Microsatellite allele analysis has been used for individual identification and paternity test. In the present study, the biological father of three puppies was determined by using microsatellite allele amplification analysis. The mother bitch of the litter was a poongsan dog. The three study dogs that could have inseminated the bitch, by being in the same residence, were a white Poosan dog, a mixed breed, and a white Jindo dog. DNA was obtained from all the relevant dogs by buccal swabbing. Four loci of tetranucleotide repeat microsatellite were PCR-amplified, and analyzed by polyacrylamicde gel electrophoresis and silver staining. The results of genotyping unambigously assigned the Poongsan dog as the biological father. There was no evidence of superfecundation. Therefore, the present study demonstrated the usefulness of microsatellite allele analysis as a simple, efficient method of paternity test in dogs.

Previous studies suggested that the inhibition of thyroxine (T4) release by alpha1-adrenoceptor and muscarinic receptor stimulation results in activated protein kinase C (PKC) from mouse and guinea pig thyroids. In the present study, the effect of carbachol, methoxamine, phorbol myristate acetate (PMA), and R59022 on the release of T4 from the mouse, rat, and guinea pig thyroids was compared to clarify the role of PKC in the regulation of the release of T4. The thyroids were incubated in the medium containing the test agents, samples of the medium were assayed for T4 by EIA kits. Forskolin, an adenylate cyclase activator, chlorophenylthio-cAMP sodium, a membrane permeable analoge of cAMP, and isobutyl-methylxanthine, a phospho-diesterase inhibitor, like TSH (thyroid stimulating hormone), enhaced the release of T4 from the mouse, rat, and guinea pig thyroids. Methoxamine, an alpha1-adrenoceptor agonist, inhibited the TSH-stimulated release of T4 in mouse, but not rat and guinea pig thyroids. In contrast, carbachol, a muscarinic receptor agonist, inhibited the release of T4 in guinea pig, but not mouse and rat thyroids. These inhibition were reversed by prazosin, an alpha1-adrenoceptor antagonist or atropine, a muscarinic antagonist or M1- and M3- muscarinic antagonists, in mouse or guinea pig thyroids. In addition, staurosporine, a PKC inhibitor, reversed methoxamine or carbachol inhibition of TSH stimulation. Furthermore, PMA, a PKC activator, and R59022, a diacylglycerol (DAG) kinase inhibitor, inhibited the TSH-stimulated release of T4 in mouse, rat, and guinea pig thyroids. These inhibition were blocked by staurosporine. these findings suggest that the activation of receptor or DAG inhibits TSH-stimulated T4 release through a PKC-dependent mechanism in thyroid gland.

To elucidate the action of the cholinergic and adrenergic nerve on isolated gastric fundus smooth muscle of rabbit, the effects of electrical transmural stimulation were investigated in the presence of atropine, cholinergic receptor blocker; phentolamine, nonselective alpha-adrenergic receptor blocker; propranolol, nonselective beta-adrenergic receptor blocker and L-arginine from the isometric contraction of physiological recording system. 1. The contractile response induced by electrical transmural stimulation was increased as the frequency(1~32Hz)-dependent manner on the isolated gastric fundus smooth muscle. 2. The contractile response induced by electrical transmural stimulation was markedly inhibited by the pretreatment of atropine(1 microM). 3. The contractile response induced by electrical transmural stimulation was inhibited by the pretreatment of phentolamine(1 microM). 4. The relaxative response induced by electrical transmural stimulation on presence of atropine (1 microM) was inhibited by the pretreatment of propranolol(1 microM). 5. The relaxative responses on precontraction induced by histamine(10 microM) with guanethidine (50 microM) and atropine(1 microM) by electrical transmural stimulation were incresed by L-arginine(1mM). These fingings suggest that it was the exciatory action of cholinergic and alpha-adrenergic nerve, and the inhibitory action of beta-adrenergic nerve and nonadrenergic noncholinergic nerve on the isolated gastric fundus smooth muscle of rabbit.

