G M1 gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal β-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 β-galactosidase cDNA first was defined. The feline β-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 β-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human β-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal β-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 β-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 β-galactosidase cDNA restored activity in transfected G M1 fibroblasts to 18-times normal. β-Galactosidase protein levels in G M1 tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant β-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. CIHR Author Manuscript CIHR Author Manuscript CIHR Author ManuscriptThe lysosomal enzyme β-D-galactosidase (βgal, EC 3.2.1.23) cleaves terminal galactose residues from a variety of molecules, including gangliosides G A1 and G M1 . Deficiency of βgal is known to cause two lysosomal storage diseases: G M1 gangliosidosis (neuronopathic) and Morquio B Disease (mucopolysaccharidosis IVB, non-neuronopathic [3,4]. Although the in vivo biochemical effect of the Arg482His mutation often is difficult to discern because it occurs most frequently in compound heterozygotes, patients homozygous for the G1445A substitution present with the infantile (most severe) form of G M1 gangliosidosis [3,7,8]. A similar mutation, Arg482Cys, also produced no residual βgal activity after expression in G M1 gangliosidosis fibroblasts [4].Feline G M1 gangliosidosis, first described in a Siamese cat in 1971 [9], models the juvenile form of the human disease. Onset of clinical neurological disease in affected cats occurs at approximately 3.5 months of age with a fine head or limb tremor. G M1 mutant cats have progressive dysmetria and ambulatory difficulties, with blindness and epileptiform seizures in the terminal disease stage at 9-10 months of age. In the current st...

In dogs, administration of 20 mg of SAMe/kg/d may mitigate the apparent pro-oxidant influences of prednisolone but did not block development of classic clinicopathologic or histologic features of vacuolar hepatopathy.

Seventy chemicals were tested for the ability to induce sex-linked recessive lethal (SLRL) mutations in postmeiotic and meiotic germ cells of male Drosophila melanogaster. As in the previous studies in this series, adult feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. Two chemicals, n-butane and propylene, were gaseous and therefore tested only by inhalation. One chemical (dimethylcarbamoyl chloride) was tested only by injection. Those chemicals that were mutagenic in the SLRL assay were further tested for the ability to induce reciprocal translocations. Sixteen of the 70 chemicals tested were mutagenic in the SLRL assay: 3-chloro-2-methylpropene, 3-(chloromethyl)pyridine HCl, dimethylcarbamoyl chloride, HC blue 1,3-iodo-1,2-propanediol, malaoxon, N,N'-methylene-bis-acrylamide, 4,4'-methylenedianiline 2HCl, ziram, cis-dichlorodiaminoplatinum II, 1,2-dibromoethane, dibromomannitol, 1,2-epoxypropane, glycidol, myleran, and toluene diisocyanate. The last seven also induced reciprocal translocations. A comparison of the results from the SLRL assay with other assays for mutagens and carcinogens suggests that the SLRL assay is highly specific, but poorly sensitive, both for mutagens and potential carcinogens.

Fifty chemicals were tested for mutagenic activity in post-meiotic and meiotic germ cells of male Drosophila melanogaster using the sex-linked recessive lethal (SLRL) assay. As in the previous studies in this series, feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. One gaseous chemical (1,3-butadiene) was tested only by inhalation. Those chemicals that were mutagenic in the sex-linked recessive lethal assay were further tested for the ability to induce reciprocal translocations. Eleven of the 50 chemicals tested were mutagenic in the SLRL assay. These included bis(2-chloroethyl) ether, 1,4-butanediol diglycidyl ether, 1-chloro-2-propanol, dimethyl methylphosphonate, dimethyl morpholinophosphoramidate, dimethyloldihydroxyethylene urea, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isatin-5-sulfonic acid (Na salt), isopropyl glycidyl ether, and urethane. Five of these, including 1,4-butanediol diglycidyl ether, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isopropyl glycidyl ether, and urethane, also induced reciprocal translocations.

