Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Surgery of hepatic cysts in a cat.

A 2-year-old female Japanese domestic cat weighing 3.6 kg was presented due to marked abdominal distention and was diagnosed as hepatic cyst. The bile acid concentration of the hepatic cyst was as low as the serum bile acid concentration. The membrane of the cysts was excised from the edge of the compressed hepatic tissue and no other surgical manipulation was performed. This surgical procedure is simple and quick compared to hepatic lobectomy, however, it is necessary to evaluate the fluid of the cyst before performing this procedure.     

Whole-Cell Fatty Acid Composition to Characterize and Differentiate Isolates of Rhizoctonia Species Associated with Turfgrass Diseases in Japan

Cellular fatty acids were analyzed to characterize and differentiate 34 isolates of Rhizoctonia species representing binucleate Rhizoctonia AG-D (I), AG-D (II), R. solani AG 2-2 IIIB, AG 2-2 LP, R. circinata var. circinata and var. oryzae associated with turfgrass diseases in Japan. Myristic, pentadecanoic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids were consistently present in varying quantities in all isolates. Heptadecanoic and 9-heptadecenoic acids were present in isolates of Rhizoctonia AG-D (I), AG-D (II), R. solani AG 2-2 IIIB and AG 2-2 LP but not in isolates of R. circinata var. circinata and var. oryzae. Palmitic, oleic and linoleic acids were the major fatty acids found, constituting 88.30-98.37% of the whole-cell fatty acid content. The remaining fatty acids were present in smaller amounts. Isolates within a single group were closely clustered, whereas isolates from different groups were clearly distinguishable based on average linkage cluster analysis of cellular fatty acids. Principal component analysis, based on all fatty acids detected, confirmed the distinct separation of isolates representing the six groups of Rhizoctonia species obtained from turfgrasses. These results suggested that fatty acid analysis is useful for the characterization and differentiation of isolates of Rhizoctonia species associated with turfgrass diseases. Received 21 May 2001/ Accepted in revised form 28 September 2001     

MCPB-ethyl疏花对富士苹果授粉受精及胚珠发育的影响

通过在花期用MCPB-ethyl处理,对富士苹果花粉的发芽、花粉管的伸长以及胚珠的发育等进行了形态方面的观察和探讨,以阐明MCPB-ethyl的疏花机制。结果表明,MCPB-ethyl对花粉的发芽及花粉管的伸长没有影响,整个受精过程与对照相同,没有发现异常。但受精后胚乳核只进行了数次分裂便停止生长,此后珠皮、珠心细胞迅速解体。根据以上结果,认为MCPB-ethyl的疏花效果不是通过影响花粉的发芽或花粉管的伸长阻碍受精所致,而是使胚和胚珠的发育停止,形成离层导致了落花。     

Effects of cyclodextrin-iodopropane complex on methane production, ruminal fermentation and microbes, digestibility and blood metabolites in steers

The effects when adding cyclodextrin‐iodopropane complex (CD‐IP) to a diet, on ruminal fermentation and microbes, digestibility, blood metabolites and methane production, were evaluated using four Holstein steers in a cross‐over design. The steers were fed Sudangrass hay plus concentrate mixture at a ratio 1.5:1, and CD‐IP (1% of dry matter) was given twice daily by mixing with concentrate mixture. Rumen and blood samples were collected at 0, 2, and 5 h after morning dosing. Ruminal pH and numbers of protozoa were unaffected by CD‐IP treatment. Ruminal molar proportion of acetate was decreased (P < 0.05), and propionate was increased (P < 0.01) at 2 h after CD‐IP dosing. Proportion of butyrate was increased (P < 0.05) and ammonia‐N was decreased (P < 0.05) at 2 and 5 h after CD‐IP dosing. Adding CD‐IP had no effect on the feed intake and digestion of nutrients. Plasma glucose was increased and urea‐N was decreased (P < 0.05) at 2 and 5 h after CD‐IP dosing. Methane production was decreased (P < 0.05) by approximately 18% in the treatment steers. Numbers of methanogenic bacteria were decreased (P < 0.05), while total viable counts, cellulolytic, sulfate reducing and acetogenic bacteria were unaffected. The present results are the first to show that CD‐IP can partially inhibit in vivo ruminal methanogenesis without adverse effects on digestion of nutrients.     

Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Previously, the kinase inhibitor dasatinib was shown to have potent growth inhibitory activity against HS cells in vitro, possibly via targeting the EPHA2 receptor. Here, the in vivo effect of dasatinib in HS cells was investigated using a xenograft mouse model. Moreover, the expression status of EPHA2 was examined in six HS cell lines, ranging from insensitive to highly sensitive to dasatinib. In the HS xenograft mouse model, dasatinib significantly suppressed tumor growth, as illustrated by a decrease in mitotic and Ki67 indices and an increase in apoptotic index in tumor tissues. On Western blot analysis, EPHA2 was only weakly detected in all HS cell lines, regardless of sensitivity to dasatinib. Dasatinib likely results in the inhibition of xenograft tumor growth via a mechanism other than targeting EPHA2. The findings of this study suggest that dasatinib is a targeted therapy drug worthy of further exploration for the treatment of canine HS.     

Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin.     

Time-course comparison of pulmonary inflammation induced by intratracheal instillation of four different nickel oxide nanoparticles in male Fischer rats

Occupational exposure to nickel oxide (NiO) is an important cause of respiratory tract cancer. Toxicity is known to be associated with the dissociated component, i.e. nickel (II) ions. To address the relationship between physicochemical properties, including solubility in artificial lysosomal fluid, of NiO and time-course changes in the pulmonary response, we conducted an intratracheal instillation study in male Fischer rats using four different well-characterized NiO products, US3352 (NiO A), NovaWireNi01 (NiO B), I small particle (NiO C), and 637130 (NiO D). The NiOs were suspended in purified water and instilled once intratracheally into male F344 rats (12 weeks old) at 0 (vehicle control), 0.67, 2, and 6 mg/kg body weight. The animals were euthanized on days 3, 28, or 91 after instillation, and blood analysis, bronchoalveolar lavage fluid (BALF) testing, and histopathological examination were performed. The most soluble product, NiO B, caused the most severe systemic toxicity, leading to a high mortality rate, but the response was transient and surviving animals recovered. The second-most-soluble material, NiO D, and the third, NiO A, caused evident pulmonary inflammation, and the responses persisted for at least 91 days with collagen proliferation. In contrast, NiO C induced barely detectable inflammation in the BALF examination, and no marked changes were noted on histopathology. These results indicate that the early phase toxic potential of NiO products, but not the persistence of pulmonary inflammation, is associated with their solubility.     

Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.