Production and characterization of monoclonal antibodies that recognize bovine Kit receptor.

Kit receptor is a transmembrane tyrosine kinase that is the receptor for stem cell factor (SCF). The extracellular domain of bovine Kit receptor (boKit) was produced by a baculovirus expression system. Six monoclonal antibody (MAb) clones designated as bK-1 to bK-6 were obtained upon immunization of mice with the recombinant protein. Immunoprecipitation and flow cytometric analysis indicated that all of the MAbs specifically bound to boKit expressed in COS-7 cells transfected with boKit cDNA. Four of the six MAbs neutralized the biological activity of recombinant bovine SCF, whereas the other two did not. The boKit-positive and boKit-negative cell fractions were sorted from cryopreserved bovine bone marrow cells by the use of MAb bK-1. Colony formation assays indicated that the cells which were able to grow in response to bovine SCF were enriched in the boKit-positive fraction. These MAbs would be valuable in studying possible boKit-positive cell species such as bovine hematopoietic cells, and in defining the biological role of Kit receptor in cattle.     

Clinicopathological study of canine oral epulides.

To clarify the clinicopathological features of canine epulides, 189 epulides were reviewed retrospectively. The incidence of the fibromatous, ossifying, acanthomatous and giant cell epulides were 56.6% (107/189), 23.3% (44/189), 18.0% (34/189) and 2.1% (4/189), respectively. The average ages of dogs with fibromatous, ossifying, acanthomatous and giant cell epulides were 8.8, 8.4, 7.8 and 8.7 years, respectively. The male/female ratio of dogs with the acanthomatous epulis (0.8) was lower than those of dogs with the fibromatous (1.9), ossifying (1.4) and giant cell epulis (3.0). There were slight breed differences among the types of epulides. The most noticeable result was that 38.2% of the acanthomatous epulis occurred in Shetland sheepdogs. 43.9% of the fibromatous epulis and 52% of the ossifying epulides arose around maxillary premolars, while 58.8% of the acanthomatous epulis arose around the mandibular canines. Dogs with the fibromatous and ossifying epulides had more severe dental plaque deposition than those with the acanthomatous epulides. Few of the fibromatous (6/104) or ossifying epulides (4/44) showed recurrence after excision, while the majority (21/23) of the acanthomatous epulides showed rapid and repeated recurrences after surgical excision. Epulides treated with hemimandibulectomy or bleomycin chemotherapy did not recur. Giant cell epulides showed no recurrence after surgical removal. These results indicate that the acanthomatous epulis differed from other types of epulides in biological and morphological features and poor prognosis.     

Estimation of the shear strength of wood by uniaxial-tension tests of off-axis specimens

To determine shear strength we conducted uniaxial-tension tests of off-axis specimens and examined the proper off-axis angles. Sitka spruce (Picea sitchensis Carr.) and katsura (Cercidiphyllum japonicum Sieb. and Zucc.) were used for the studies. Uniaxial tension tests of the specimens with various off-axis angles were conducted, and the shear stress at failure was obtained. Independent of the tension tests, torsion tests were conducted, and the shear strengths were obtained. Comparing the data of the uniaxial tension and torsion tests, we examined the validity of estimating shear strength by the off-axis tension test. The shear strengths obtained from the tension tests coincided well with those measured by the torsion tests when the specimen had an off-axis angle of 15°–30°. In this off-axis angle range, the tensile stress perpendicular to the grain might have a serious influence on the shear strength, and we thought that the shear strength predicted by uniaxial tension tests should be treated as an approximate value despite the simplicity of the tension test. Other test methods should be adopted to obtain the precise shear strength of wood.     

Efficacy of colostral immunoglobulins for therapeutic and preventive treatments of calf diarrhea.

Pooled colostral milk samples were collected from apparently healthy Holstein cows. Immunoglobulin (Ig) was partially purified from the colostral milk and concentrated by freeze drying. The Ig powders had neutralizing antibody titers of 1:6,250 to 1:31,250 against bovine rotavirus (RV), and reacted with four RV proteins of VP2, VP4, VP6, and VP7. No therapeutic effect of Ig powder showed on clinically serious diarrhea with RV in dairy and beef calves. However, the Ig powder had a preventive effect on the diarrhea in beef calves when it was given soon after calving.     

Development of immune assay system for both CpG and non-CpG DNA from lactic acid bacteria using a transfectant of swine Toll-like receptor 9

Bacterial DNA is expected to be a potent immune stimulating agent to Toll‐like receptor (TLR) 9 expressed cells such as macrophages, monocytes, B lymphocytes, NK cells and dendritic cells. In the present study, we constructed a transfectant of swine TLR9 with mammalian cells. We demonstrated that the transfectant, recognizing both CpG and non‐CpG oligonucleotides from lactic acid bacteria, induced NF‐κB activation by gene reporter assay. These findings indicate that the swine TLR9 transfectant will be highly useful for the screening of immunostimulatory DNA from lactic acid bacteria.     

