Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium.

Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G. In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.

Induced expression of a Drosophila hsp70 promoter-fusion transgene is reduced after repeated heat shocks.

Levels of transcripts produced by a heat shock protein 70 (hsp70)-antisense white transgene in Drosophila were measured after single and multiple heat shocks to determine whether the hsp70 promoter could produce sustained high levels of transgene transcripts. A single heat shock resulted in typical highly inducible levels of RNA, but the amount of antisense RNA was substantially reduced after multiple heat shocks. Endogenous hsp70 mRNA levels were also less abundant after multiple heat shocks as compared to a single heat shock. The hsp70 promoter is unsuitable for use in fusion gene constructs for long term expression studies where repeated heat shocks are required.

Coronary artery bypass graft failure--an autoimmune phenomenon?

Plasma anticardiolipin antibody (ACA) was measured in 83 patients having coronary artery bypass graft surgery and results were correlated with the incidence of early (1-2 weeks) and late (12 months) graft occlusion, as judged by angiography. There was an association between preoperative ACA level and the incidence of late graft occlusion in relation to both number of patients with an occlusion and number of distal anastomoses occluded. 8 of 15 patients (53.3%) whose maximum ACA level exceeded 4 SD of the mean of controls had a late graft occlusion. When the ACA titre was 2-4, 0-2, or less than 0 SD above the mean of the controls, the occlusion rates were 3 of 13 (23.1%), 3 of 33 (9.1%), and 1 of 15 (6.7%), respectively (p less than 0.03). The incidence of a postoperative rise in ACA level was higher in patients who had had a myocardial infarction in the past than in those with a history of angina only (chi 2 = 4.08, p less than 0.05). This observation supports the notion that one mechanism of ACA production is an immune response to myocardial injury. These results raise the further possibility that the ACA, or related antiphospholipid antibodies, may play a part in progressive coronary vessel disease.

Small bowel mucosa from celiac patients generates 15-hydroxyeicosatetraenoic acid (15-HETE) after in vitro challenge with gluten.

Celiac disease (gluten-sensitive enteropathy [GSE]) is a disorder characterized by small intestinal mucosal injury caused by dietary exposure to wheat gluten and similar proteins. There is evidence that the mucosal injury is immunologically mediated and there is an inflammatory infiltrate present in the mucosa. It is postulated that release of lipid-derived inflammatory mediators may be involved in the pathogenesis of the mucosal injury. Jejunal mucosal biopsy samples from patients with GSE and from a group of patients who were subsequently shown to have normal jejunal mucosa were incubated with tritiated arachidonate and a peptic/tryptic digest of either gluten or casein. Generation of lipid-derived inflammatory mediators was measured by beta-scintillation counting after separation of metabolites by reverse-phase high performance liquid chromatography with two different buffer systems. The predominant arachidonic acid metabolite generated was 15-hydroxyeicosatetraenoic acid (15-HETE). Mucosa from newly diagnosed GSE patients on a normal diet generated more 15-HETE than either control patients or GSE patients maintained on a gluten-free diet. In addition, gluten acted as a specific stimulus to 15-HETE production by mucosa from the GSE patients on a normal diet. 15-HETE has a number of biologic effects that could contribute to the mucosal changes seen in GSE, and the specific release of 15-HETE by gluten suggests involvement in the pathogenesis of the disorder.