Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis.

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.

An inversion of 25 base pairs causes feline GM2 gangliosidosis variant.

In G(M2) gangliosidosis variant 0, a defect in the beta-subunit of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52) causes abnormal accumulation of G(M2) ganglioside and severe neurodegeneration. Distinct feline models of G(M2) gangliosidosis variant 0 have been described in both domestic shorthair and Korat cats. In this study, we determined that the causative mutation of G(M2) gangliosidosis in the domestic shorthair cat is a 25-base-pair inversion at the extreme 3' end of the beta-subunit (HEXB) coding sequence, which introduces three amino acid substitutions at the carboxyl terminus of the protein and a translational stop that is eight amino acids premature. Cats homozygous for the 25-base-pair inversion express levels of beta-subunit mRNA approximately 190% of normal and protein levels only 10-20% of normal. Because the 25-base-pair inversion is similar to mutations in the terminal exon of human HEXB, the domestic shorthair cat should serve as an appropriate model to study the molecular pathogenesis of human G(M2) gangliosidosis variant 0 (Sandhoff disease).

Effects of dietary omega 3 fatty acids on vascular contractility in preanoxic and postanoxic aortic rings.

BACKGROUND: Vasomotor reactivity may contribute to the pathophysiology of ischemic injury. The atherosclerotic vessel may be particularly susceptible to vasoconstriction because of the damaged endothelial layer with resultant loss of vasodilatory factors. While dietary omega 3 fatty acids have been proposed to protect against vascular occlusion, it is not clear to what extent this results from alterations in the function of platelets or from changes intrinsic to the blood vessel itself. METHODS AND RESULTS: The effects of dietary supplementation with fish oils on vascular contractility were examined in endothelialized and de-endothelialized aortic rings under pre- and postanoxic conditions. De-endothelialization was defined functionally by the loss of acetylcholine-induced vasodilation in norepinephrine-preconstricted aortic rings from rats fed normal rat chow. Three groups of rats were fed diets containing either 20% menhaden oil or 20% beef tallow, both supplemented with 3% corn oil or 23% corn oil for longer than 4 weeks. All animals received vitamin E. Under well-oxygenated conditions, de-endothelialized aortic rings from rats fed fish oil and corn oil contracted to similar extents with norepinephrine and vasopressin and less than rings from rats fed beef tallow. Endothelialized (intact) and de-endothelialized rings from rats fed fish oil relaxed more in response to acetylcholine than rings from rats fed beef tallow and corn oil. After anoxic exposure and reoxygenation, KCl-induced contraction of intact rings from rats fed fish oil and corn oil was similar and less than rings from rats fed beef tallow. Intact and de-endothelialized rings from rats fed fish oil relaxed more to acetylcholine than did rings from rats fed beef tallow and corn oil. CONCLUSIONS: Under preanoxic or postanoxic conditions, rings from rats fed fish oil and corn oil contracted less than rings from rats fed beef tallow. The relaxation response to acetylcholine, however, was greater in rings from rats fed fish oil than from rats fed either corn oil or beef tallow. These vascular effects of fish oil feeding may result in increased blood flow to ischemic and reperfused tissues in vivo.

n-3 fatty acids increase postischemic blood flow but do not reduce myocardial necrosis.

The effects of a fish oil-supplemented diet on infarct size and regional myocardial blood flow were examined in a rat model of acute ischemia followed by reperfusion. Thirty-five rats were fed a diet containing 20% by weight: fish oil (FO), rich in n-3 polyunsaturated fatty acids; corn oil (CO), with predominantly n-6 polyunsaturated fatty acids; or beef tallow (BT), containing large amounts of saturated fatty acids. After 6-12 wk on the diet, animals underwent 40 min of left coronary artery occlusion followed by 2 h of reperfusion. Regional transmural myocardial blood flow was determined with radioactive microspheres at 30 min of occlusion and again 30 min after reperfusion. Infarct size was determined with triphenyltetrazolium chloride. Blood flow was virtually undetectable within the ischemic zone in all groups during occlusion. With reperfusion, however, ischemic zone absolute blood flow and relative flow (normalized to nonischemic zone flow) were significantly greater in the fish oil group [2.4 +/- 0.25 ml.min-1.g-1, 44 +/- 4% vs. 1.7 +/- 0.3, 29 +/- 5% for CO (P less than 0.05 vs. FO), and 1.4 +/- 0.3, 29 +/- 5% for BT (P less than 0.05 vs. FO)]. Despite differences in reperfusion blood flow, average percent transmural extent of infarction was nearly identical (68 +/- 4, 68 +/- 5, and 64 +/- 3%) and overall infarct size was similar (38 +/- 3, 36 +/- 4, and 29 +/- 3%) for FO, CO, and BT groups, respectively. In conclusion, dietary supplementation with fish oils increases postischemic blood flow but has no effect on extent of myocardial infarction in this ischemia-reperfusion model in rats.