Oxidative damage to the membrane of canine erythrocytes with inherited high Na, K-ATPase activity.

Oxidative damage to the membrane in canine erythrocytes with inherited high Na, K-ATPase activity (HK cells) was compared with that in normal canine cells (LK cells). When 30 mM beta-acetylphenylhydrazine (APH) was applied to HK and LK cells, lipid peroxidation and hemoglobin denaturation occurred. Lipid peroxidation determined from malondialdehyde (MDA) formation was significantly lower in HK than in LK cells so far as endogenous glutathione (GSH) concentration was maintained at appropriate levels. With the depletion of GSH, MDA formation was accelerated and difference between HK and LK cells was not significant. Denatured hemoglobin bound to the membrane protein was less in HK than in LK cells. During incubation with APH, osmotic fragility increased markedly in LK cells, while HK cells showed very little change. The amounts of total lipid, total and free cholesterol, glycolipid, phospholipid and fatty acids were essentially the same in both cell types. Fatty acid compositions showed very small differences. The membrane of HK cells thus appear to have greater protection against oxidative damage induced by APH, owing to the presence of excess GSH in HK cells. The capability of HK cells to withstand oxidative damage would not be due to differences in membrane lipid compositions.     

Tetracycline-resistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema

The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined. The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema. Five (22%) of 23 C. septicum strains, five (71%) of seven C. perfringens strains and all strains of C. sordellii showed resistance to oxytetracycline. These oxytetracycline-resistant strains carried tetracycline-resistance genes [tetA(P), tetA408(P), tetB(P) and tetM]. The sequences of the tetracycline-resistance genes of some C. septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species. This is the first report of resistance of C. septicum to tetracycline.     

Quantitative assessment of muscle mass and gene expression analysis in dogs with glucocorticoid-induced muscle atrophy

The present study aimed to quantitatively evaluate muscle mass and gene expression in dogs with glucocorticoid-induced muscle atrophy. Five healthy beagles received oral prednisolone for 4 weeks (1 mg/kg/day), and muscle mass was then evaluated via computed tomography. Histological and gene expression analyses were performed using biopsy samples from the biceps femoris before and after prednisolone administration. The cross-sectional area of the third lumbar paraspinal and mid-femoral muscles significantly decreased after glucocorticoid administration (from 27.5 ± 1.9 to 22.6 ± 2.0 cm² and from 55.1 ± 4.7 to 50.7 ± 4.1 cm², respectively; P<0.01). The fast- and slow-twitch muscle fibers were both atrophied (from 2,779 ± 369 to 1,581 ± 207 μm² and from 2,871 ± 211 to 1,971 ± 169 μm², respectively; P<0.05). The expression of the growth factor receptor-bound protein 10 (GRB10) significantly increased after prednisolone administration (P<0.05). Because GRB10 suppresses insulin signaling and the subsequent mammalian target of rapamycin complex 1 activity, increased expression of GRB10 may have resulted in a decrease in protein anabolism. Taken together, 1 mg/kg/day oral prednisolone for 4 weeks induced significant muscle atrophy in dogs, and GRB10 might participate in the pathology of glucocorticoid-induced muscle atrophy in canines.     

Production of transgenic rabbits using centrifuged pronuclear zygotes

Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.     

Comparison of the blood coagulation profiles of ferrets and rats

The aim of this study was to examine the blood coagulation profiles of ferrets and compare them with those of rats. The ferret activated partial thromboplastin time (aPTT) was slightly longer than the rat aPTT. In contrast, the ferret prothrombin time and thrombin time were profoundly shorter than the corresponding rat values. The fibrinogen level in ferret plasma was 2 times higher than that in rats. Heparin prolonged all blood coagulation times in a concentration-dependent manner in both ferret and rat plasma. A significantly (P<0.01) higher concentration of heparin was required to double the aPTT in ferrets than rats. These blood coagulation data for ferrets will be useful in experimental animal studies.     

Detecting endotoxin activity in bovine serum using an automated testing system

The aim of the present study was to compare the ability of the commercially available portable test system (PTS^TM) to detect endotoxin activity in bovine serum, with that of the traditional LAL-kinetic turbidimetric (KT) and chromogenic (KC) assays. Prior to testing, serum samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS^TM was not significantly different from that of the traditional LAL-based assays. The results using PTS^TM correlated with those using KT (r²=0.963, P<0.001) or KC assays (r²=0.982, P<0.001). Based on these findings, the PTS^TM could be applied as a simplified system to assess endotoxin activity in bovine serum.     

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.     

Electrocorticographic and histological findings in a Shetland sheepdog with intractable epilepsy

A Shetland sheepdog with epilepsy refractory to antiepileptic drugs was brought to the division of Veterinary Radiology at Nippon Veterinary and Animal Science University. Scalp electroencephalography and computed tomography was performed, but no abnormality was detected in either examination. To obtain detailed information, electrodes were implanted on the dura mater, and the electrocorticogram (ECoG) was recorded. In the ECoG, sporadic spikes were detected in the left parietal region, suggesting the presence of the epileptic focus in this region. After the dog's death, abnormalities of gyri were found in the region where spikes were detected in the ECoG. On histopathological examination, laminar malacia of the cingulate gyrus was observed. Furthermore, in the hippocampus, neuronal loss of pyramidal cells was observed.