Molecular strategies for increasing seed oil content in canola

xix, 245 leaves : ill. ; 28 cm.Previous research has shown that microsomal DGAT activity from cultures of Brassica napus was stimulated by human acylation stimulating protein (ASP) and bovine serum albumin (BSA). Genetic constructs were engineered to facilitate the expression of ASP or BSA in developing seeds to test the stimulatory effect of these proteins at the site of TAG formation. As well, genetic constructs were designed to produce a truncation of the BSA polypeptide in an attempt to localize the portion fo the macromolecule responsible for stimulation of DGAT activity. An oleosin promoter was used for seed specific expression and to express the polypeptides at a precisely cooridnated time when oil was accumulating in the developing seeds. Lipid analysis coordinated time when oil was accumlating in the developing seeds. Lipid analysis was performed on the seeds of transgenic plants designed to cytosolically express these mammalian proteins and the seeds of control plants. The first generation data revealed that the total lipid within the mature seeds of ASP and BSA plants was not significantly different from the total lipid of negative control plants using both gravimetric and low resolution-nuclear magnetic resonance methods of analysis. The seeds from ASP 8 and ASP 10 plants, however, did produce significantly more lipid on a per seed basis as compared to negative control plants. The levels of the fatty acid composition for total acyl lipids were measured in these first generation transgenic plants. ASP 3 had significantly lower levels of linoleic acid, ASP 14 had significantly lower levels of a-linolenic acid and BSA 11 had significantly higher levels of both of these fatty acids in comparison to negative control plants

Enhanced vascular reactivity of small mesenteric arteries from diabetic mice is associated with enhanced oxidative stress and cyclooxygenase products

1. Vascular reactivity to the alpha-adrenoceptor agonist phenylephrine (PE) was enhanced in small mesenteric arteries (SMA) from diabetic (db/db) mice under both high and low in vitro oxygen conditions. 2. Mechanical removal of the endothelium significantly attenuated the enhanced vascular reactivity of SMA from db/db mice. 3. Acute incubation of the SMA with sepiapterin, a precursor of tetrahydrobiopterin, and N(ω)-nitro L-arginine (L-NA), an inhibitor of nitric oxide (NO) synthase (NOS), resulted in no significant change in the enhanced vascular reactivity to PE in db/db mice. Endothelial nitric oxide synthase (eNOS) mRNA and protein levels in SMA were not different between db/+ and db/db mice. 4. Acute incubation of SMA with a combination of polyethylene glycol superoxide dismutase and catalase significantly reduced the enhanced contraction to PE in db/db mice. There were higher levels of malondialdehyde, a marker of lipid peroxidation and basal superoxide as measured by dihydroethidium staining, in SMA from db/db mice compared to db/+ mice. 5. Acute incubation with indomethacin, a nonselective inhibitor of cyclooxygenase, SQ 29548, a selective thromboxane receptor antagonist and furegrelate, a thromboxane synthesis inhibitor, significantly attenuated the enhanced contraction to PE in SMA from db/db mice. 6. This study demonstrates that the enhanced contractility of SMA from db/db mice to PE was endothelium dependent and involves elevated reactive oxygen species, cyclooxygenase activity and thromboxane synthesis, but not changes in the eNOS/NO pathway

Endothelial dysfunction in the streptozotocin-induced diabetic apoE-deficient mouse

1. Endothelial dysfunction plays a role in the development of atherosclerosis and diabetes-associated vascular disease and, in the streptozotocin (STZ)-induced apoE-deficient diabetic mouse, we report that, when compared to the citrate (CIT)-treated nondiabetic apoE-deficient control, acetylcholine (Ach)-mediated endothelium-dependent relaxation was reduced in the small mesenteric arteries (SMA) and the plaque-prone regions of the aorta from the STZ-diabetic mouse. 2. In the SMA the component of Ach-mediated relaxation that was attributed to nitric oxide (NO) from STZ-treated diabetic apoE-deficient mice was enhanced; however, the endothelium-derived hyperpolarizing factor (EDHF)-mediated component was reduced. The EDHF component was assessed by determining the component of the Ach-mediated response that was resistant to the combination of the NO synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester, cyclooxygenase inhibitor, indomethacin, and soluble guanylate cyclase inhibitor, ODQ, and inhibited by the combination of the intermediate conductance K(Ca) (IK(Ca)) inhibitor TRAM-34 and the small-conductance K(Ca) (SK(Ca)) inhibitor apamin. 3. Endothelial NOS was increased but SK2, SK3 and connexin (Cx) 37 mRNA expressions were significantly (P<0.05) decreased in the SMA from STZ-treated apoE-deficient mice compared to the CIT-treated controls. There was no difference in the IK(Ca) expression or in Cx 40, 43 and 45 mRNA levels between STZ- and CIT-treated mice. 4. The microvasculature of STZ-induced apoE-deficient mice developed endothelial dysfunction, which may be linked to a decrease in the contribution of the EDHF component due to a decrease in SK2 and 3 and Cx 37 expression