation

Populations of Myriophyllum alterniflorum L. as bioindicators of pollution in acidic to neutral rivers in the Limousin region

Myriophyllum alterniflorum D.C. (Haloragaceae) was studied in the acidic to neutral rivers Vienne and Gartempe (Limousin region, France). Two complementary levels of organisation were evaluated to determine their usefulness for pollution assessment: morphological traits of river populations, and eco-physiological responses to ammonium enrichment in indoor experiments. Inter-node length, stem diameter and leaf length were measured and their ratio calculated for 100 selected plants collected in 20 homogeneous areas. Using multidimensional analysis, their ecological significance for trophic assessment was demonstrated: stem diameter was linked to conductivity, and leaf length/stem diameter ratio to phosphates. Nitrate reductase activity was measured in three populations (upstream, median and downstream populations) after experimental enrichment with ammonium. Ammonium enrichment decreased activity starting at very low concentrations. However, inhibition levels depended on tested populations, with upstream populations being less sensitive due to natural ammonium content in water. The differences could be explained by an eco-adaptation to the trophic level of water. The relationships between the two levels of organisation are discussed. These results could help to distinguish different ecotypes using Myriophyllum alterniflorum

Fate and forms of Cu in a reservoir ecosystem following copper sulfate treatment

Copper sulfate (CuSO4) addition to freshwater for phytoplankton control has been practiced for decades, and remains the most effective algicidal treatment for numerous managed water bodies. A reservoir in the centre of France was the site for an investigation of copper distribution in aquatic systems after a copper sulfate treatment Results of copper monitoring showed a rapid conversion of dissolved Cu to particulate forms, with significant accumulation in the sediments(83% of total copper added). Total sediment Cu content increased from approximately 37.7 to 45.4 mug.g(-1) dry weight after the first treatment. Sequential extraction suggested that a significant portion of the sediment-borne Cu was associated with the organic fraction which may release Cu to the water column, although significant release would occur only under extreme changes in water chemistry. Based upon measured Cu concentrations, flows at the down-stream water, and known mass applied during treatment, mass balance calculations indicated that approximately 17% of the Cu was exported from the reservoir over a 70 day period following a 196 mug.L-1 Cu2+ (as CuSO4, 5 H2O) treatment. The largest amount of copper was probably adsorbed on downstream sediment or lost in running water. Copper bioaccumulation by a moss, Fontinalis antipyretica, in the down-stream water showed that it was possible to distinguish between a treated and an untreated area. The impact of copper treatment in the down-stream reservoir could be followed using mosses. The bioaccumulation data further showed that there is a distance effect which could be exploited to determine potential copper impact on receiving water bodies. Thirty days after copper sulfate addition, Fontinalis still indicated copper exposure