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

In order to study the effects of Jengjengamiyjintang on the duodenal ulcer induced by HCI-aspirin in rats, the changes of histological profiles, goblet cells(PAS-positive cells), and the distribution and frequency of cholecystokinin(CCK)-8 and serotonin-producing gastro-entero-endocrine cells were observed after oral administration of Jengjengamiyjintang. Histologically, very severe injury to duodenal epithelium were observed in control groups and theses injuries were increased with time intervals. But in the Jengjengamiyjintang administrated groups, no gross lesion of ulcer were demonstrated and histologically minor injury to the mucosal epithelium were observed. PAS-positive cells were increased in the Jengjengamiyjintang administrated groups compared to that of control groups. Severe degranulation of CCK-8- and serotonin-immunoreactive cells were observed in control groups but these phenomenon was seldom in the Jengjengamiyjintang administrated groups. Serotonin-immunoreactive cells were significantly decreased in control groups but increased in Jengjengamiyjintang administrated groups compared with control groups. According to these result, it is suggested that Jengjengamiyjintang would accelarat the healing of the duodenal ulcer but the functional mechanisms were unknown.

DNA hybridization assay using probes prepared from liver was carried out to identify species characterization of the domestic animals. Gel electrophoresis showed that the target DNA extracted from raw muscle were 1kb and uniform pattern while fragments size of heated muscle were irrgular. Hybridizatino was performed by adding 200ng/ml probe in hybridization solution and incubating for 12 hours at 68 degrees centigrade. To obtain good discrimination, applied washing buffer and washing step differently depending on the species. The probes of pig, horse and dog formed the specific hybrids with each target DNA respectively. Although cross reaction ws detected in cattle, goat and sheep but signal intensity among these species made the discrimination possible each other. Such pattern was the same in the cases of chicken, turkey and duck. The hybridization pattern of heated muscle was similar to that of raw muscle in general, but the signal intensity was inferior to that of raw muscle. Species identification between closely related animal species, hybridized using the target DNA of such closely related animal species as a blocking agent, remarkable increase of discrimination from the evident decrease of non specific reaction compared with the control group. In addition, in the admixture where certain meat was included in the beef, pork, chicken meat, we colud find whether any unjust meat was admixed or not. In this case, detection limit of certain meat in admixture was 1%.

Swine shipworm(Trichuris suis) is cosmopolitan nematode which can cause serious pathology in immature stage(larva2~larva5) of infected pigs, such as anorexia, diarrhea, anemia, and death in heavy infections. In this larval stages, it is very difficult to diagnose the infection of whipworm and to differentiate from other common swinegstrointestinal disorders such as 21 day scours which are associated with TGE virus, rota virus, coccidium, and the stress of weaning. In this experiment, the isolated antigens of Trichuris spp. were carried out to examine the structure and specificity of antigens and to select the resonable antigens which would be used in serological diagnosis by electrophoresis, Western blotting, ELISA. The results of this experiment were as follews: 1. The common fractions of each Trichuris suis antigen were identified 28,32,45, 80kDa by SDS-PAGE with silver stain and four major fractions could be detected in positive swine sera by Western blot analysis. 2. The OD(optical density) values of somatic and excretory-secretory antigens which were reacted against positive(negative) sera from pigs infected with Trichuris suis by ELISA reader were; 1) OD values(mean+_SD) of adult somatic antigen against positive(negative) sera were O.30+_0.12(0.09+_0.006) and third-stage larva of somatic antigen were 0.28+_0.038(0.10+_0.005). And OD values of excretory-secretory antigens of adult and third-stage larva were 0.24+_0.031(0.11+_0.005) and 0.08+_0.013(0.10+_0.003), respectively. 2) OD values of adult somatic, larval somatic antigen and adult excretory-secretory antigen response to positive sera were significantly (p0.01) associated with negative swine sera. And the Cut-off OD values(minimum positive value) were determined to be mean negative value plus 3 SD that would minimized the risk of false positives. 3. The OD values of somatic antigens of T suis and T vulpis against swine positive(negative) sera were 0.30+_0.120(0.09+_0.006) and 0.25+_0.141(0.09+_0.003). These data mean that the somatic antigens of T suis and T vulpis were able to diagnose T vulpis infection in dogs as well as T suis infection in pigs. These results suggest that somatic antigen of third-stage larva and excretory-secretory antigen of adult T suis could be used the diagnostic antigen by serological test(ELISA) in immature Trichuris spp. infection.