This study was conducted to evaluate the ELISA kit for measuring the level of cortisol in the urine. The CV of within-run variation and day to day variation were 0.4-2.8 and 1.8-5.7, respectively. the minimum limitation of measurement was 1ng/ml. the cross reaction was high (CR50(%)=11.4-43.2) in prednisolone, 11-deoxycortisol, 21-deoxycortisol and predinosone. There was low and no cross reaction in other steroid. To develop the ELISA kit we measured the cortisol level in diluted urine with PBS (procedure I), extracted urine with methylene chloride (procedure II) and extracted methylene chloride-extracted urine from thin-layer chromatography (procedure III). The CV value of procedure I, II, III was 9.4-28.3%, 7.2-8.9% and 2.5-5.7%, respectively. There was significant difference between procedure I with II, and pro-cedure I with III(p0.01), but no difference between procedure II with III significantly(p0.01). The mean UCCR of urine collected through am 8 to 10 ws 9.5+_7.6(0.14-28.0) in 12-month-old dog(n

Galectin-3 plays an important role in cell development, differentiation and cancer metastasis, including cell-cell/extracellular matrix (ECM) interactions and is supposed to have an effect of apoptosis on T-cells in thymic clonal selection. In this study, to know the effect of galectin-3 on thymocyte development, we used recombinant human galectin-3 (rHgal-3) from Escherichia coli, JM105, which was inserted with human gal-3 gene-transformed plasmid vector (prGal-3) to express human galectin-3. Expressed rHgal-3 was confirmed by western blot using the culture supernant of hybridoma (M3/38) producing monoclonal antibody to human galectin-3. Sepharose gel affinity chromatography was used to purify the expressed rHgal-3. Thymocytes and hepatocytes from 6-week-old male BALB/c mice were incubated with rHgal-3 and showed marked increase of apoptotic population on analysis using flow cytometry with 7-AAD in a dosedependent manner. However, rHgal-3 failed to induce apoptosis on hepatocytes. Interestingly, this apoptotic effect was not inhibited by lactose, a specific lectin domain inhibitor. From these results, we concluded that extrinsic galectin-3 induces apoptosis on mouse thymocytes, and galectin-3 may have an apoptotic effect on T-cells in thymic clonal selection.

The present experiments were performed to examine the effects of acetylcholine (ACh) and carbachol (CC) on thyroxine (T4) release and any possible relation between inhibition of T4 relese and sighaling pathway in guinea pig thyroids. The thyroids were incubated in the medium containing the test agents, samples of the medium wer assayed for T4 by EIA kits. ACh and CC inhibited the TSH-stimulated T4 release. These inhibition were reversed by atropine, but not by d-tubocurarine. The ingibitory effects of ACh on T4 release were prevented by M1- and M3-muscarinic antagonists and its inhibition was also slightly reversed by M2- and M4- muscarinic antagonists. R59022, like ACh and CC, also inhibited the TSH-stimulated T4 release. This inhibition was reversed by protein kinase C inhibitor and Ca2+ channel blocker. The present study suggests that cholinergic inhibition of T4 release from thyroids can be induced mainly by ctivation of the M1- or M3-receptors and that it is mediated throught the muscarinic receptor-stimulated protein kinase C activation

The aim of these experiments was to investigate the effects of bovine oviduct epithelial cells(OEC) derived from different segments to bind sperm binding and maintain their motility in vitro. In experiment 1, the number of sperm attached to OEC derived from isthmus or ampulla, the motility of unattached sperm during co-culture and fertilizing ability were assessed. In experiment 2, heparin treated sperm 9hsp) or no treated sperm (nsp) were used to evaluate OEC binding ability ofcapacitated sperm. In experiment 1, regardless ofthier origin, approximately 65% of the sperm were attached to OEC within 2h. From 6h of co-culture, the numbers of unattached sperm on ampullary OEC were significantly higher than those on isthmic OEC (p0.005). From 12h of co-culture, the motility of unattached sperm on isthmic OEC were significantly higher than those on ampullary OEC(p0.05). The cleavage rate of oocytes inseminated on OEC derived from isthmic segment was also significantly higher than those from ampullary segment (p0.01). In experiment 2, the numbers of unattached hsp on OEC were significantly higher than those of controls(p0.01), between 2~24h examination. From 12h of co-culture, the motility of unattached nsp were sighificantly greater than those of hsp(p0.01). These results show that bovine OEC derived from the isthmus play more important role(s) for sperm binding, maintaining motility and fertilization in vitro than those from the ampulla, and heparin induced capacitation may change sperm binding ability on OEC in vitro.

