Biological effect of tyrosine kinase inhibitors on three canine mast cell tumor cell lines with various KIT statuses

Tyrosine kinase inhibitors (TKIs) can be important in the treatment of canine mast cell tumor (cMCT). Meanwhile, some TKIs have been identified as substrates for ABCB1. The inhibitory effect of four TKIs (axitinib, imatinib, masitinib, and vatalanib) for proliferation and phosphorylation of c-Kit receptor as well as the expression and function of ABCB1 were investigated in three cMCT cell lines (HRMC, VIMC1, and CMMC1). The IC(50) values of the TKIs in HRMC, the only cell line with wild-type KIT, were clearly higher than those in CMMC1 and VIMC1. In HRMC and CMMC1, both the growth and phosphorylation of c-Kit receptor were suppressed proportionally by the TKIs. VIMC1 required higher concentrations for the inhibition of c-Kit receptor phosphorylation than those in cell growth. The treatment with cyclosporine increased the effects of the TKIs on VIMC1 since ABCB1 was expressed in VIMC1. The results indicated that cMCT cell lines harboring wild-type KIT had lower sensitivity to TKIs. The growth of VIMC1 was seemingly reduced by TKIs through the inhibition of other tyrosine kinases than c-Kit receptor. There was little influence of ABCB1 on TKI effects to the proliferation of VIMC1. These results will be helpful to understand the different sensitivity to TKIs in cMCT patients.     

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.     

Supplementary report on experimental autumn crocus (Colchicum autumnale L.) poisoning in cattle: morphological evidence of apoptosis.

Previously we reported that tissue destruction characterized by the presence of karyopyknotic, karyorrhectic and mitotically arrested cells was seen in alimentary epithelial cells and lymphocytes in the lymphoid and hemopoietic systems of cattle experimentally administered with autumn crocus (Colchicum autumnale L.). This report deals with the mechanism of acute cellular injury following experimental autumn crocus poisoning in cattle as demonstrated by the in situ DNA strand break analysis and electron microscopy. The analyses revealed that cellular injury caused by autumn crocus was closely associated with apoptosis.     

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.     

Protein synthesis measured in the whole body of chick embryos cultured in vitro

1. Whole body protein synthesis was measured in chick embryos cultured in vitro. On day 7 of incubation chick embryos were cultured for 60 min in synthetic serum‐free medium containing 4‐[³H]phenylalanine. Specific radioactivities in free and protein‐bound phenylaline in the whole embryo were measured, starting 2 min after commencement of the culture process.

2. The values for fractional synthesis rate (FSR) estimated in vitro at 20, 30, 45 and 60 min during the embryo culture agreed well, ranging from 35 to 40%/d, suggesting that the method would serve as a useful model for studying the effect of growth promoters in chick embryos.

3. Bovine insulin in the synthetic medium did not affect FSR of protein in chick embryos cultured in vitro.     

Genetic correlation between serum insulin-like growth factor-1 concentration and performance and meat quality traits in Duroc pigs

This study was intended to examine whether serum IGF-I concentration is appropriate for use as a physiological predictor for genetic improvement of meat production and meat quality traits in pigs. Heritabilities and genetic correlations were estimated for these traits. The Duroc breed used in this study was selected for seven generations for average daily BW gain (DG) from 30 to 105 kg of BW, loin-eye muscle area (EM), backfat thickness (BF), and intramuscular fat (IMF) content. Serum IGF-I concentration of boars and gilts at the fourth generation of selection and that of boars, gilts, and barrows from the fifth to seventh generations of selection were measured at 8 wk (IGFI-8W) for 832 animals and again at the time they reached 105 kg of BW (IGFI-105KG) for 834 animals. A multivariate REML procedure was used to estimate genetic parameters with a model incorporating generation of selection, sex, common environmental effect of litter, and individual additive genetic effects. Heritability estimates for IGFI-8W and IGFI-105KG were 0.23 +/- 0.02 and 0.26 +/- 0.03, respectively. The estimates of common environmental effect for IGFI-8W and IGFI-105KG were 0.20 +/- 0.02 and 0.03 +/- 0.01, respectively. Positive genetic correlations were estimated between IGFI-8W and DG (0.26 +/- 0.08), EM (0.22 +/- 0.10), and IMF (0.32 +/- 0.10). Moreover, the positive genetic correlation between IGFI-105KG and EM was 0.42 +/- 0.08. These results indicate that serum IGF-I concentration at an early stage of growth was effective for prediction of IMF, but it was not a reliable physiological predictor of genetic merit of meat production traits.     

Endocrine status and development of porcine testicular tissues in host mice

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.     

Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.