Plant parasite control and soil fauna diversity

The use of pesticides to control plant parasites and diseases has generated serious problems of public health and environmental quality, leading to the promotion of alternative Integrated Pest Management strategies that tend to rely more on natural processes and the active participation of farmers as observers and experimenters in their own fields. We present three case studies that point at different options provided by locally available populations of soil organisms, the maintenance of diverse populations of pests or increased resistance of plants to pest attacks by their interactions with earthworms and other useful soil organisms. These examples demonstrate the diversity of options offered by the non-planned agro-ecosystem diversity in pest control and the need to identify management options that maintain this biodiversity.     

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.     

Thermal selection of PGM allozymes in newly founded populations of the thermotolerant vent polychaete Alvinella pompejana

Alvinella pompejana lives on the top of chimneys at deep-sea hydrothermal vents of the East Pacific Rise. It is thought to be one of the most thermotolerant and eurythermal metazoans. Our experimental approach combines methods of population genetics and biochemistry, considering temperature as a potential selective factor. Phosphoglucomutase (Pgm-1 locus) is one of the most polymorphic loci of A. pompejana and exhibits four alleles, from which alleles 90 and 100 dominate with frequencies of approximately 0.5 in populations. Results from previous studies suggested that allele 90 might be more thermostable than allele 100. Significant genetic differentiation was found by comparing contrasted microhabitats, especially the young, still hot, versus older and colder chimneys, with allele 90 being at highest frequency on young chimneys. Moreover the frequency of allele 90 was positively correlated with mean temperature at the opening of Alvinella tubes. In parallel, thermostability and thermal optimum experiments demonstrated that allele 90 is more thermostable and more active at higher temperatures than allele 100. This dataset supports an additive model of diversifying selection in which allele 90 is favoured on young hot chimneys but counterbalanced over the whole metapopulation by the dynamics of the vent ecosystem.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Identification of 13 novel human modification guide RNAs

Members of the two expanding RNA subclasses termed C/D and H/ACA RNAs guide the 2'-O-methylations and pseudouridylations, respectively, of rRNA and spliceosomal RNAs (snRNAs). Here, we report on the identification of 13 novel human intron-encoded small RNAs (U94-U106) belonging to the two subclasses of modification guides. Seven of them are predicted to direct 2'-O-methylations in rRNA or snRNAs, while the remainder represent novel orphan RNA modification guides. From these, U100, which is exclusively detected in Cajal bodies (CBs), is predicted to direct modification of a U6 snRNA uridine, U(9), which to date has not been found to be pseudouridylated. Hence, within CBs, U100 might function in the folding pathway or other aspects of U6 snRNA metabolism rather than acting as a pseudouridylation guide. U106 C/D snoRNA might also possess an RNA chaperone activity only since its two conserved antisense elements match two rRNA sequences devoid of methylated nucleotides and located remarkably close to each other within the 18S rRNA secondary structure. Finally, we have identified a retrogene for U99 snoRNA located within an intron of the Siat5 gene, supporting the notion that retro-transposition events might have played a substantial role in the mobility and diversification of snoRNA genes during evolution.     

Differential effects of mechanical ventilatory strategy on lung injury and systemic organ inflammation in mice

Patients with acute respiratory distress syndrome are at increased risk for developing multiorgan system dysfunction. The goal of this study was to establish an in vivo murine model to assess the differential effects of ventilation-protective strategies on the development of acute lung injury and systemic organ inflammation. C57B/6 mice were randomized to mechanical ventilation (MV) with conventional, high (17 ml/kg) or protective, low (6 ml/kg) tidal volume (VT) after intratracheal hydrochloric acid or no intervention. Mean arterial pressure was continuously monitored during MV and did not differ between groups. After 4 h, lung injury was assessed by measurement of wet/dry lung weight, lung lavage protein concentration and cell count, and histology. Concentration of IL-6, TNF-alpha, VEGF, and VEGF receptor-2 (VEGFR2) was measured in lung, liver, kidney, and heart. Results were compared with control, spontaneously breathing mice. Lung injury and altered pulmonary cytokine expression were not detected after MV of healthy mice with low or high VT. Although MV did not significantly alter IL-6 or TNF-alpha in systemic organs, VEGF concentration significantly increased in liver and kidney. After acid aspiration, mice ventilated with high VT manifested lung injury and increased IL-6 and VEGFR2 in lung, liver, and kidney, whereas VEGF increased only in liver and kidney. MV with low VT after acid aspiration attenuated lung injury, both IL-6 and VEGFR2 expression in lung and systemic organs, and hepatic, but not renal, increased VEGF. Our data suggest that MV strategy has differential effects on systemic inflammatory changes and thus may selectively predispose to systemic organ dysfunction.     

In vivo impact of CpG1826 oligodeoxynucleotide on CD8 T cell primary responses and survival

CpG oligodeoxynucleotide (ODN) promotes maturation of APCs in vivo and induces strong type 1 T cell responses in mice. In this study, we have investigated the ability of CpG1826 to modulate peptide-specific CD8 T cell responses in a context where CD4 T cells are likely to play a minor role. The effects of CpG1826 were evaluated in a system where a population of NP68-specific F5 TCR transgenic CD8 T cells is diluted into a polyclonal host following adoptive transfer into C57BL/10 syngeneic recipients. Using this approach, we found that CpG1826 enhanced the ability of F5 CD8 T cells to undergo multiple divisions in vivo, to express IFN-gamma ex vivo, and to up-regulate memory-associated cell surface markers such as CD122 (IL-2Rbeta) and Ly-6C. Moreover, CpG1826 greatly increased in vivo cytotoxic activity. Using tetramer detection, we found that CpG1826 promoted long-term survival of Ag-specific CD8 T cells after immunization while no NP68-specific cells were detected when the cognate peptide was injected alone. These results indicate that CpG1826 acts as an adjuvant which increases CD8 T cell effector responses and promotes long-term survival of NP68 peptide-specific cells in vivo. They also suggest that this adjuvant can modulate CD8 T cell responses in a system which is likely to be independent of CD4 T cell help.     

Functional analysis of a divergent system II protein,Ccs1, involved in c-type cytochrome biogenesis

The Ccs1 gene, encoding a highly divergent novel component of a system II type c-type cytochrome biogenesis pathway, is encoded by the previously defined CCS1 locus in Chlamydomonas reinhardtii. phoA and lacZalpha bacterial topological reporters were used to deduce a topological model of the Synechocystis sp. 6803 Ccs1 homologue, CcsB. CcsB, and therefore by analogy Ccs1, possesses a large soluble lumenal domain at its C terminus that is tethered in the thylakoid membrane by three closely spaced transmembrane domains in the N-terminal portion of the protein. Molecular analysis of ccs1 alleles reveals that the entire C-terminal soluble domain is essential for Ccs1 function and that a stromal loop appears to be important in vivo, at least for maintenance of Ccs1. Site-directed mutational analysis reveals that a single histidine (His(274)) within the last transmembrane domain, preceding the large lumenal domain, is required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys(199)) is shown to be non-essential. Ccs1 is proposed to interact with other Ccs components based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.