This study was conducted to examine the outbreak rate and the causative agents of otitis externa in 26 dogs(49 ears; 23 dogs = bilateral, 3 dogs = unilateral), and the normal microfloras of external ear canal in 68 dogs(133 ears; 65 dogs = bilateral, 3 dogs = unilateral) in Taegu, 1997. The breed, living environment, sex, age and season distribution of otitic dogs were as follows: Dogs with erect and hairy ears(42.3%), pendulous and hairy ears(38.5%), indoor(92.3%), female(65.4%) and below one year old(38.5%) were more prevalent. According to season, otitis externa was mainly occurred between July and October. The major causative agents of canine otitis externa were Malassezia pachydermatis(32.7%), Staphylococcus aureus(26.5%) and S intermedius(16.3%). The major causative agents of canine otitis externa were Malasszia pachydermatis(32.7%), Staphylococcus aureus(26.5%) and S intermedius(16.3%). In the microorganism isolated 39 otitic ear canals, single infection was 53.8% and mixed infection was 46.2%. The normal microfloras of canine external ear canal were fungi including M pachydermaits, Aspergillus spp, Microsporum canis, Alternaria spp, Verticillium spp and Yeast, and bacteria including Staphylococcus spp(10 species including S xylosus), Bacillus spp, Corynebacterium spp, Listeria spp, Actinomyces pyogenes and Escherichia coli. No growth was 34.6%.

The purpose of the present study was to investigate the effect of bovine amniotic membrane grafts on healing of full-thickness skin wound in dogs. Two 3cm*3cm area-matched full-thickness skin wounds were induced bilaterally on the dorsolateral aspect of the trunk of 15 dogs Chlorhexidine-treated amnion, dried amnion, silver sulfadiazine and 0.9% sterile saline solution were applied on the wound area and examined grossly and histopathologically. Begining 14 days after wounding, amnion applied group had appreciably less amount of inflammatory exudate and hemorrhage than sulfadiazine and saline treated groups. From 14 days after wounding, the degree of wound contraction in amnion groups, especially in the dried amnion group was greater than that of the sulfadiazine and saline treated groups. The percentages of wounds completely healed on 28 days after wounding in saline treated group, chlorhexidine-treated amnion group, dried amnion group and sulfadiazine treated group were 33%, 50%, 83% and 50%, respectively. Microscopically neovascularization and fibrosis were first noticed on 5 days after wounding in the dried amnion group and sulfadiazie treated group, on 7 days in the chlorhexidine-treated amnion group and on 14 days in the saline treated group. Epithlialization in thedried amion and sulfadizine treated groups ws first noticed on 9 days after wounding, which was faster than that in the other groups. The present study suggests that bovine amniotic membrane, especially dried bovine amnion is effective on healing of full-thickness skin wound in dogs through both wound contraction and epithelialization.

The study was designated to investigate the electron microscopic findings following diethylnitrosamine (DEN) treatment in rats. Forty four male (Srague Dawley) rats were continuously given water containing 0.01% DEN for 13 weeks and livers of five rats with more tumor lesions at 16 and 17 weeks after initial treatment were used as EM materials. In transmission electron microscopic findings, most small-sized hepatocytes were active cells containing large mount of organelles, but light (pale staining) hepatocyte among small-sized hepatocytes were injured cells containg disorganized organelles. Tumor cells among small-sized hepatocytes were irregularly arranged and have ;leomorphic nuclei containing electron dense chromatin but the organelles in cytoplasm were swelled. Large-sized hepatocytes were active cells with condensed chromatin but the cytoplasm of these cells were pale due to be injured and dilated organelles. Dark hepatocytes were apoptotic cells with homogenous pyknotic nuclei and cytoplasm, and the cytoplasm of these cells contained dilated smooth endoplasmic reticulum (sER) but these sER were non-vesiculated. Cholangiocarninoma cells were crowed and were pale by far less number of organelles in cytoplasm and nuclei. In scanning electron microscopic findings, the lumens of portal veins, bile canaliculi, bile ductules, bile ducts and sinusoids were dilated and have irregular folded inner surface by protruded parenchyma.