Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin

Nearly one-third of the world's population, mostly women and children, suffer from iron malnutrition and its consequences, such as anaemia or impaired mental development. Iron fortification of food is difficult because soluble iron is either unstable or unpalatable, and non-soluble iron is not bioavailable. Genetic engineering of crop plants to increase iron content has therefore emerged as an alternative for iron biofortification. To date, strategies to increase iron content have relied on single genes, with limited success. Our work focuses on rice as a model plant, because it feeds one-half of the world's population, including the majority of the iron-malnourished population. Using the targeted expression of two transgenes, nicotianamine synthase and ferritin, we increased the iron content of rice endosperm by more than six-fold. Analysis of transgenic rice lines confirmed that, in combination, they provide a synergistic effect on iron uptake and storage. Laser ablation-inductively coupled plasma-mass spectrometry showed that the iron in the endosperm of the transgenic rice lines accumulated in spots, most probably as a consequence of spatially restricted ferritin accumulation. Agronomic evaluation of the high-iron rice lines did not reveal a yield penalty or significant changes in trait characters, except for a tendency to earlier flowering. Overall, we have demonstrated that rice can be engineered with a small number of genes to achieve iron biofortification at a dietary significant level.     

The intrinsic factor-vitamin B12 receptor, cubilin, is assembled into trimers via a coiled-coil alpha-helix

A large protein was purified from bovine kidney, using selective extraction with EDTA to solubilize proteins anchored by divalent cation-dependent interactions. An antiserum raised against the purified protein labeled the apical cell surface of the epithelial cells in proximal tubules and the luminal surface of small intestine. Ten peptide sequences, derived from the protein, all matched the recently published sequences for rat (Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for intrinsic factor-vitamin B12 complexes, identifying the protein as bovine cubilin. In electron microscopy, a three-armed structure was seen, indicating an oligomerization of three identical subunits. This model was supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its subunits obtained by analytical ultracentrifugation. In a search for a potential assembly domain, we identified a region of heptad repeats in the N-terminal part of the cubilin sequence. Computer-assisted analysis supported the presence of a coiled-coil alpha-helix between amino acids 103 and 132 of the human cubilin sequence and predicted the formation of a triple coiled-coil. We therefore conclude that cubilin forms a noncovalent trimer of identical subunits connected by an N-terminal coiled-coil alpha-helix.     

Distribution,density and migration dynamics of Ixodes ricinus in an area of the Jurassic mountains of Switzerland

In an area of the Jurassic mountains of Switzerland ecological aspects of the tick species Ixodes ricinus were studied. In particular, the influence of temperature, humidity and vegetation parameters on tick density and distribution were investigated several times in six different habitat types. This was carried out on two scales; on a small scale, within a habitat type and on a larger scale, where all six habitat types were included. By a marking experiment, aspects of the migration dynamics of the ticks were studied. The densities of the six habitat types differed significantly in most of the cases. An analysis of the distribution pattern within a habitat type showed a contagious distribution mostly for the forests and the forest edge, whereas a random distribution was found as well, mainly for the meadows. But these distribution patterns tend towards randomness with time. On a small scale none of the factors showed an influence on tick distribution, therefore the most important factor on this scale might be the hosts of Ixodes ricinus. On a larger scale, temperature and humidity do in some cases influence tick densities. The marking experiment suggests an extremely high turnover of the ticks in an area with an average host density, probably due to passive transportation by hosts and to activation of resting individuals.     

Continuous production of lactic acid from whey permeate by Lactobacillus helveticus in two chemostats in series

The effect of dilution rate on the production of lactic acid from whey permeate by Lactobacillus helveticus has been investigated. In the first chemostat of a two-stage system, total conversion (98.1%) and maximum lactic acid concentration (43.7 g l⁻¹) were obtained at a dilution rate (D_Itot) of 0.06 h⁻¹. Maximum volumetric productivities of lactic acid (8.27 g l⁻¹ h⁻¹) and biomass (1.90 g l⁻¹ h⁻¹) occurred at D_Itot of 0.40 h⁻¹. The fraction of -lactate in the product was found to increase with dilution rate and reached a maximum of 66% at the same dilution rate. The maximum specific growth rate (μ_max) on this medium was 0.7 h⁻¹. A Y_ATP (max) value of 22.4 g dry weight (mol ATP)⁻¹ and a maintenance coefficient of 8.0 mmol ATP (g dry weight h)⁻¹ were determined. The second stage, in series with the first, confirmed these results and further showed that the total residence time could be reduced by 50%, compared with a single chemostat for the same nearly complete level of substrate conversion.     

Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia

Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different. In dermatitis herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6 are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6 can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2 can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced autoimmune responses of DH and GA.     

Decorin regulates endothelial cell motility on collagen I through activation of insulin-like growth factor I receptor and modulation of alpha2beta1 integrin activity

The proteoglycan decorin is expressed by sprouting but not quiescent endothelial cells, and angiogenesis is dysregulated in its absence. Previously, we have shown that decorin core protein can bind to and activate insulin-like growth factor-I receptor (IGF-IR) in endothelial cells. In this study, we show that decorin promotes alpha2beta1 integrin-dependent endothelial cell adhesion and migration on fibrillar collagen type I. We provide evidence that decorin modulates cell-matrix interaction in this context by stimulating cytoskeletal and focal adhesion reorganization through activation of the IGF-IR and the small GTPase Rac. Further, the glycosaminoglycan moiety of decorin interacts with alpha2beta1, but not alpha1beta1 integrin, at a site distinct from the collagen I-binding A-domain, to allosterically modulate collagen I-binding activity of the integrin. We propose that induction of decorin expression in angiogenic, as opposed to quiescent, endothelial cells promotes a motile phenotype in an interstitial collagen I-rich environment by both signaling through IGF-IR and influencing alpha2beta1 integrin activity.     

Potentiometric sensor for the measurement of Cd2+ transport in yeast and plants

Research on heavy metals, and especially on transport of Cd(2+), has attracted much interest during the past decade. An optimized Cd(2+)-selective electrode for the continuous potentiometric monitoring of Cd(2+) fluxes in biological systems is presented. The selectivity of the electrode for Cd(2+) was further improved, and it now has very good long-term stability. The utility of this simple and inexpensive method is demonstrated by studying the Cd(2+) transport with model organisms, such as the yeast Saccharomyces cerevisiae and Arabidopsis cell cultures, frequently used in plant science. Its lower detection limits in the presence of commonly used growth media for yeast and plant cells are improved by approximately three orders of magnitude and are 10(-10) and 10(-8)M Cd(2+), respectively. Control experiments using atomic absorption spectrophotometry confirm that the decrease in Cd(2+) activities in the cell cultures is indeed due to the uptake of these metal ions by the cells. Both model systems can be easily transformed; therefore, in combination with the new electrode, they are very promising tools for the investigation of any protein of interest that might be involved in Cd(2+) transport.     

Preference of dairy cows for ryegrass, white clover and red clover, and its effects on nutrient supply and milk quality

Two experiments were conducted with 30 dairy cows each, to study the preference for fresh (Experiment 1) and ensiled (Experiment 2) ryegrass, white and red clover. Both experiments consisted of three choice diets with white or red clover or both, offered with ryegrass, and two diets with ryegrass mixed with white or red clover (40% clover). Cows consumed diets with 37.7% fresh white and 45.9% red clover, and no preference was observed when the cows were offered all three forages. By contrast, cows preferred white and red clover silage (73.0 and 69.2%, respectively) over ryegrass silage (of lower nutritive quality). When offered three forages, cows preferred white (59.8%) over red clover (17.5%) and ryegrass (22.7%). Choice diets resulted in diets similar (fresh forages) or higher in nutrient content and digestibility (silages). Treatments did not affect feed intake and performance. Choices compared to mixed diets with red clover silage were preferable regarding the fatty acid composition of the milk fat. Obviously, only large differences in nutrient and energy concentration facilitate preferences for clovers over ryegrass, which could, depending on clover type, be beneficial in terms of the milk's fatty acid composition.