Cloning and characterization of excitatory amino acid transporters GLT-1 and EAAC1 in canine brain.

Excitatory amino acid transporters play important roles in termination of glutamatergic neurotransmission and protection of neurons from the excitotoxicity of glutamate in the central nervous system. We herein report isolation of cDNA clones of two distinct excitatory amino acid transporters, GLT-1 and EAAC1, from canine brain cortex by PCR-based cloning, and characterization of these transporter subtypes. Canine GLT-1 and EAAC1 exhibited Na+-dependent glutamate transport with high affinities in a Xenopus oocyte expression system. Despite the similarity in transport kinetics and in the predicted primary structures, GLT-1, EAAC1, and the previously identified GLAST showed different sensitivities to several structural analogues of L-glutamate. In addition, transcripts of these transporter subtypes showed distinct regional distribution in the brain in RT-PCR analysis, suggesting that excitatory amino acid transporters have distinct physiological and pathophysiological roles in the brain.

Differentiation of stromal-vascular cells isolated from canine adipose tissues in primary culture.

A culture condition supporting adipocyte differentiation of stromal-vascular (S-V) cells isolated from canine adipose tissues was established. Morphological observation and determination of glycerol-3-phosphate dehydrogenase (GPDH) activity were used as the criteria for adipocyte differentiation. After reaching confluence, the cells were able to undergo terminal adipocyte differentiation by treatment with 100 microM indomethacin, 10 microg/ml insulin and 0.5 mM 1-methyl-3-isobutylxanthine (MIX) in medium supplemented with 5% fetal calf serum (FCS). In the absence of either indomethacin or insulin, the S-V cells did not undergo adipose conversion and GPDH activity was not increased, indicating that both indomethacin and insulin play essential roles in this culture system. The S-V cells from inguinal adipose tissues exhibited the greatest increase in GPDH activity among the four depots (inguinal > abdominal-subcutaneous > perirenal > omental). demonstrating that adipocyte differentiation was also intensely dependent on anatomic sites from which the S-V cells were derived. Interestingly, dimethylsulfoxide (DMSO) was found to accelerate adipocyte differentiation in combination with indomethacin and insulin. Under this condition, up to 90% of the cells displayed adipocyte phenotypes and the GPDH activity reached 1288 +/- 441 mU/mg protein. This culture system may be useful for investigating other adipogenic factors as well as anti-adipogenic factors involved in the regulation of canine adipose tissue development.

Effects of lipid-related factors on adipocyte differentiation of bovine stromal-vascular cells in primary culture.

The effects of several factors related to lipids on bovine adipocyte differentiation were investigated in primary culture. Adipocyte differentiation was assessed by development of glycerol-3-phosphate dehydrogenase (GPDH) activity and morphological observation. Addition of triglyceride mixture (Intralipid), caprylic acid and very low-, low- and high-density lipoproteins (VLDL, LDL and HDL) stimulated bovine preadipocyte differentiation in serum-free condition. Especially, VLDL strongly increased both cell protein contents and GPDH activity, suggesting that it stimulated both proliferation and differentiation of bovine preadipocytes. Under Intralipid-induced condition, differentiation of preadipocytes from subcutaneous adipose tissues was more evident than those from omental adipose tissues. However, such depot difference was not observed in medium supplemented with indomethacin, which is a peroxisome proliferator-activated receptor (PPAR) gamma agonist. This suggests that the differentiation capacity of bovine preadipocytes was different between depots and such difference is dependent on the ability to utilize lipids as endogenous PPARgamma ligands. Therefore, lipid metabolites have the stimulatory effects on bovine adipocyte differentiation in vitro, and lipoproteins, especially VLDL, may play an important role in development of bovine adipose tissues in vivo.

Connective tissue-type mast cell leukemia in a dog.

An unusual case diagnosed as connective tissue-type mast cell leukemia with marked mastocyte infiltration into visceral organs in a seven-year-old female Curly-Coated retriever is presented. Acute circulatory collapse, emesis, diarrhea, abdominal enlargement, icterus, cyanosis, dyspnea, pulmonary edema, hepatomegary, ascites, and right ventricular enlargement were observed. Hematologic and biochemical examinations revealed mast cell leukemia, mature neutrophilia, monocytosis, thrombocytopenia, hemolytic hyperbilirubinemia, hyperhistaminemia, renal and hepatic injuries. Mast cells were distributed systemically, but predominantly in the diaphragm and liver with a large mass among the serosa of ileum, cecum and colon. Mast cells were stained intensely by both safranin and berberine sulfate.

Inherited defects of sodium-dependent glutamate transport mediated by glutamate/aspartate transporter in canine red cells due to a decreased level of transporter protein expression.