Results from Drosophila mutagenicity tests of 45 chemical compounds assayed for the National Toxicology Program are presented. Nine compounds were judged positive and four equivocal in the sex-linked recessive lethal test. The nine positive compounds were acetin, allyl glycidyl ether, cyclophosphamide, 1,2-dibromo-3-chloropropane, 2,3-dibromo-1-propanol, dimethylcarbamyl chloride, 1,2-epoxy-butane, lasiocarpine, and N-nitrosopiperidine. The results for chloral hydrate, maleic hydrazide, propantheline bromide, and trifluralin were equivocal. Of the nine compounds positive in recessive lethal induction, only two--allyl glycidyl ether and dimethylcarbamyl chloride--failed to induce translocations. The remaining 32 were judged to be nonmutagenic under the conditions used.

A 4-year-old, neutered male domestic shorthair cat presented for evaluation of ataxia and visual deficits. Neurological examination revealed severe cerebellar ataxia with symmetrical hypermetria and spasticity, a coarse whole-body tremor, positional vertical nystagmus, and frequent loss of balance. A menace response was absent bilaterally, and the pupils were widely dilated in room light. A funduscopic examination revealed markedly attenuated to absent retinal vessels and pronounced tapetal hyperreflectivity, findings consistent with end-stage retinal degeneration. Blood work evaluation included retroviral testing, a complete blood count, serum biochemistry analysis, taurine levels, and toxoplasma immunoglobulin G and immunoglobulin M titers. All were within reference ranges. The patient was euthanized, and a necropsy was performed. Microscopically, lesions of the nervous system were confined to the cerebellum and were consistent with cerebellar cortical abiotrophy. Selective photoreceptor degeneration was seen on histopathological examination of the retina with a reduction in the number of rods and cones. The combination of clinical findings and histopathological lesions seen here has not been previously reported in the cat.

S-adenosylmethionine (SAMe), an important hepatic metabolite and glutathione (GSH) donor, has been studied mechanistically in vitro, in humans with clinical liver disease, and in experimental animal models of liver disease. Collective findings encourage its therapeutic use in necroinflammatory and cholestatic liver disorders. A chronic longitudinal study (pre-and posttreatment parameters compared) was undertaken with 15 clinically healthy cats given a stable 1,4-butanedisulfonate (SS isomer) SAMe salt (enteric coated tablets providing 180 mg SAMe), dosage 48 mg/kg PO q24h, on an empty stomach for 113 days. Routine physical and clinicopathologic assessments, red blood cell (RBC) osmotic fragility, liver function and histology, hepatic concentrations of reduced GSH (RGSH) and its oxidized disulfide form (GSSG), protein, glycogen, and deoxyribonucleic acid, GSH concentrations in RBCs, total bile acids in serum and bile, oxidative membrane products (TBARS) in RBCs and liver, and plasma SAMe concentrations were evaluated. SAMe administered PO significantly increased plasma SAMe concentrations, and peak concentrations usually occurred 2-4 hours after dosing. Chronic SAMe administration did not change peak or cumulative plasma SAMe concentrations and did cause overt signs of toxicity. A positive influence on RBC and hepatic redox status (RBC TBARS reduced 21.1% [P .002], liver GSH increased 35% [P .002], liver RGSH : GSSG ratio increased 69% [P .03]) and improved RBC resilience to osmotic challenge (P .03) were observed. Results prove that this SAMe PO product is enterically available and suggest that it imparts biologic effects that might be useful for attenuating systemic or hepatic oxidant challenge.

Background Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs. Objectives To report 3 new breed‐specific PK‐LR gene mutations and a retrospective survey of PK mutations in a small and selected group of Beagles and West Highland White Terriers (WHWT). Animals Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other), WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1). Methods Exons of the PK‐LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia. Results A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed‐specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK‐deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK‐deficient and 3% were carriers (0.37). Conclusions and Clinical Importance Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed‐specific mutation assays simplify the diagnosis of PK deficiency.