Long-term preservation of mouse spermatozoa as frozen testicular sections

We previously demonstrated that testicular spermatozoa can be preserved as frozen testicular sections, allowing us to preserve male gametes in less space than conventional methods. However, it remains unclear whether the testicular spermatozoa can be preserved for a long period using this procedure. In this study, we examined the function of testicular spermatozoa preserved as frozen testicular sections for l year at -30 or -80 C. Testicular spermatozoa were successfully retrieved from frozen testicular sections preserved at either -30 or -80 C, and their function was assessed using intracytoplasmic sperm injection (ICSI). Over 90% of the oocytes injected with long-term preserved testicular spermatozoa formed pronuclei, which was a frequency similar to that obtained with spermatozoa preserved for a short term, indicating that the testicular spermatozoa retained oocyte activation factor(s). Approximately 70% of the fertilized oocytes developed to 2-cell stage embryos, and 9.3 to 12.8% of the embryos developed to term after transfer into pseudopregnant females, regardless of the preservation temperatures examined. These results indicate that the birthrates of progeny did not differ between the preservation temperatures examined. They also indicate that male gametes can be preserved in testicular frozen sections for at least 1 year without loss of function.     

Molecular cloning and expression profiling of procollagen α1 (I) of cultured Pacific bluefin tuna

Type I collagen is widely distributed in most organs in teleosts. It plays a role not only in intercellular adhesion, but also in molecular signaling. In this study, Pacific bluefin tuna (PBT) procollagen α1 (I) cDNA was cloned and characterized. The nine fragments of a procollagen α1 (I) chain cDNA clone were prepared and spliced together to create the complete coding region. The resulting amino acid sequence was homologous with that of other teleosts. The mRNA expression profile of PBT procollagen α1 (I) in various tissues and the phylogenetic analysis with other vertebrate procollagen α1 (I) chains suggest that PBT procollagen α1 (I) could be a precursor form of the PBT type I collagen α1 chain. In addition, its level of expression in PBT larvae and early juveniles gradually increased with somatic growth. This increase was related to the standard length, wet body weight, and protein content of each individual fish. Therefore, the expression profile of procollagen α1 (I) may be a useful indicator for somatic growth in fish larvae and juveniles.     

Mapping the relative risk of surface water acidification based on cumulative acid deposition over the past 25 years in Japan

Sensitivity maps of atmospheric acid deposition in Japan have not been updated in 20 years. Here, we propose new relative risk maps of surface water acidification in forests based on a weighted overlay of cumulative potential acid deposition (CPAD) simulated for a 25-year period (1981–2005), including the sensitivities of soil and bedrock to acidification. We assumed that relative acidification risk is high in areas that exhibit high sensitivities of soil and bedrock to acid and have received a large amount of cumulative acid deposition over the past several decades. We aggregated fine soil and bedrock maps into a 20-km mesh for overlay onto an 80-km mesh map of CPAD by considering their spatial structures in Japan. Allocation of the weights among CPAD and soil and bedrock sensitivities was performed based on observational trends in river pH over the past 30 years. The resulting risk map for surface water acidification showed that large areas of western Japan, as well as smaller areas of Hokkaido, Tohoku, Kanto, and Kyushu, are at high risk of surface water acidification. Seventy-seven percent of all rivers for which a declining trend in pH was observed from 2001 to 2009 were also high-risk areas. Acid deposition might be one factor controlling surface water acidification in areas with high bedrock sensitivity, in addition to high CPAD and soil sensitivity, although the risk of soil acidification remains unclear.     

Quality improvement of the thermal gel of horse mackerel by sodium citrate and its inhibition of the insoluble metalloproteinase

Modori-inducing proteinases (MIPs) in horse mackerel and the effect of sodium citrate on the modori phenomenon were investigated. Cysteine protease caused the modori phenomenon only at 60 ℃, while metalloproteinase caused it at both 60 ℃ and 70 ℃. Moreover, the autolysis of myofibrillar proteins was mainly inhibited by ethylene diamine tetra-acetic acid (EDTA), verifying the existence of insoluble metalloproteinase. From the results comparing thermal gel properties and protease activities, we confirmed that increased activity of insoluble metalloproteinase corresponded to the weakening of the thermal gel. Thus, insoluble metalloproteinase was the major MIP in horse mackerel. Moreover, sodium citrate could markedly inhibit insoluble metalloproteinase activity at 60 ℃. The addition of sodium citrate significantly improved the breaking force of the thermal gel at 60 ℃ and 70 ℃, suggesting that sodium citrate could suppress the modori phenomenon by inhibiting insoluble metalloproteinase. Therefore, it is desirable to use sodium citrate in horse mackerel surimi-based products to prevent the modori phenomenon.

     

Reliable enzyme-linked immunosorbent assay for the determination of walnut proteins in processed foods

Among food allergens of tree nuts, walnuts are a frequent cause of adverse food reactions in allergic patients. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of walnut soluble proteins in processed foods was developed. The sandwich ELISA is highly specific for walnut soluble proteins. The recovery ranged from 83.4 to 123%, whereas the intra- and interassay coefficients of variation were less than 8.8 and 7.2%, respectively. This study showed that the proposed method is a reliable tool for detection in the presence of hidden walnut proteins in processed foods.