Canine red cells have a high affinity Na(+)/K(+)-dependent glutamate transporter. We herein demonstrate that this transport is mediated by the canine homologue of glutamate/aspartate transporter (GLAST), one of the glutamate transporter subtypes abundant in the central nervous system. We also demonstrate that GLAST is the most ubiquitous glutamate transporter among the transporter subtypes that have been cloned to date. The GLAST protein content was extremely reduced in variant red cells, low glutamate transport (LGlut) red cells characterized by an inherited remarkable decrease in glutamate transport activity. All LGluT dogs carried a missense mutation of Gly(492) to Ser (G492S) in either the heterozygous or homozygous state. The GLAST protein with G492S mutation was fully functional in glutamate transport in Xenopus oocytes. However, G492S GLAST exhibited a marked decrease in activity after the addition of cycloheximide, while the wild type showed no significant change, indicating that G492S GLAST was unstable compared with the wild-type transporter. Moreover, LGluT dogs, but not normal dogs, heterozygous for the G492S mutation showed a selective decrease in the accumulation of GLAST mRNA from the normal allele. Based on these findings, we conclude that a complicated heterologous combination of G492S mutation and some transcriptional defect contributes to the pathogenesis of the LGluT red cell phenotype.

Hypercholesterolemia in Shetland sheepdogs.

Plasma lipoprotein cholesterol in 64 clinically healthy Shetland sheepdogs was evaluated to assess whether the breed is more susceptible to hypercholesterolemia. The incidence of hypercholesterolemia was clearly higher in Shetland sheepdogs and mean plasma cholesterol level was significantly higher in Shetland sheepdogs than in control dogs. Blood biochemical examinations did not evidence the abnormalities, which imply the causative disorders, and thyroid hormone levels were not significantly different from the controls. These results suggest that the cholesterolemia is a primary disorder. Cholesterol fractionation by agarose gel electrophoresis and ultracentrifugation revealed that accumulation of alpha2-migrating lipoproteins was the common characteristic of dogs showing cholesterol level over 250 mg/dl in the breed. Increase in prebeta-beta-lipoproteins was also found in Shetland sheepdogs with marked hypercholesterolemia over 500 mg/dl. Therefore. Shetland sheepdogs may include more dogs with primary disorders in lipoprotein metabolism, which cause hypercholesterolemia. at least in Japan.

Biliary cirrhosis secondary to obstruction of the common bile duct by ectopic pancreas in a cow.

A 10-year-old Japanese Black cow showed icterus with gradual emaciation for a period of 3 months. Ultrasonography demonstrated increased "echogenicity" of the entire liver, with marked dilatation of the gall-bladder. At necropsy, the common bile duct was found to be obstructed by focally thickened fibrous tissue adjacent to a flesh-like mass (9 x 4 x 4 mm) attached to the wall of the duct. The mass consisted of normal pancreatic tissue. The liver showed moderate fibrosis, with nodular regeneration of the hepatic tissue and bile duct hyperplasia. A diagnosis of biliary cirrhosis secondary to ectopic pancreas in the common bile duct was made.

Efficacy of doramectin against intestinal nematodes and sarcoptic manage mites in naturally infected swine.

Endectocidal efficacy of doramectin administered intramuscularly at a dosage of 300 micrograms/kg was evaluated in 464 pigs naturally infected with intestinal nematodes or mange mites on 14 commercial farms in Japan. By doramectin treatment, fecal egg counts were reduced > 99% for Ascaris suum, Strongyloides ransomi, Oesophagostomum dentatum, and Trichuris suis; worm counts of T. suis and mite counts of Sarcoptes scabiei reduced 90.1% on Day 21 and 99.5% on Day 28 following treatment, respectively.

Non-functional pituitary chromophobe adenoma in a calf.

A 5-month-old male calf showed neurological signs, including circling, in-co-ordination, depression, blindness and eventual recumbency. On necropsy, a mass measuring 3.5 x 4.5 x 5 cm was found at the base of the brain, at the level of the pituitary gland. The hypothalamus and optic nerves were compressed by the dorsally expanding mass. Non-functional pituitary chromophobe adenoma was diagnosed from histochemical, immunohistochemical and electron microscopical findings.

Electrocardiographic features and incidence of atrial fibrillation in apparently healthy dairy cows.

Two hundred and eighty-five apparently healthy Holstein dairy cows were evaluated serially for atrial fibrillation (AF) over an 18-month period. AF was noted in 7 cows with an incidence of 2.5%. No particular disease conditions were noted in any case at the time of onset of AF and thereafter. In Cases 1, 2 and 3, arrhythmia developed 30 to 40 days postpartum and persisted thereafter. In Case 4, AF developed 28 days postpartum and lasted for 2 days before spontaneous conversion to normal sinus rhythm. In Cases 5 and 6, AF occurred 2 days after the animals ceased lactation and disappeared spontaneously within a few days. In Case 7, AF developed just before calving and reverted to normal sinus rhythm spontaneously on the 2nd day postpartum. Thus, 3 animals (Cases 1, 2 and 3) exhibited established AF whereas the remaining 4 animals (Cases 4, 5, 6 and 7) demonstrated paroxysmal AF, suggesting that different causative factors may be involved in the genesis of AF in the 7 cases presented here. The genesis of the AF in these cases which had no underlying disease conditions may be interpreted as an increased load imposed on the heart, increased sympathetic tone, or increased vagal tone.

