Influence of organically or conventionally produced wheat on health, performance and mycotoxin residues in tissues and bile of growing pigs

From 1999-2001 three different varieties of wheat [Contur (susceptible to Fusarium), Batis and Petrus (less susceptible to Fusarium)] were cultivated under organic and conventional conditions in order to determine mycotoxin burden. Soil quality, preceding crop and weather conditions were comparable in the different production systems. The wheat batches were analysed for moulds, and the contents of zearalenone (ZEN) and deoxynivalenol (DON). Feeding trials were carried out with growing pigs (n = 96; average initial live weight 22.2 +/- 1.5 kg [mean +/- SD]) to examine a possible influence on the animal performance and on mycotoxin residues. The data recorded were clinical conditions, performance, biochemical and hematological data. Residues of ZEN, alpha- and beta-zearalenol (ZEL) and of DON were determined in bile, liver and muscle after slaughtering. Conventionally cultivated wheat was more frequently contaminated with Fusarium and contained more frequently ZEN and DON in higher concentrations than the organically produced wheat. Hematological and biochemical parameters of pigs fed with organically cultivated diets were not different from those of conventionally fed pigs. Pigs fed with organically produced wheat showed a slightly higher daily weight gain, but a lower carcass yield than the conventionally fed animals. The highest residues of DON and total-ZEN (ZEN + alpha-ZEL + beta-ZEL) were found in bile. Bile samples of organically fed pigs contained lower concentrations of total-ZEN than those of conventionally fed pigs. Altogether, these data suggest that wheat from an organic farming does not have higher mycotoxin-contamination than wheat from the conventional farming system.     

Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2+ T cells

Chemokines play an important role in the recruitment of leukocytes and have recently been shown to also attract regulatory T cells. Using blocking mAbs, we analyzed the role of the chemokine receptor CCR2 during initiation and progression of collagen-induced arthritis in mice. Blockade of CCR2 from days 0 to 15 markedly improved clinical signs of arthritis and histological scores measuring leukocyte infiltration, synovial hyperplasia, and bone and cartilage erosion. CCR2 blockade during disease initiation significantly reduced plasma titers of collagen Abs in vivo. In vitro CCR2 blockade also interfered with collagen-specific activation and proliferation of T cells. Surprisingly, CCR2 blockade from days 21 to 36 markedly aggravated clinical and histological signs of arthritis and increased the humoral immune response against collagen. We show that CCR2 is expressed on regulatory T cells. Purified CCR2+ T cells are fully anergic toward polyclonal and collagen-specific activation and potently suppress activation of other T and B cells. The subpopulation of CCR2+ CD25+ regulatory T cells increases approximately 5-fold in the progression phase, while CCR2 expression on other leukocyte populations remains unchanged. These findings identify CCR2+ T cells as regulatory T cells and indicate that CCR2 also plays an important role in down-modulating an inflammatory response.     

Lamins A and C are differentially dysfunctional in autosomal dominant Emery-Dreifuss muscular dystrophy

Mutations in the LMNA gene, which encodes nuclear lamins A and C by alternative splicing, can give rise to Emery-Dreifuss muscular dystrophy. The mechanism by which lamins A and C separately contribute to this molecular phenotype is unknown. To address this question we examined ten LMNA mutations exogenously expressed as lamins A and C in COS-7 cells. Eight of the mutations when expressed in lamin A, exhibited a range of nuclear mislocalisation patterns. However, two mutations (T150P and delQ355) almost completely relocated exogenous lamin A from the nuclear envelope to the cytoplasm, disrupted nuclear envelope reassembly following cell division and altered the protein composition of the mid-body. In contrast, exogenously expressed DsRed2-tagged mutant lamin C constructs were only inserted into the nuclear lamina if co-expressed with any EGFP-tagged lamin A construct, except with one carrying the T150P mutation. The T150P, R527P and L530P mutations reduced the ability of lamin A, but not lamin C from binding to emerin. These data identify specific functional roles for the emerin-lamin C- and emerin-lamin A- containing protein complexes and is the first report to suggest that the A-type lamin mutations may be differentially dysfunctional for the same LMNA mutation.     