Sal enteritidis thin fimbriae, SEF14, were found to be restricted to the predominantly poultry-associated members of the Salmonella serogroup D1 that are considered as the important pathogens in poultry industry. SefA together with sefB and sdfC encode the proteins involved in SEF14 biosynthesis. In order to develop the rapid and specific detection methods for Salmonella serogroup D1, a PCR technique for the am;lification of sefA gene was established, and its specificity and sensitivity were investigated with various microorganisms. The bacterial genomic DNA was extracted by colony-picking and rapid boiled-lysate technique. In comparison of SefI and SefII primers used in the PCR. SefI primer for sefA gene of 513bp showed higher specificity than that of SefII. The established PCR was s sensitive as to detect 1pg of Sal enteritidis DNA. When 73 strains in 28 genera including the reference strains and the field isolates of various Salmonella serotypes, Bacillus subtilis, Bordetella bronchiseptica, E coli, Listeria spp., Micrococcus luteus, Rhodococcus equi, Staphylococcus spp., Streptococcus spp., Vibrio parahemolyticus, Yersinia spp. were studied, the established PCR yielded specifically positive results with only Salmonella serogroup D1. The results suggested that the PCR for sefA gene could be a potential candidate among the specific detection methods for Salmonella serogroup D1.

The purpose of this study was to establish a rapid, reliable diagnostic method detecting Encephalomyocarditis virus(EMCV) RNA in formalin-fixed, paraffin-embedded tissues of EMCV naturally infected pigs by cDNA probe of EMC K3, the EMCV strain isolated from Korea. Using a biotin-labelled nick translated probe for the cDNA marker. We made up for some defects of radiolabeled method. In situ hybridization(ISH) technique, differently from theother nucleic acid hybridization methods, is able to detect the virus genome specifically in the state of the intact shapes of cells and/or tissues. We succeeded in performing the experiment to detect the EMCV within 1~2 hours usign the MicroProbeTM capaillary action system. In this study, we ovserved highly specific positive sighals of red color by staining the paraffinembedded tissue sections of naturally EMCV-infected pig organs or tissues, including brain, heart, kidney and lacrimal gland with the Fast Red TR salt/Naphtol phosphate chromogen. The results suggested that this ISH method is considered as a highly sensitive and reliable tool for molecular biologic diagnosis of the EMC viral disease.

Ischemic damage in the selectively vulnerable populations of neurons is thought to be caused by an abnormal accumulation of intracellular calcium. It has been reported that the neurons, expressing specific calcium binding proteins, might effectively control intracellular calcium concentrations because of a high capacity to buffer intracellular calcium in the brain ischemic condition. It is uncertain that parvalbumin, one of the calcium binding proteins, can protect the neurons from the cerebral ischemic damage. Recently, treatment of kappa opioid agonists increased survival rate, improved neurological function, and decreased tissue damage under the cerebral ischemic condition. Many evidences indicate that these therapeutic effects might result from regulation of calcium concentration. This study was desighed to analyze the changes of number in parvalbumin-positive neurons after cerebral ischemic damage according to timepoints agter cerebral ischemic inductionl In addition, we evaluated the effect of GR89696 (kappa opioid agonist) or naltrexone(non selective opioid antagonist) on the changes of number in parvalbumin expressing neurons under ischemic condition. Cerebral ischemia was induced by occluding the common carotid artery of experimental animals. The hippocampal areas were morphometrically analyzed at different time point after ischemic induction(1, 3, 5 days) by using immuno-histochemical technique and imaging analysis system. The number of parvalbumin-positive neurons in hippocampus was sighificantly reduced at 1 day after ischemia(p0.05). Furthermore, the number of parvalbumin-immunoreactive neurons was dramatically reduced at 3 and 5 days after cerebral ischemic induction(p0.05) as compared to 1 day group after ischemia, as well as sham control group. Sighificant reduction of parvalbumin positive neurons in CA1 region of hippocampus was observed at 1 day after cerebral ischemic induction. However, sighificant loss of MAP2 immunoreactivity was observed at 3 day after cerebral ischemia. The loss of parvalbumin-positive neurons and MAP2 immunoreactivity in CA1 region was prevented by pre-administration of GR89696 compared to that of saline-treated ischemic group. Furthermore, protective effect of GR89696 partially reversed by pre[treatment of naltrexone. These data indicate that parvalbumin-positive neurons more sensitively responded to cerebral ischemic damage than MAP2 protein. Moreover, this loss of parvalbumin-positive neurons was effectively prevented by the pretreatment of kappa opioid agonist. It was also suggested that the changes of number in parvalbumin-positive neurons could be used as the specific marker to analyze the degree of ischemic neuronal damage.