Clinostomum complanatum (Trematoda) infection in freshwater fish from fish dealers in Tottori, Japan.

During the period January 1989 through February 1991, 327 freshwater fish of 3 different species (Carassius cuvieri, Cobitis anguillicaudatus and Hypomesus transpacificus) purchased from 7 fish dealers in Tottori City were investigated for the metacercarial infection of Clinostomum complanatum. Among them, 20.5% (16/78) of C. cuvieri were infected with this fluke. Each infected fish harbored 1 to 30 metacercariae. The sites and rates of parasitization of metacercariae in the infected fish were 42.4% in the body muscles, 30.3% in the muscles at the pectoral fin, 21.2% in the tissues around the gills and 6.1% in the cheek muscles.

Type C Niemann-Pick disease in a boxer dog.

Pathological and biochemical studies were performed on a 9-month-old boxer dog with progressive neurological abnormality. Histological examination revealed marked neuronal storage throughout the central nervous system and histiocytic storage in the reticuloendothelial system. Ultrastructurally, the neuronal storage consisted of accumulation of concentric membranous inclusions and clusters of dense bodies. The biochemically unesterified cholesterol content was high in the liver and spleen. The brain showed increased levels of lactosylceramide and two gangliosides, GM3 and GM2. These findings indicate that this dog was affected with a heterogeneous lipid storage disease similar to the human Niemann-Pick type C disease.

Clinostomum complanatum (Trematoda: Clinostomatidae) in five new fish hosts in Japan.

Metacercariae of Clinostomum complanatum were found in six species of natural freshwater fishes collected at Koyama Pond in Tottori City of Tottori Prefecture, Japan. Five of these fish species are reported here as new records for second intermediate hosts of C. complanatum in Japan: the silver crucian carp (Carassius gibelio langsdorfi), the deepbodied crucian carp (Carassius cuvieri), the carp (Cyprinus carpio), the topmouth gudgeon (Pseudorasbora parva) and the rose bitterling (Rhodeus ocelatus).

Natural infections of Clinostomum complanatum (Trematoda: Clinostomatidae) in wild herons and egrets, Tottori Prefecture, Japan.

Nycticorax nycticorax, Ardea cinerea, Egretta garzetta, and Egretta intermedia were naturally infected with Clinostomum complanatum (Trematoda: Clinostomatidae) among fourteen wild herons, seven wild egrets and one wild bittern evaluated at the Veterinary Hospital of Tottori University, Tottori, Japan. The latter three species of heron and egrets are reported for the first time as definitive hosts of this parasite in Japan.

Myocardial infarction secondary to a disseminated coagulopathy in a cow.

A 3-year-old Holstein cow was presented with a history of high fever, jaundice and subsequent recumbency. The animal died 3 hours after arrival at our department; continuous electrocardiographic examination was performed during those last 3 hours. There were ventricular premature beats (VPBs) with high frequency and complex patterns (bigeminal, trigeminal, repetitive or early cycle VPBs) and paroxysms of ventricular tachycardia (VT). Serum CK-MB was markedly elevated. On histopathological examination, large areas of myocardial necrosis were found in the anterior aspects of the left ventricle. Large, partially organized thrombi occluded the intramural coronary arteries within and adjacent to the lesion. From the histopathological findings, it appeared that a disseminated coagulopathy caused the thromboembolism in the intramural coronary arteries, resulting in acute myocardial infarction (AMI). Various clinical findings, such as arrhythmias, were similar to those in man.

Incorporation of nitrogen-15 from dietary [15N] diammonium citrate into amino acids of liver and muscle proteins in germfree and specific-pathogen-free neonatal pigs.

Incorporation of 15N from dietary [15N]diammonium citrate ([15N]DAC) into amino acids isolated from hydrolyzed tissue proteins was investigated in the presence or absence of intestinal flora in neonatal pigs. The 15N was incorporated into all of the essential and nonessential amino acids from liver and muscles in one of two germfree pigs and two specific-pathogen-free pigs which were killed on the 5th day after the administration of [15N]DAC. But, a higher 15N concentration than natural abundance of 15N was not detected in histidine, lysine, and threonine from these tissues in another germfree pig killed the next day. Nitrogen transfer from DAC into all amino acids, including these three essential amino acids, may be possible in the specific-pathogen-free pig and even in the germfree pig.