Cytotoxicity for porcine islet cells by complement of six animal species

Complement-mediated cytotoxicity for porcine islet cells (PICs) was evaluated using sera of six animal species. Then soluble complement receptor type-1 (sCR1) as an anti-complement agent was added to those sera, and the changes in 50% hemolytic unit of complement serum (CH50) and cytotoxic effect of those sera on PICs were examined. All the sera except for that of pig showed cytotoxicity. However, the extent of toxicity was considerably different between species. In the rat and human serum, sCR1 significantly reduced CH50 and cytotoxicity, however in the dog serum, sCR1 had no suppressive effects. These results may suggest that complement contribute to humoral cytotoxicity for PICs as a main factor, and the compatibility of complement with PICs differs between animal species.     

Matrix-metalloproteinases-2 and -9 production in bovine endometrial cell culture

In vitro cell culture is a convenient tool for studying cellular mechanisms. In the present study, production of matrix-metalloproteinases (MMPs) in bovine endometrial (containing both epithelial and stromal cells) monolayer cells was examined. Blastocysts attached to the endometrial cells in a monolayer culture were examined for their effects on MMP-2 production. Initial attachment of blastocysts to the monolayer inhibited MMP-2 production by endometrial cells. But once trophoblast cells began to migrate into the endometrial cell layer, MMP-2 production increased, and at the same time MMP-9 production also became evident in the medium. In order to understand how blastocysts affected MMP-2 production, we examined the effect of progesterone, estradiol, insulin-like growth factors (IGFs), tumor necrosis factors (TNFs), and interferon-tau (IFN-tau) supplementation. It was IFN-tau that inhibited the production of MMP-2. In addition, progesterone at a lower dose appeared to inhibit MMP-2 production. Both TNF-alpha and TNF-beta strongly stimulated the production of MMP-2 and MMP-9, whereas IGFs had no effect. Based on these findings, it appears that conceptus has the capacity to inhibit MMP activity.     

Need for flagella for complete virulence of Pseudomonas syringae pv. tabaci: genetic analysis with flagella-defective mutants ?fliC and ?fliD in host tobacco plants

The flagellins purified from Pseudomonas syringae pv. tabaci induce a hypersensitive reaction in nonhost tomato cells. To investigate the role of flagella and flagellin in the compatible interaction, we generated two types of flagella-defective mutant. The fliC mutant lost the fliC gene that encodes flagellin protein, whereas the fliD mutant lost the fliD gene that encodes HAP2-capping protein. The two mutants had markedly reduced ability to cause disease symptoms in tobacco leaves. Furthermore, propagation of the mutants in tobacco leaves was less than that in wild-type pv. tabaci. Compared to the inoculation with wild-type pv. tabaci, inoculation with the two mutants did not markedly induce the expression of typical defense response-related genes such as PAL and hsr203J. Complementation of each fliC and fliD gene to the corresponding deficient mutant restored motility and virulence. These results indicate that flagella of P. syringae pv. tabaci are indispensable organelles for complete virulence on host tobacco plants.     

Morphological analogy of the rostral midline area in the chicken midbrain to the anteromedian nucleus in the mammalian midbrain

Substance P-immunoreactive neurons projecting from the midbrain to the spinal cord of the chicken were examined by the use of the retrograde tract-tracing method combined with immunohistochemical techniques. Many small neurons were densely clustered in the rostral midline area of the midbrain (RMA), and showed substance P-immunoreactivity. These substance P-immunoreactive neurons sent axons to the intermediomedial cell column (avian autonomic preganglion) and its vicinity in the lumbar spinal segments. On the basis of the strong neuroanatomical analogy in the cytoarchitectural features, immunoreactivity, and fiber connections, the RMA was assumed to be the avian homologue of the anteromedian nucleus in the mammalian midbrain.     

Effect of modified oligopeptides from the beetle Allomyrina dichotoma on Escherichia coli infection in mice

The novel peptides based on a putative active site of defensin, an anti-bacterial peptide from the beetle Allomyrina dichotoma, were synthesized. These synthetic oligopeptides exhibited strong anti-bacterial activity in vitro, even against antibiotic-resistant pathogenic bacteria. Then, anti-bacterial activity of two newly synthesized peptides, RLYLRIGRR-NH(2) (peptide A) and RLRLRIGRR-NH(2) (peptide B) was also examined by macroscopic and histopathologic assessment during the course of infection in mice inoculated with antibiotic-resistant pathogenic Escherichia coli (E. coli) in vivo. Peptide B decreased the mortality of mice inoculated with antibiotic-resistant pathogenic E. coli. The results of macroscopic and histopathologic examinations revealed that peptide B could protect the mice from infection. In contrast, peptide A failed to protect mice from infection with antibiotic-resistant pathogenic E. coli. Also, modified peptides A and B produced no toxicity or side effects in mice. These results suggest that peptide B is useful for developing novel antibiotics against antibiotic-resistant pathogenic bacteria.     

The incidence of disseminated intravascular coagulation in dogs with malignant tumor

The incidence of DIC in 208 dogs with a malignant tumor was evaluated. The incidence of DIC was 9.6% in dogs with a malignant tumor which was a solid tumor in all. In 164 dogs with a malignant solid tumor, the incidence of DIC was 12.2%. The incidence of DIC in dogs with hemangiosarcoma, mammary gland carcinoma and adenocarcinoma of the lung was significantly higher than that in dogs with other malignant tumors. These results suggested that special care in looking for DIC should be taken in dogs with a malignant solid tumor.     

Establishment and characterization of four canine melanoma cell lines

Four canine melanoma cell lines were established from the subcutaneous, oral gingival and mucosal melanoma tissues at the primary and metastatic sites. These cell lines were designated as CMeC-1, CMeC-2, KMeC and LMeC. The cells were spindles in shape, similar to that of primary tumor cells. The doubling times of these cells ranged from 34.1 +/- 5.61 to 57.9 +/- 3.28 hr and their chromosome number ranged from 46 to 80. When transplanted into nude mice, CMeC-1 and LMeC produced tumors, whereas CMeC-2 and KMeC did not. The morphology of the tissue formed by xenotransplantation of these cells was similar to their primary tumors.     

Evaluation of fetal growth and estimation of fetal age based on skeletal growth in Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884)

We investigated fetal development and the estimation of fetal age of 127 Hokkaido sika deer fetuses, categorizing them into three groups according to the nutritional condition of populations. The order and time of the appearance of ossification centers were clarified, and fetal age was determined based on bone length and the appearance of ossification centers. Then we observed the differences in fetal growth among three populations, and discussed the effect of poor nutrition on the fetal growth. The results suggest that fetal diaphysial length of the femur was affected very little by nutritional conditions, whereas conception dates were delayed and fetal weight was restricted as the nutritional condition became poorer. Although it is impossible to know the exact accurate fetal age in wild populations, it was possible to create a standard to estimate fetal age more precisely by the method described in this study. Both the bone length and the appearance of ossification centers are reliable indices to estimate fetal age precisely in measurements available from fetuses of unknown age, and can be applied to estimate the fetal age of other populations of sika deer, whereas estimation of fetal age based on weight is prone to great errors.     

Immunohistochemical analysis of expression patterns of tumor necrosis factor receptors on lymphoma cells in enzootic bovine leukosis

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5+/CD11b+), B-1b (CD5-/CD11b+) and B-2 (conventional B) (CD5-/CD11b-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.     

Establishment and characterization of eight feline mammary adenocarcinoma cell lines

Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.