This study was conducted to detect the toxoplasma-specific DNA in peripheral blood collected from cats experimentally infected with Toxoplasma gondii (RH strain) and from domiciled cats by B1 gene-base polymerase chain reaction (PCR). The sensitivity of oligonucleotide primer, T-1 & T-2, designed from toxoplasma B1 gene amplification method was compared with parasite detection by mouse inoculation(MI). And also, latex agglutination test(LAT) and indirect fluorescent antibody test(IFAT) were conducted to detect the fluctuation of serum antibodies compared with the detection of toxoplasma by PCR and MI. Toxoplasma B1 gene PCR was shown consistently high sensitivity and the results obtained by PCR agreed completely with those from MI. All blood samples collected before infection with T gondii gave negative results by PCR and MI. Also, toxoplasma B1 gene PCR was not cross reaction with Neospora caninum DNA and normal cat leucocyte as cnotrols. The toxoplasma-specific DNA was detected by PCR in blood of 5 cats experimentally infected with T gondii 6 days after infection and the detection of this specific-DNA was long lastedin blood for 64 days after infeciton. The detection of toxoplasma-specific DNA by PCR could be identified as few as 10 tachyzoites and the isolation of T gondii by MI could be isolated as few as 1 tachyzoite from tenfold serial dilution of T gondii with normal cat blood, respectively. In healthy domiciled cats, the toxoplasma-specific DNA and the parasite were detected and isolated in blood from 3 of 56(5.3%) cats by both PCR and MI, respectively. In the results of antibody test from the total 56 heads of healthy domiciled cats, the positive rates are 15(26.7%) by LAT and 19(33.9%) by IFAT. These results suggest that PCR detection of toxoplasma can be applied as a sensitive and specific diagnostic and research tool.

The contents of histidine dipeptides, a metabolic products of muscle protein, were investigated to compare muscle composition among the 9 domestic animals including cattle. In major domestic animal, analyzed the effects of age, part and sex of the animals on their muscle composition. Large amounts of carnosine(68.63+_17.41 micro mol/g) were detected in cattle and it was higher than other animals. Wheres the content of anserine showed high level in order of turkey, chickens and duck. The ratio of carnosine and anserine(C/A ratio) was different depending on the animal species. Statistical data indicated that difference among species was significant(p0.05). The contents of histidine dipeptides in hearted muscle by boiling for 40 minutes at 110 degrees centigrade was similar to those of raw muscle. C/A ration in heated muscle was not different from that of raw muscle, indicating that no change has been made after heating process. The contents of camosine and anserine were different according to the parts of their muscle. Especially chuck of the mammalian showed extremely low level of histidine dipeptides compared with other parts, while C/A ratio maintained certain level regardless of age, part, sex. Therefore, based on the content of histidine depeptides, could be found the difference of muscle composition among the species. Also C/A ratio of horse, pig, cattle, duck, sheep nd turkey were characteristic respectively.