Cell wall biochemical alterations during Agrobacterium‐mediated expression of haemagglutinin‐based influenza virus‐like vaccine particles in tobacco

Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.     

Landscape genetics structure of European sweet chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill): indications for conservation priorities

Sweet chestnut is a tree of great economic (fruit and wood production), ecological, and cultural importance in Europe. A large-scale landscape genetic analysis of natural populations of sweet chestnut across Europe is applied to (1) evaluate the geographic patterns of genetic diversity, (2) identify spatial coincidences between genetic discontinuities and geographic barriers, and (3) propose certain chestnut populations as reservoirs of genetic diversity for conservation and breeding programs. Six polymorphic microsatellite markers were used for genotyping 1608 wild trees sampled in 73 European sites. The Geostatistical IDW technique (ArcGIS 9.3) was used to produce maps of genetic diversity parameters (He, Ar, PAr) and a synthetic map of the population membership (Q value) to the different gene pools. Genetic barriers were investigated using BARRIER 2.2 software and their locations were overlaid on a Digital Elevation Model (GTOPO30). The DIVA-GIS software was used to propose priority areas for conservation. High values of genetic diversity (He) and allelic richness (Ar) were observed in the central area of C. sativa’s European distribution range. The highest values of private allelic richness (PAr) were found in the eastern area. Three main gene pools and a significant genetic barrier separating the eastern from the central and western populations were identified. Areas with high priority for genetic conservation were indicated in Georgia, eastern Turkey, and Italy. Our results increase knowledge of the biogeographic history of C. sativa in Europe, indicate the geographic location of different gene pools, and identify potential priority reservoirs of genetic diversity.     

Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

Cell–cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h− isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h− cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h− cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h− cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell–cell fusion.     

Microbial Communities and Exopolysaccharides from Polynesian Mats

Microbial mats present in two shallow atolls of French Polynesia were characterized by high amounts of exopolysaccharides associated with cyanobacteria as the predominating species. Cyanobacteria were found in the first centimeters of the gelatinous mats, whereas deeper layers showing the occurrence of the sulfate reducers Desulfovibrio and Desulfobacter species as determined by the presence of specific biomarkers. Exopolysaccharides were extracted from these mats and partially characterized. All fractions contained both neutral sugars and uronic acids with a predominance of the former. The large diversity in monosaccharides can be interpreted as the result of exopolymer biosynthesis by either different or unidentified cyanobacterial species. Received July 25, 2000; accepted October 21, 2000     

Adaptation of an insect cell line of Spodoptera frugiperda to grow at 37°C: Characterization of an endodiploid clone

Summary Sf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37°C. After some months of adaptation at 37°C we obtained two new cell lines, Sf21-HT and SF9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37°C than the wild type at 28°C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.     

Activation of the D prostanoid receptor 1 regulates immune and skin allergic responses

The mobilization of Langerhans cells (LCs) from epithelia to the draining lymph nodes is an essential process to initiate primary immune responses. We have recently shown that in mice, PGD2 is a potent inhibitor of epidermal LC emigration. In this study, we demonstrate that activation of the D prostanoid receptor 1 (DP1) impedes the TNF-alpha-induced migration of human LCs from skin explants and strongly inhibits the chemotactic responses of human LC precursors and of maturing LCs to CC chemokine ligands 20 and 19, respectively. Using a murine model of atopic dermatitis, a chronic Th2-type allergic inflammatory disease, we demonstrate that the potent DP1 agonist BW245C dramatically decreases the Ag-specific T cell activation in the skin draining lymph nodes and markedly prevents the skin lesions following repeated epicutaneous sensitization with OVA. Interestingly, analysis of the local response indicates that BW245C treatment strongly reduces the recruitment of inflammatory cells into the dermis and disrupts the Th1/Th2 balance, probably through the increased production of the immunoregulatory cytokine IL-10, in the skin of sensitized mice. Taken together, our results suggest a new function for DP1 in the regulation of the immune and inflammatory responses. We propose that DP1 activation by specific agonists may represent a strategy to control cutaneous inflammatory Th2-associated diseases.     

Role of Hippoboscidae flies as potential vectors of Bartonella spp. infecting wild and domestic ruminants

The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.     

Calmodulin spatial dynamics in RBL-2H3 mast cells

A line of rat basophilic leukaemia (RBL) cells, a model of mast cells, stably expressing EGFP-tagged calmodulin secreted normally in response to standard agonists. As reported for other cell types, calmodulin was concentrated in the mitotic spindle poles of dividing cells. In unstimulated interphase cells calmodulin was concentrated in the cell cortex and at a single central location. Disruption of cortical actin eliminated the concentration of calmodulin at the cortex while the central calmodulin concentration was associated with an enrichment of tubulin and is likely to represent the centrosome. Following stimulation with either an agonist that crosslinks Fc receptors or co-application of phorbol ester and a calcium ionophore the interior of the cells lost calmodulin while cortical fluorescence became more pronounced but also less uniform. After stimulation discrete bright puncta of calmodulin-EGFP (CaM-EGFP) appeared in the cell interior. Puncta colocalised with moving lysotracker-labelled granules, suggesting that calmodulin may play a role in organising their transport. Our results show that in interphase RBL cells a large fraction of the calmodulin pool is associated with targets in the actin cytoskeleton and demonstrate the utility of this model system for studying calmodulin biology.