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

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.     

Role of NF-kappaB in constitutive expression of MAIL in epidermal keratinocytes

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein that is also known as interleukin-1-inducible nuclear ankyrin repeat protein and inhibitor of nuclear factor kappaBzeta (IkappaBzeta). We previously observed that MAIL-deficient mice were affected by atopic dermatitis-like skin lesions and demonstrated the importance of MAIL in the skin. In this study, we investigated MAIL expression in mouse keratinocytes. MAIL mRNA was constitutively expressed in the skin epidermis. MAIL expression was also confirmed in primary keratinocytes and the PAM212 keratinocyte cell line. The inhibitors of nuclear factor kappaB (NF-kappaB)-Bay11-7082 and the IkappaBalphaM supersuppressor-considerably downregulated MAIL expression in the keratinocytes. Immunoreactivity for NF-kappaB components was localized in the cytoplasm and nucleus of normal unstimulated keratinocytes. The expression level of MAIL in the skin did not change following lipopolysaccharide (LPS) administration to mice. Interestingly, in accordance with the in vivo findings, the MAIL expression level did not change following LPS stimulation even in primary keratinocytes; however, MAIL expression was strongly increased by interleukin-1 stimulation. These results collectively suggest that the constitutive expression of MAIL in keratinocytes is controlled, at least in part, by NF-kappaB and that there may be LPS-specific repressive mechanisms that inhibit MAIL induction.     

Yield estimation of 1-year-old asparagus grown using rootstock-planting forcing culture

Rootstock-planting forcing culture was developed in asparagus to harvest spears even during the seasons when the plants become dormant, but the demand for them high. In this study, cumulative hours during which the air temperature remained lower than 5°C, i.e. chilling hours (CHs), were calculated to determine dormancy breakage for asparagus cultures. We also measured CIELab colour values for cut stems immediately before rootstock digging, and determined whether they could be substituted and/or compensated for CHs while evaluating asparagus plant productivity in different low-temperature backgrounds, and obtained regression equations for yield estimation. Asparagus seedlings were cultivated in seven different regions across Japan and brought to the study site for harvesting. Our regression equation based on CHs and rootstock weight for yield estimation had relatively high fitness (adjusted R² = 0.5795). The colour values of cut stalks at rootstock digging can also be used to evaluate their productivity. These values can be useful in regions where CHs cannot be determined, although their effectiveness was slightly lower than that of CHs of areas adjacent to the study sites.     

A Generalized Two-Dimensional Gaussian Model of Disease Foci of Head Blight of Wheat Caused by Gibberella zeae

ABSTRACT A generalized two-dimensional Gaussian model is proposed to describe disease foci of head blight of wheat in plots (100 to 2,500 m(2)) originating from small areas (1 to 16 m(2)) inoculated with Gibberella zeae-colonized corn kernels. These anisotropic, asymmetrical foci arose from ascospores produced in perithecia. The model is Z = exp[-(AX(2) + BY(2) + CXY + DX + EY + F)], in which Z = the incidence of seed or spikelet infection at point (X,Y) located in the plot, exp = the exponential function, X = the abscissa or spatial coordinate of the point along a given axis (approximately parallel to the average wind vector during the period of spore release in these experiments), Y = the ordinate or spatial coordinate of the point along the axis perpendicular to the X axis (approximately perpendicular to the wind direction in these experiments), A and B = the quadratic coefficients of the second-order polynomial AX(2) + BY(2) + CXY + DX + EY + F, C = the bilinear coefficient, D and E = the linear coefficients, and exp(-F) = the incidence of seed or spikelet infection at the focus peak in which X = 0 and Y = 0. The generalized two-dimensional Gaussian model was tested on data from a circular or isotropic focus, an elliptical or anisotropic focus with two axes of symmetry, and two anisotropic foci with one and zero axis of symmetry. Its goodness-of-fit (r(2) and adjusted r(2)) was compared with the inverse power, modified inverse power, exponential, and classical Gaussian models. Submodels using only the linear terms, only the quadratic terms, or combinations selected from stepwise regression procedures using various probabilities to enter and to stay and a procedure maximizing the adjusted r (2) were also considered. Spatial analysis of the residuals was performed using Geary's c coefficient at the first distance class. For the circular and elliptical foci, our model provided a fit similar to the modified inverse power and exponential models. However, for anisotropic foci with one or zero axis of symmetry arising from ascospores influenced by wind direction, the generalized two-dimensional Gaussian model provided a better fit. For these anisotropic foci, the linear term X but not the quadratic term X(2) was generally retained in the model, indicating an exponential gradient in the direction parallel to the wind. In all models, the quadratic term Y(2) was retained, along with Y in some cases, indicating that the gradient in the direction roughly perpendicular to the wind was Gaussian or Gaussian-exponential in shape. The bilinear term XY provided an indication of the orientation of the focus in relation to the axes of the sampling grid. This model has the versatility and parameters (quadratic, bilinear, and linear) to better describe the anisotropy of foci from wind-dispersed spores.     

Age-related changes of genital systems in the female Crj:CD (SD) IGS rats during sexual maturation

The age-related changes of vaginal opening, body weight, the weights of the uterus and ovary, together with histological examination, serum 17beta-estradiol (E2) and progesterone levels were examined in intact female Crj:CD (SD) IGS rats between 21 and 36 days of age to understand the basic biological profile of changes of the female genital system during sexual maturation in the rat for female pubertal assays. With the beginning of the elevation of serum E2 level from 28 days of age, all parameters except body weight started to show drastic change until 31 days of age. The highest incidence of vaginal opening was recorded at 34 days of age. On macroscopic examinations, a number of rats showed uterine imbibition but vaginal opening. Immediately after the confirmation of the vaginal opening, the genital systems of three rats were observed microscopically. Both ovaries already had multiple corpora lutea, and degeneration of endometrial epithelial cells was observed. In conclusion, we obtained essential data on genital tract development of female Crj:CD (SD) IGS rats for in vivo screening assays that will contribute to detect potential endocrine active chemicals. In addition, it is assumed that the first ovulation precedes or occurs simultaneously with vaginal opening.     

Post-exposure treatment of cats with mouse-cat chimeric antibodies against feline herpesvirus type 1 and feline calicivirus

In order to confirm the in vivo effectiveness of anti- feline herpesvirus type 1 (FHV-1) mouse-cat chimeric antibody (FJH2), and anti-feline calicivirus (FCV) mouse-cat chimeric antibody (F1D7), cats that had been experimentally infected with FHV-1 or FCV were administered intravenously with the chimeric antibodies, and observed for clinical manifestations. The symptoms due to FHV-1 or FCV infection in the cats administered FJH2 or F1D7 were obviously decreased when compared with those of the non-administered control cats. From these results, it was confirmed that both FJH2 and F1D7 were effective at reducing the appearance of symptoms due to FHV-1 and FCV infection, respectively.     

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

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.