Overexpression of the p53 gene product in canine mammary tumors.

p53, a tumor suppressor gene, is a target of genetic alternations in many human and animal cancers. Compared to normal tissues, cancer tissues overexpress mutant p53 protein thus allowing their detection by a number of immunochemical procedures. To what extent the expression of mutant p53 correlates with dog mammary tumorigenesis has not been fully studied. In the present study, 20 spontaneously arising canine mammary tumors were examined for overexpression of mutant p53. Two different monoclonal antibodies, BP53-12 and PAb122, which recognize different epitopes of the p53 product, were used. The canine tumors in the present study exhibited five different histological types: i) osteosarcoma (n=7); ii) carcinosarcoma (n=4); iii) solid carcinoma (n=5); iv) complex carcinoma (n=3); and v) tubulopapillar carcinoma (n=1). The positive ratios against BP53-12 and PAb122 antibodies were 50% (10/20) and 60% (12/20) respectively. Among these positive samples, 35% (7/20) reacted to both antibodies. Finally, 15 out of 20 tumors showed positivity against one of the monoclonal antibodies. Mostly, as in human mammary tumor cells, BP53-12 staining was observed in the nuclei of tumor cells. PAb122 staining, however, was confined to cytoplasm of osteosarcoma or carcinosarcoma cells. To confirm the location of the staining, immunoelectron microscopy was done. The results showed that the cytoplasm of cartilage cells in the sarcomas had positive staining. These results indicate that anti-p53 antibodies BP53-12 and PAb122, generated against human p53 are cross reacting with the same molecule in canine cells and that the role of p53 in tumorigenesis is not only confined to tumors in human. Our finding suggests that a combination of p53 monoclonal antibodies should be used to screen, not only canine mammary tumors but also human mammary tumors, to obtain a better tumor prognosis.

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.