Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.     

Osamu Yamato  Hiroyuki Satoh  Naoaki Matsuki  Kenichiro Ono  Masahiro Yamasaki  Yoshimitsu Maede 《Journal of veterinary diagnostic investigation》2004,16(1):39-44

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.  相似文献   

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni     

Yamasaki M  Asano H  Otsuka Y  Yamato O  Tajima M  Maede Y 《American journal of veterinary research》2000,61(12):1520-1524

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.  相似文献   

GM1 gangliosidosis in shiba dogs     

Yamato O  Ochiai K  Masuoka Y  Hayashida E  Tajima M  Omae S  Iijima M  Umemura T  Maede Y 《The Veterinary record》2000,146(17):493-496

A six-month-old shiba dog with a one-month history of progressive motor dysfunction showed clinical signs of a cerebellar disorder, including ataxia, dysmetria and intention tremor of the head. Histopathological and ultrastructural studies revealed distended neurons packed with membranous cytoplasmic bodies throughout the central nervous system. The activities of lysosomal acid beta-galactosidase in its leucocytes and liver were less than 2 per cent of the control levels, and the compound accumulated in the brain was identified as GM1 ganglioside. A sibling which died immediately after birth was shown to have a beta-galactosidase deficiency in the brain and visceral organs. A family study revealed that the sire and dam of the probands were heterozygotes with approximately half of the normal level of beta-galactosidase activity, suggesting an autosomal recessive pattern of inheritance.  相似文献   

Rapid and simple mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.     

Osamu Yamato  Eun-Og Jo  Toru Shoda  Masahiro Yamasaki  Yoshimitsu Maede 《Journal of veterinary diagnostic investigation》2004,16(5):469-472

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.  相似文献   

A Practical Technique for Electron Microscopy of Buffy Coats in Dogs and Cats          下载免费PDF全文

A. Yabuki  M. Sawa  H.‐S. Chang  O. Yamato 《Anatomia, histologia, embryologia》2015,44(4):317-320

Plastic hematocrit tubes (PHTs) are convenient tools for electron microscopy (EM) of peripheral blood buffy coats, and the PHT‐EM technique is expected to be a practical method for veterinary clinical medicine. In this study, fixatives composed of various concentrations of sucrose, glutaraldehyde, and phosphate buffer (PB) were tested for preparing canine and feline buffy coats. The highest quality images were obtained using a fixative consisting of 2.5% glutaraldehyde in 0.1 m PB, and it was concluded that this method allows clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM.  相似文献   

Development and application of multiple immunofluorescence staining for diagnostic cytology of canine and feline lymphoma          下载免费PDF全文

Mariko Sawa  Akira Yabuki  Asuka Setoguchi  Osamu Yamato 《Veterinary clinical pathology / American Society for Veterinary Clinical Pathology》2015,44(4):580-585

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Ectomycorrhizal fungal communities of Dipterocarpus alatus seedlings introduced by soil inocula from a natural forest and a plantation     

Tharnrat Kaewgrajang  Uthaiwan Sangwanit  Motoichiro Kodama  Masahide Yamato 《Journal of Forest Research》2014,19(2):260-267

We attempted to introduce ectomycorrhizal (ECM) fungi onto seedlings of Dipterocarpus alatus (Dipterocarpaceae) by soil inocula collected from tree stands of D. alatus. Top soils collected from beneath the trees of D. alatus in a dry evergreen forest and a 15-year-old plantation were inoculated onto germinating seeds of D. alatus. After 7 months of seedling cultivation, ECM fungal communities in the seedlings were investigated based on the sequences of ITS rDNA. The ECM fungi detected were divided into 19 phylotypes by molecular analysis. Most of the phylotypes were identified as ECM fungal taxa, i.e., Clavulina, Laccaria, Lactarius, Tomentella, Pyronemataceae, and Tricholomataceae. Accordingly, we can confirm that soil inoculation is a simple method to induce ECM formation with diverse fungi in pot cultured seedlings, which would be useful for introducing diverse ECM fungi to dipterocarp plantations.  相似文献   

Structure of the rotor of the V-Type Na+-ATPase from Enterococcus hirae     

Murata T  Yamato I  Kakinuma Y  Leslie AG  Walker JE 《Science (New York, N.Y.)》2005,308(5722):654-659

The membrane rotor ring from the vacuolar-type (V-type) sodium ion-pumping adenosine triphosphatase (Na+-ATPase) from Enterococcus hirae consists of 10 NtpK subunits, which are homologs of the 16-kilodalton and 8-kilodalton proteolipids found in other V-ATPases and in F1Fo- or F-ATPases, respectively. Each NtpK subunit has four transmembrane alpha helices, with a sodium ion bound between helices 2 and 4 at a site buried deeply in the membrane that includes the essential residue glutamate-139. This site is probably connected to the membrane surface by two half-channels in subunit NtpI, against which the ring rotates. Symmetry mismatch between the rotor and catalytic domains appears to be an intrinsic feature of both V- and F-ATPases.  相似文献   

Magnetic resonance imaging of GM2-gangliosidosis in a golden retriever     

Matsuki N  Yamato O  Kusuda M  Maede Y  Tsujimoto H  Ono K 《The Canadian veterinary journal. La revue veterinaire canadienne》2005,46(3):275-278

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Inhibitory effect of pyrimidine and purine nucleotides on the multiplication of Babesia gibsoni: possible cause of low parasitemia and simultaneous reticulocytosis in canine babesiosis     

Hossain MA  Yamato O  Yamasaki M  Maede Y 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2004,66(4):389-395

The present study was conducted to determine the cause of low parasitemia and simultaneous reticulocytosis in canine babesiosis. The parasitemia was significantly decreased in in vitro cultures of Babesia gibsoni by the pretreatment of host canine erythrocytes with lead acetate, which is a specific inhibitor of pyrimidine 5'-nucleotidase subclass I (P5N-I). The serum from dogs chronically infected with B. gibsoni did not decrease the activities of hexokinase, glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase in canine reticulocytes, although it was previously reported that this serum had inhibitory effects on both the maturation of reticulocytes and the canine P5N-I and purine-specific 5'-nucleotidase activities. Furthermore, the in vitro multiplication of B. gibsoni was significantly inhibited by pyrimidine nucleotides such as cytidine 5'-monophosphate (5'-CMP), which is preferentially catalyzed by P5N-I and also inhibits the morphological maturation of canine reticulocytes. Purine nucleotides such as inosine 5'-monophosphate (5'-IMP) also had an inhibitory effect on the multiplication of this parasite. These results suggest that nucleotides such as 5'-CMP and 5'-IMP might accumulate in young erythrocytes and/or serum in dogs infected with B. gibsoni as a result of the decreased activity of erythrocyte 5'-nucleotidase, and the accumulation of these nucleotides might inhibit the multiplication of this parasite and simultaneously retard the maturation of reticulocytes. The results obtained from the in vitro examinations in the present study may partially clarify the relationship between low parasitemia and simultaneous reticulocytosis in vivo in canine babesiosis.  相似文献