Shigeyuki Tanabe

Effects of extracellular Ca2+ on phagocytosis and intracellular Ca2+ concentrations in polymorphonuclear leukocytes of postpartum dairy cows

The aim of this study was to determine the effects of extracellular Ca(2+) concentration ([Ca(2+)](e)) on phagocytosis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine polymorphonuclear leukocytes (PMNs). The experiments were performed by using blood samples from parturient paretic and clinically normal parturient cows and manipulating the [Ca(2+)](e) in vitro. Phagocytosis by PMNs (with and without stimulation with phorbol myristate acetate and inhibition with cytochalasin B) and resting [Ca(2+)](i) were significantly lower in parturient paretic cows. Repletion of Ca(2+) in the extracellular media for the samples from these animals increased phagocytosis and resting [Ca(2+)](i). In the blood of clinically normal parturient cows, decreasing the [Ca(2+)](e) decreased phagocytosis and resting [Ca(2+)](i) in PMNs, but increasing the [Ca(2+)](e) did not affect phagocytosis. These results suggest that the hypocalcemic condition of parturient paretic cows in vivo causes decreased phagocytosis and resting [Ca(2+)](i) in PMNs, which may partly contribute to greater susceptibility to infection.

Expression of mRNA of chemokine receptor CXCR4 in feline mammary adenocarcinoma.

The expression of MRNA of the chemokine receptor CXCR4 in 65 surgically resected mammary adenocarcinomas from cats was investigated by in situ hybridisation. No expression of the receptors mRNA was detectable in the mammary tissue of healthy cats, but it was expressed in areas adjacent to necrosis, surrounding blood vessels and cells infiltrating the lymphatics of 47 (72.3 per cent) of the 65 samples. There was a significant relationship between lymphatic infiltration by neoplastic cells and the expression of the receptors MRNA (P<0.005), but there was no significant relationship between its expression and the one-year survival of the cats.

IDENTIFICATION AND THE ROLE OF SOLUBLE ANTIGENS DETECTED IN BILE FROM EIMERIA STIEDAI-INFECTED RABBITS

Antibodies against Eimeria stiedai sporozoites and merozoites were detected in the sera of rabbits immunized with bile obtained from infected rabbits on the 15th day post-infection. The trails made by gliding sporozoites were also detected by the sera. After penetration into the host cell, an antibody-binding region was observed on the parasitophorous vacuole membranes of the parasites. Rabbits administered a combination of the bile and cholera toxin shed fewer oocysts in the feces after infection than control rabbits. The immunized rabbits developed a high level of IgA antibody against soluble antigens in the bile. By immunoblotting, antigens with molecular masses of 32, 37, and 49 kDa were detected in the bile obtained from infected rabbits on the 15th day postinfection. Absorption treatment with sporozoites reduced or abolished the antibody reactivity to the 32-kDa antigen of merozoites and the bile antigens. However, antibody reactivity to the 37- and 49-kDa antigens still remained. These results indicate that soluble antigens are present in the bile of rabbits in the acute phase of infection, and these may be produced and released by merozoites during the host cell invasion process.

Molecular cloning of the canine nicotinic acetylcholine receptor α-subunit gene and development of the ELISA method to diagnose myasthenia gravis

We investigated the molecular structure of canine nicotinic acetylcholine receptor (AChR) alpha-subunit gene and developed an enzyme-linked immunosorbent assay (ELISA) as an immunological method to diagnose myasthenia gravis (MG). Canine AChR alpha-subunit cDNA was constructed from mRNA isolated from skeletal muscle of five dogs using the reverse transcriptase-polymerase chain reaction and its molecular structure was determined. The canine AChR alpha-subunit gene had 1371 base pairs encoding 457 amino acids and had a 96.1% homology to the human AChR alpha-subunit gene at the amino acid level. From the results of sequencing the DNA, specific antibodies to the acetylcholine binding domain of the canine AChR alpha-subunit were produced by immunizing rabbits with synthetic oligopeptides (alpha-subunit 183-200 amino acids). The specificity of the rabbit anti-oligopeptide serum was examined by Western blot analysis using an E. coli-expressed AChR alpha-subunit protein and an AChR alpha-subunit protein fraction prepared from canine skeletal muscle as an antigen. An ELISA assay was developed using oligopeptides corresponding to the binding domain to diagnose canine MG; specific antibodies were detected from two dogs with MG, one diabetic dog and two healthy dogs among 25 dogs examined. Further examinations of the ELISA using a large number of samples of clinically MG-positive and MG-negative dogs are needed to establish its usefulness in MG diagnosis.

Trail antigen in Eimeria stiedai sporozoites associated with a thrombospondin-related motif and the entry of cultured cells.

In order to examine the antigenic similarity and specificity of the trail antigen of Eimeria stiedai and Etp 100, a microneme protein of Eimeria tenella, monoclonal antibodies to the trail antigen of E. stiedai sporozoites were selected by an indirect immunofluorescent antibody method. The monoclonal antibody of one clone, 3D10, reacted with the anterior portion of non-fixed sporozoites. By immunoblotting, the monoclonal antibody was found to react with a 100 kDa antigen of E. stiedai sporozoites, and a 117 kDa antigen of E. tenella sporozoites and merozoites. It was also found to react with a recombinant protein with thrombospondin-/properdin-like motifs homologous to E. tenella microneme protein Etp 100. The monoclonal antibody significantly inhibited the penetration of E. stiedai sporozoites into cultured rabbit hepatobiliary epithelial cells. These results suggest that E. stiedai sporozoites have a trail antigen, located in the anterior region on the outer surface of the sporozoites, which has an epitope with thrombospondin-/properdin-like motifs similar to E. tenella microneme protein Etp 100. This protein may play an important functional role in the process of penetration of host cells.

Comparison of nonmetal and metal hydrophilic photosensitizer, ATX-S10 (Na) and ATN-2, binding with human serum proteins using spectrophotometry.

Intermolecular interactions of human serum proteins with a hydrophilic nonmetalloporphyrin, 13,17‐bis(1‐carboxy‐propionyl)carbomoylethyl‐8‐ethenyl‐2‐hydroxy‐3‐hydroxy‐iminoethylidene‐2,7,12,18‐tetramethylporphyrin sodium salt (ATX‐S10 (Na)), or a hydrophilic gallium‐metalloporphyrin, diethylenetriamine pentaacetic acid ester of 2‐[1‐(2‐hydroxyethoxy)ethyl]‐4‐vinyl‐deuteroporphyrin (IX) Ga complex (ATN‐2), were investigated using spectrophotometry. ATX‐S10 (Na) caused a bathochromic shift with albumin, high‐density lipoprotein and low‐density lipoprotein, but little or no shift was observed with hemopexin, transferrin and immuno‐globulin G. In contrast, ATN‐2 displayed a bathochromic shift only with hemopexin. These results suggest that the association energy of ATX‐S10 (Na) with albumin might be slightly greater than that with lipoproteins and that of ATN‐2 with hemopexin might be greater than that with other serum proteins.

Expression of cyclooxygenase-2 in dogs with cystitis

S. Azuma, DVM, Department of Veterinary Clinical Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan S. Tanabe, DVM, PhD, Y. Uzuka, DVM, PhD, T. Sarashina, DVM, PhD, Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Science, Y. Kobayashi, DVM, PhD, H. Furuoka, DVM, PhD, T. Matsui, DVM, PhD, Laboratory of Veterinary Pathology, Department of Pathobiological Science, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido 080-8555, Japan T. Oomachi, DVM, PhD, Patholab, 7-333-12-405 Sagamidai, Sagamihara, Kanagawa 228-0821, Japan