Immunohistochemical expression of p63, Ki67 and ß-catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder.

Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or ß-catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and ß-catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.

Endothelin-1 in smooth muscle cells and mast cells of mouse uterus after parturition.

To elucidate the physiological importance of endothelin-1 (ET-1) in mouse uterus, we investigated quantitative changes in ET-1 mRNA levels in the uterus during the estrous cycle, pregnancy and post-parturient period by use of the real-time PCR technique and we examined the cellular distribution of the ET-1 peptide by use of immunohistochemical techniques. Low and constant mRNA levels were observed in the uterus from cyclic or pregnant mice. However, a significant increase in mRNA levels was found immediately after parturition (day 0 postpartum) which then decreased gradually to a basal level at day 14 postpartum. Discernible immunopositivity was found in myometrial cells as well as in endometrial epithelial cells in the post-parturient uterus. Myometrial cells showed the strongest staining at day 0 postpartum, and some large cells in the myometrial layers, intensely positive for ET-1, were characterized as mast cells. These findings suggest the possibility that in mouse uterus ET-1 may play a role in recovery from the uterine changes caused by pregnancy and parturition.

Rapid quantification of murine endothelin-1 and vasoactive intestinal contractor gene expression levels by a real-time PCR system.

A rapid quantitative analysis method for murine endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC) gene expression levels was established using a real-time polymerase chain reaction (PCR). We designed primer pairs and TaqMan probes specific for murine prepro-ET-1 (PPET-1) and prepro-VIC (PPVIC) genes, based on the cDNA sequence region common to both mouse and rat. The dynamic range for detection in this system spanned 100000-fold of the starting molecule. The gene expression levels of PPET-1 and PPVIC were estimated as gene expression rates normalized by the expression of the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase. To examine the reproducibility of this assay system, we calculated the intra-assay and interassay coefficients of variation of the gene expression rate, which ranged from 16.2 to 55.0% and from 24.2 to 56. 5%, respectively. Using this system, we examined gene expression levels of PPET-1 and PPVIC in mouse tissues. PPET-1 gene expression was found in all tissues at relatively high levels, whereas high levels of PPVIC gene expression were observed only in stomach, intestine, uterus, and ovary. The gene expression patterns agreed well with those determined by RNase protection assay and conventional PCR. These results show that this new rapid method is accurate and reproducible.

Doxorubicin affects the cardiac muscarinic system in the rat.

During the study on the mechanism of doxorubicin-induced cardiotoxicity, we observed that a long incubation (4 hr) with doxorubicin reduced the maximal negative inotropic effects of a muscarinic receptor agonist, carbachol. The mechanism responsible for this doxorubicin-induced reduction of the efficacy of carbachol was examined in isolated guinea pig hearts. In isolated left atrial muscle preparations, 1 hr incubation with 100 microM doxorubicin caused a parallel right-ward shift of the concentration-response curves for carbachol, but a longer (4 hr) incubation with this agent (30, 100 or 200 microM), caused a significant reduction of the magnitude of the negative inotropic effect of carbachol in addition to the concentration-dependent parallel right-ward shift. The 4-hr incubation with these concentrations of doxorubicin also reduced the maximal negative inotropic effect of an adenosine A1 receptor agonist, R-phenylisopropyl adenosine (R-PIA), without affecting the potency of this agonist. Doxorubicin (1 to 100 microM) reduced [3H]quinuclidinyl benzilate (QNB) binding in a concentration dependent manner, but failed to alter [3HIR-PIA binding. The decrease in the magnitude of the maximal negative inotropic effect by doxorubicin was caused by changes in the muscarinic system at steps common to the transduction of muscarinic and adenosine A1 receptor mechanisms.

Structure of mouse preproendothelin-3 and phylogenetic analysis of the endothelins.

Endothelin (ET)-related peptide hormones, ET-1, vasoactive intestinal contractor (VIC), ET-2 and ET-3, have multiple physiological roles including vasoconstriction. To reveal the structural diversity of the precursor proteins of the ET family, cDNAs, cross-hybridizing with ET-1 and VIC probes, were cloned from the mouse intestine library. The deduced protein is a 214-amino acid precursor of mouse ET-3, preproendothelin-3 (PPET-3), which is the counterpart of the hypothalamus-, not placenta-, derived human PPET-3. Sequence identities of PPET-3 amino acids of mouse with human and rat are 65% over 194 amino acids and 65% over 214 amino acids. Phylogenetic analysis of the precursor proteins for ET-1, VIC and ET-3 suggest that members of the ET family are distantly related and probably descended from a common ancestral gene.