Performance of seedlings from natural and afforested populations of Cupressus sempervirens under different temperature and moisture regimes

For successful afforestation programs seed quality is crucial, but seedlings are susceptible to climatic stress. Therefore, to improve afforestation success it is necessary to compare performance of seedlings from natural and cultivated populations under different climatic conditions. We investigated growth performance in seedlings of three natural and four afforested Syrian Cupressus sempervirens L. populations under different temperature and moisture regimes. A “warm” climate chamber approximately simulated current mean annual temperatures (day/night: 20/10°C) while a “hot” chamber simulated an average increase of 5°C (day/night: 25/15°C). Seedlings were irrigated twice (drier) or thrice (moist) weekly. Seedlings from natural provenances outperformed those from afforested stands in all growth variables in both chambers. In the warm chamber, root length and biomass were not affected by irrigation for both population types, but shoot height decreased for afforested seedlings under drier treatment while it slightly increased in natural seedlings. In the hot chamber, shoot height decreased but root length and biomass increased for population types under the drier treatment. Comparison between the two chambers showed that under the drier treatment shoot height and biomass decreased at higher temperatures, but root length and biomass were not significantly different. The same response to higher temperatures was observed under the moist treatment, but root biomass decreased too. Our results emphasize the necessity to protect the remaining natural forest of C. sempervirens in Syria and recommend systematic collection of seed material from natural stands for afforestation programs. This might also hold for ex situ cultivation of retrieving rare and endangered plant species.     

Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction

Harpin HrpZ is one of the most abundant proteins secreted through the pathogenesis-associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane-binding and pore-forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant proteins. We found that oligomerization is mainly mediated by a region near the C-terminus of the protein, and that the same region is also essential for membrane pore formation. Phosphatidic acid binding seems to be mediated by two regions separate in the primary structure. Tobacco, a nonhost plant, recognizes, as a defence elicitor, a 24-amino-acid HrpZ fragment which resides in the region indispensable for the oligomerization and pore formation functions of HrpZ.     

Short-term TNFα shedding is independent of cytoplasmic phosphorylation or furin cleavage of ADAM17

Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.     

Extreme genetic depauperation and differentiation of both populations and species in Eurasian feather grasses (Stipa)

A highly selfing breeding system affects gene flow, which may have consequences for patterns of genetic variation and differentiation on both the population and species level. Feather grasses (Stipa spp.) are dominant elements of Eurasian steppes that persist in Central Europe in scattered isolated populations that are of great conservation interest. Cleistogamy is common in the Stipa pennata group, the phylogeny of which is largely unresolved. Intraspecific patterns of genetic variation can be characterised by lack of gene flow due to selfing, but also by large-scale historical migrations and long-term isolation. We analysed both 5 species within the S. pennata group and 33 populations of Stipa pulcherrima sampled across a large part of its range. Using AFLP markers we assessed phylogenetic relationships of the S. pennata group and patterns of genetic variation within and among populations. The S. pennata group formed a consistent clade separated from S. capillata. Stipa pulcherrima was sister to S. eriocaulis, but the relationships among S. pennata s. str., S. borysthenica., and S. tirsa remained unresolved. Within-population genetic variation was extremely low in all species of the S. pennata group (H _e = 0.0–0.013). In S. pulcherrima, genetic variation was consistently relatively high in the east (Romania, Russia) and declined toward western populations, with many populations at the western range edge lacking genetic variation entirely. Populations were strongly differentiated (F _ST = 0.762), and this differentiation did not follow a classical pattern of isolation by distance. Bayesian cluster analysis revealed nine gene pools in S. pulcherrima, which were mostly geographically clustered. Overall the results suggest that S. pulcherrima and species of the S. pennata group are characterised by a cleistogamous breeding system leading to extremely low levels of genetic variation and high levels of population differentiation at both the population and species level. Postglacial colonisation, current population isolation, and population bottlenecks at the western range periphery have further reduced genetic variation and obviated gene exchange. Thus, genetic variation can only be preserved by the conservation of multiple populations.