Functional properties of flours from field grown transgenic wheat lines expressing the HMW glutenin subunit 1Ax1 and 1Dx5 genes

The high molecular weight glutenin subunits (HMW-GS) of wheat are major determinants of the viscoelastic properties of gluten and dough. The bread making quality of field grown transgenic lines of bread wheat expressing the HMW-GS 1Ax1 or 1Dx5 genes were evaluated over a two year period. Subunit 1Ax1 represented about 29% and 48% of the total HMW-GS in lines 1-2 and 2-2, respectively, while subunit 1Dx5 represented 65.4% and 62% of the total HMW-GS in transgenic lines 6-2 and 9, respectively. The expression of subunits 1Ax1 or 1Dx5 in transgenic wheat led to corresponding decreases in the proportions of endogenous HMW-GS. HMW-GS 1Ax1 and 1Dx5 had contrasting effects on dough quality determined by the Alveograph and sedimentation test. Subunit 1Ax1 increased the tenacity (P), extensibility (L), deformation work (W), and sedimentation value, with the increase being related to the level of expression. In contrast, subunit 1Dx5 led to a smaller increment in the tenacity (P), but to drastic decrease in both extensibility (L), deformation work (W), and the sedimentation value. Expression of subunit 1Ax1 in transgenic wheat resulted in lines with improved rheological properties whereas the lines expressing subunit 1Dx5 resulted in unsuitable breadmaking-related characteristics.     

Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment.

Ten current European wheat varieties were transformed at efficiencies ranging from 1-17% (mean 4% across varieties) following modifications in particle bombardment and tissue culture procedures. All plants surviving phosphinothricin selection were screened for uidA and bar gene activity, and for the presence of marker gene sequences by PCR analysis. A minimum of 35% plant 'escape' frequency was achieved with selection on 4 mg l(-1) gluphosinate ammonium after shoot initiation. Mean co-transformation frequency with various genes-of-interest was 66%. The estimated number of insertions of the uidA gene in 25 lines were; 1-2 in 32%, 3-5 in 52%, and 6-8 in 16% of lines. In T(1) progenies, marker genes segregated in a Mendelian fashion in 50% of 39 lines analysed, as determined by transgene activity assays. Based on PCR analysis, it appeared that in some lines the occurrence of distorted segregation was due to poor transmission of the transgenes.     

Field evaluation and agronomic performance of transgenic wheat

Seven transgenic lines of wheat have been evaluated under field conditions during 2 agonomic years. Four lines contained the transgenes for beta-glucuronidase ( uidA), herbicide resistance ( bar) and for one high-molecular-weight (HMW) subunit, and three lines contained only one transgene for one HMW glutenin subunit and no marker genes. Agronomic traits and yield components were studied in transgenic lines and compared with the non-transgenic parent and null segregant lines. Although phenotypic differences for many traits have been found, only heading date and the number of spikelets per spike showed clear genotypic differences for both field trials. All transgenic lines had a longer heading date than parent lines whereas the number of spikelets per spike in transgenic lines was around that for L88-31 and higher for L88-6 than the corresponding parent lines. No differences were found between lines constitutively expressing the uidA and bar genes from those which only expressed the HMW genes. We conclude that differences between transgenic lines and their parents are small, and could be eliminated by backcrossing transgenic lines with their parents and selecting for the wanted genotype.     

An in vivo model of hyperacute rejection: characterization and evaluation of the effect of transgenic human complement inhibitors

Hyperacute rejection (HAR) occurring after transplantation within phylogenetically distant species is a severe reaction triggered by preexisting xenoreactive antibodies and complement activation, leading to the destruction of the donor organ. Expression of human complement inhibitors in transgenic pig organs prolongs the survival of xenograft in experimental models. Moreover, the extent of protection from hyperacute rejection is dependent on the level and site of expression of the transgenic molecules and, probably, on the combination of different molecules. In this regard a small animal model to test the efficacy of expression vectors and different human molecules could be very advantageous. A murine model developed in our laboratory was characterized by measurement of several parameters characteristic of HAR in the livers of control and transgenic mice expressing transgenic human DAF (CD55) or MCP (CD46) at the end of 2h of perfusion with human plasma and after 1 day. The parameters studied were heamatological values of hepatic functions (GOT and GPT), induction of pro-inflammatory molecules and histopathological evaluation. Cytokines (IL-1, IL-1, IL-6) induction and exposure of P-selectin on the endothelial cell surface, was only observed in control animals after 2h of perfusion, as an early event. GOT and GPT values increase drammatically after 2h perfusion and 1 day after the treatment according to the histopathological observation of liver damage. On the contrary, the livers of hDAF or hMCP transgenic mice, under the same treatment were significantly protected although the extent of this protection is dependent on the level of expression of transgenic human molecules.     

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

Background  

Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.     

Chromosomal variation in dividing protoplasts derived from cell suspensions of barley (Hordeum vulgare L.)

Summary Numerical and structural chromosome variation was analysed in dividing protoplasts isolated from suspension cells of barley. Five cell lines exhibited distribution patterns in chromosome number with different peaks and ranges. Embryogenic/morphogenic cell lines showed a peak at 2n = 14 (ca. 50%) after 6–7 months in culture, while older non-embryogenic cell lines had peaks at aneuploid or polyploid chromosome numbers. Culture duration had a clear effect on numerical and structural chromosome variation in embryogenic cell lines. With ageing of the cultures chromosome variation accumulated and the proportion of 2n = 14 cells decreased. The effect of protoplast isolation and culture on chromosome variation was examined; more cells with normal chromosome sets (12%) were maintained in protoplast-derived colonies than in source suspension cells (4%) of the same culture age.Abbreviations DC Dicentric - F fragment - T telocentric     

HIGH-INTENSITY EXERCISE TRAINING INDUCES MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN SKELETAL MUSCLES

In the present study we investigated the effect of two different exercise protocols on fibre composition and metabolism of two specific muscles of mice: the quadriceps and the gastrocnemius. Mice were run daily on a motorized treadmill, at a velocity corresponding to 60% or 90% of the maximal running velocity. Blood lactate and body weight were measured during exercise training. We found that at the end of training the body weight significantly increased in high-intensity exercise mice compared to the control group (P=0.0268), whereas it decreased in low-intensity exercise mice compared to controls (P=0.30). In contrast, the food intake was greater in both trained mice compared to controls (P < 0.0001 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively). These effects were accompanied by a progressive reduction in blood lactate levels at the end of training in both the exercised mice compared with controls (P=0.03 and P < 0.0001 for low-intensity and high-intensity exercise mice, respectively); in particular, blood lactate levels after high-intensity exercise were significantly lower than those measured in low-intensity exercise mice (P=0.0044). Immunoblotting analysis demonstrated that high-intensity exercise training produced a significant increase in the expression of mitochondrial enzymes contained within gastrocnemius and quadriceps muscles. These changes were associated with an increase in the amount of slow fibres in both these muscles of high-intensity exercise mice, as revealed by the counts of slow fibres stained with specific antibodies (P < 0.0001 for the gastrocnemius; P=0.0002 for the quadriceps). Our results demonstrate that high-intensity exercise, in addition to metabolic changes consisting of a decrease in blood lactate and body weight, induces an increase in the mitochondrial enzymes and slow fibres in different skeletal muscles of mice, which indicates an exercise-induced increase in the aerobic metabolism.