Quantitative analysis of endothelin-1 and vasoactive intestinal contractor/endothelin-2 gene expression in rats by real-time reverse transcriptase polymerase chain reaction.

We established a real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) system for the analysis of rat endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 gene expression. We used this technique to examine the expression levels in rat in 16 different organs. ET-1 gene expression was observed in all organs examined, while VIC mRNA was detected in some organs such as heart, lung, ovary, stomach, and intestine. Ovary and intestine express both ET-1 and VIC mRNA at high levels, suggesting the importance of both peptides in these organs. In addition, we examined the gene expression levels in intestinal epithelial and mesenchymal tissues from rat fetuses at 16.5 and 19.5 days postcoitus (E16.5 and E19.5). We observed distinct differences in the temporal gene expression patterns for ET-1 and VIC in fetal intestinal epithelial tissue. In fetal mesenchymal tissue the expression level of ET-1 is significantly higher than that of VIC, and the levels of both genes remain unchanged over the time period observed. These findings suggest distinct biological roles and gene regulation mechanisms for ET-1 and VIC in intestinal epithelial and mesenchymal tissues.

The endothelin-2/vasoactive intestinal contractor gene: expression and promoter activity in PC12 rat pheochromocytoma cells.

In order to understand the physiological roles of vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2), we examined the expression of this peptide by specific reverse transcriptase polymerase chain reaction (RT-PCR) analysis and found that PC12 rat pheochromocytoma cells express the VIC gene. The 5'-flanking 1.0 kilo base pair (kb) region of the mouse VIC gene is sufficient to express a secreted alkaline phosphatase (SEAP) reporter gene in transiently transfected PC12 cells. The 1.0 kb promoter region may contain cis-acting elements that determine the rate of the VIC gene transcription in PC12 cells.

Anti-muscarinic actions of mitoxantrone in isolated heart muscles of guinea pigs.

A hypotheses that mitoxantrone is a competitive antagonist at muscarinic cholinergic receptors was examined in guinea-pig hearts. In isolated left atrial muscle preparations, electrically paced at 2 Hz, the muscarinic agonist, carbachol, caused a concentration-dependent decrease in developed tension. Mitoxantrone caused a parallel right-ward shift of the concentration-response curve for carbachol. Schild plots for the effect of mitoxantrone on the carbachol concentration-response relationship were linear with a slope of 0.88 which was not significantly different from the unity. The right-ward shift of the carbachol concentration-response relationship by mitoxantrone significantly reversed after an additional incubation with a mitoxantrone-free solution, although the reversal was incomplete after a 2-h incubation in the mitoxantrone-free solution. Mitoxantrone caused a concentration-dependent displacement of specific [3H]quinuclidinyl benzilate binding to membrane preparations obtained from ventricular muscles of guinea-pig hearts. These results indicate that mitoxantrone acts as a competitive antagonist for the muscarinic receptors.

Fluctuating gene expression and localized cellular distribution of vasoactive intestinal contractor (VIC) in mouse uterus.

To understand the physiological roles of vasoactive intestinal contractor (VIC) and endothelin-2 (ET-2) in the uterus, we examined the expression levels of VIC mRNA by real-time quantitative reverse transcription-linked polymerase chain reaction (RT-PCR) and characterized the cellular distribution of VIC peptide and mRNA by immunostaining and in situ hybridization in mouse uterus. In pregnant mouse uterus, VIC mRNA expression changed considerably between Days 10.5 and 12.5 of pregnancy. The expression levels were significantly (p<0.05) higher (approximately fivefold) in the later stage of pregnancy (Days 12.5-17.5) than in the earlier stage (Days 7.5-10.5). In nonpregnant uterus, VIC mRNA expression was significantly (p <0.05) higher (approximately threefold) in proestrus and estrus than in diestrus. Immunohistochemical studies demonstrated the presence of VIC peptide in endometrial epithelial cells, myometrial cells, and vascular smooth muscle cells during the estrous cycle and pregnancy and after parturition. Notably, myometrial cells showed dominant immunostaining in proestrus and estrus, in the later pregnancy stage, and in the early postpartum period, analogous to the expression pattern of VIC mRNA. In situ hybridization confirmed localization of VIC mRNA in myometrial cells. These findings suggest that VIC may play an important role in the function of myometrial cells.

The prepro vasoactive intestinal contractor (VIC)/endothelin-2 gene (EDN2): structure, evolution, production, and embryonic expression.

Murine vasoactive intestinal contractor (VIC) and its human analog endothelin-2 (ET2) are potent vasoactive hormones composed of 21 amino acids. To study the structural characteristics of the VIC/ET2 gene (HGMW-approved symbol EDN2), we isolated the full length of the mouse VIC gene. Sequence analysis indicates that a biologically active mature VIC peptide is produced from a 175-residue precursor protein; preproVIC (PPVIC). Several remarkable similarities of the PPVIC gene to the human preproendothelin-1 gene strongly suggest that the two genes have arisen from a common progenitor by gene duplication. Transfection of ACHN adenocarcinoma cells with the cDNA resulted in the production of VIC peptide. VIC production was increased by the deletion of the 3'-untranslated region, which contains an AU-rich mRNA destabilizing sequence. Increased PPVIC gene expression during the late embryonic stage suggests an important function in development. This study provides the basis for disruption and regulation analysis of the gene, which may lead to a better understanding of VIC/ET2's physiological significance.

Utilization of intestinal triglyceride-rich lipoproteins in mammary gland of cows.

Elution profiles of total lipoproteins, apolipoprotein B (apoB) concentrations in lipoproteins, and plasma triglyceride (TG) levels were examined in early-, late-, and non-lactating cows. Additionally, arteriovenous (A-V) differences were also measured to elucidate the uptake of TG and apoB-containing lipoproteins in mammary gland. Non-lactating cows showed three major peaks corresponding to triglyceride-rich lipoprotein (TRL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fraction, whereas both early- and late-lactating cows revealed two peaks corresponding to TRL and HDL. The peak area of TRL in early- and late-lactating cows were significantly (p < 0.05) smaller than that in non-lactating cows. The plasma TG levels and apoB-48 concentrations of TRL in early- and late-lactating cows were also significantly (p < 0.01) lower. Furthermore, early lactating cows showed significantly (p < 0.05) larger A-V differences in both plasma TG and apoB-48 concentration of TRL than those in late- and non-lactating cows. These results suggested that TG in exogenous (intestinal) TRL was utilized for milk fat synthesis in lactating mammary gland of cows by the receptor-mediated uptake.

Gene expression of vasoactive intestinal contractor/endothelin-2 in ovary, uterus and embryo: comprehensive gene expression profiles of the endothelin ligand-receptor system revealed by semi-quantitative reverse transcription-polymerase chain reaction analysis in adult mouse tissues and during late embryonic development.

Vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2) is a 21 amino acid intestinal peptide characterized as a potent vasoactive and intestinal smooth muscle-contracting compound. To investigate the physiological roles of VIC/ET-2 further, we characterized the specificity of VIC gene expression relative to that of other members of the endothelin (ET) ligand-receptor system in adult mouse tissues and during embryonic development. Gene expression of ET-1, ET-3, ETA and ETB was ubiquitous in almost all tissues we examined while gene expression of VIC was localized to certain tissues. A high level of VIC gene expression was observed in ovary and uterus. The gene expression of VIC, relative to that of glyceraldehyde-3-phosphate dehydrogenase, was approximately 2.0%, 0.4%, and 2.3% in ovary, uterus, and intestine respectively, and was approximately 1.6 and 7. 1 times higher than that of ET-1 in ovary and intestine respectively. Thus, VIC may have some physiological role in adult ovary and uterus as well as intestine. In embryonic development, VIC gene expression sharply increased between 11 and 15 days post coitus and decreased after birth, suggesting an involvement in the later stages of embryonic development.

Apolipoprotein B (apoB) concentrations in lipoproteins in cows.

The concentrations of apolipoprotein B (apoB)-48 and apoB-100 in triglyceride-rich lipoproteins (TRL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) fractions separated by gel permeation chromatography were determined in Holstein and Japanese black cows by enzyme-linked immunosorbent assay (ELISA). A significant correlation (p < 0.01) was observed between apoB-48 in TRL and plasma triglyceride (TG) levels in both Holstein and Japanese black cows. Additionally, apoB-48 in TRL and plasma TG levels in Holstein cows were significantly lower (p < 0.01) than those in Japanese black cows. These results suggested that TG derived from intestinal (exogenous) TRL rather than from liver (endogenous) TRL was the major source of milk fat.