• Asian-Australasian Journal of Animal Sciences
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• v.30 no.10
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• pp.1471-1477
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• 2017

Objective: Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. Methods: We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). Results: Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. Conclusion: It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an $NF-{\kappa}B$ signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.4999-5005
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• 2013

Objective: This study aimed to investigate expression of the proto-oncogene POK erythroid myeloid ontogenic factor (Pokemon) in colorectal cancer (CRC), and assess inhibitory effects of a small interference RNA (siRNA) expression vector in SW480 and SW620 cells. Methods: Semi-quantitative reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry were performed to determine mRNA and protein expression levels of Pokemon in CRC tissues. Indirect immunofluorescence staining was applied to investigate the location of Pokemon in SW480 and SW620 cells. The siRNA expression vectors that were constructed to express a short hairpin RNA against Pokemon were transfected to the SW480 and SW620 cells with a liposome. Expression levels of Pokemon mRNA and protein were examined by real-time quantitative-fluorescent PCR and western blot analysis. The effects of Pokemon silencing on proliferation of SW480 and SW620 cells were evaluated with reference to growth curves with MTT assays. Results: The mRNA expression level of Pokemon in tumor tissues ($0.845{\pm}0.344$) was significantly higher than that in adjacent tumor specimens ($0.321{\pm}0.197$). The positive expression ratio of Pokemon protein in CRC (87.0%) was significantly higher than that in the adjacent tissues (19.6%). Strong fluorescence staining of Pokemon protein was observed in the cytoplasm of the SW480 and SW620 cells. The inhibition ratios of Pokemon mRNA and protein in the SW480 cells were 83.1% and 73.5% at 48 and 72 h, respectively, compared with those of the negative control cells with the siRNA. In the SW620 cells, the inhibition ratios of Pokemon mRNA and protein were 76.3% and 68.7% at 48 and 72 h, respectively. MTT showed that Pokemon gene silencing inhibited the proliferation of SW480 and SW620 cells. Conclusion: Overexpression of Pokemon in CRC may have a function in carcinogenesis and progression. siRNA expression vectors could effectively inhibit mRNA and protein expression of Pokemon in SW480 and SW620 cells, thereby reducing malignant cell proliferation.

• Restorative Dentistry and Endodontics
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• v.25 no.4
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• pp.587-598
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• 2000

Urea in the oral cavity is hydrolyzed mainly by bacterial ureases to ammonia, which in turn, raises pH of the oral environment, maintaining oral pH homeostasis, thereby inhibiting dental caries. Streptococcus salivarius has been shown to be a major contribution to oral ureolysis. Synthesis of urease by S. salivarius appears to be constitutive, but can be greatly enhanced in the acidic environment. It has been presumed that ureolytic activity of S. salivarius strains isolated from caries-active site is greater than that of strains from caries-free site. However, no in vivo study has supported the presumption. The present study was performed to observe the ureolytic activity of S. salivarius strains isolated from different environments in the same individual, finding out whether the ureolytic activity is related to dental caries. For the purpose, S. salivarius strains were isolated from caries-active site (>C2), a caries-free site of the tooth, and the dorsum of the tongue of each of 50 patients having decayed teeth. The strains isolated from the patients who harbored S. salivarius in more than two sites were selected and then their ureolytic activities were measured. In order to examine clonal diversity of the strains, their ureC genes were amplified by polymerase chain reaction (PCR) and then restricted with EcoRV, and the protein profiles of the strains were compared by SDS-PAGE. The results were as follows: 1. Of 50 patients, 13 patients harbored S. salivarius in more than two sites; a total of 61 S. salivarius strain were isolated from the patients and selected for the study. 2. Of 17 isolates from the caries-active site of 9 patients harboring S. salivarius in more than two sites including carious lesion, 10 (58.8%) showed a high ureolytic activity (> 200 ${\mu}mol/min/mg$). While, 19 out of 44 isolates (43.2%) from the caries-free site of the teeth and the dorsum of the tongues of 13 patients were the strains with a high ureolytic activity. 3. Of 9 patients harboring S. salivarius in more than two sites including caries-active site. 6 patients were found to have the strains in the caries-active site showing a lower ureolytic activity than the strains in the other sites. 4. Of 34 isolates with ureolytic activity higher than 40 ${\mu}mol/min/mg$, 32 isolates produced 0.54-Kbp PCR products regardless of the sites of bacterial collection. In contrast, of 27 isolates with ureolytic activity lower than 40${\mu}mol/min/mg$, 26 isolates yielded 1.3-Kbp PCR products or none regardless of the sites. 5. Different clonal types of S. salivarius with relatively higher and lower ureolytic activities were found in the same individuals and even in the same sites. 6. None of strains showing different ureolytic activity appeared to be the same clonal type. The overall results suggest that ureolytic activity of the isolates does not appear to be related to differences of the environments but related to their own genetic traits.

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.3
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• pp.483-489
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• 2002

APA-01, which is an aqueous extract of five Chinese herbs, is a modified formula of Huoxiang-Zhengqi-San. The effect of new herb extract on immune response was investigated. The parameter examined to assess apparent immune response of APA-01 in mice included changes of body weight, relative weight of immune organs, cell proliferation and cytokine gene expression. The body weight and relative weight of immune organs were not significantly changed among the tested groups. In the spleen cell prolijeration assay, APA-01 increased the cell proliferation in a dose-dependent manner. Methotrexate (MTX), an agent of immune suppression, inhibited the spleen cell proliferation (IC/sub 50/: 800㎍/㎖). However, APA-01 significantly inhibited the suppression of mouse spleen cell proliferation. Therefore, it seems that APA-01 has a reducing effect of immune suppression. Immunomodulatory effect of APA-01 was further investigated using reverse transcription polymerase chain reaction (RT-PCR) in mouse spleen cells. In RT-PCR test, APA-01 enhanced the expression of cyclooxygenase-2 (COX-2) mRNA in a dose-dependent manner. In spite of immune suppression by MTX, COX-2 mRNA was induced by co-treatment with APA-01. These results suggest that APA-01 stimulates the proliferation of spleen cells, regulates the expression of COX-2 mRNA, and accelerates the recovery of inhibition of spleen cell proliferation induced by MTX, thus providing the immunological basis for clinical benefit of APA-01.

• Journal of Veterinary Clinics
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• v.27 no.1
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• pp.1-5
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• 2010

Enterocytozoon bieneusi, a microsporidian species, has emerged as an opportunistic pathogen in AIDS patients. This organism has also been identified in a wide range of animals, and the zoonotic potential of human infections is of particular interest. This study revealed that this organism was found with relatively high prevalence in feces of asymptomatic cattle in Korea. Fecal specimens were obtained from a total of 1,720 cattle in a slaughterhouse located in Chungnam province, Daejeon city and Chonbuk province. After removal of fecal debris by sieving and density gradient centrifugation, samples were examined by microscopic examination and then nested polymerase chain reaction (PCR). Microscopic examination with the modified trichrome staining for the fecal specimens revealed 194 (11.28%) positive calves for microsporidia spore. PCR using the specific primer for E. bieneusi revealed 79 (4.59%) positive calves. The infection ratio of microsporidia was higher in March than other season.

• Journal of Pharmacopuncture
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• v.4 no.1
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• pp.123-124
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• 2001

Purpose. Free radicals are implicated in the pathophysiology of aging, ischemic injury and neurodegenerative disorders. To deform]no whether Hominis Placenta extract prevents $H_2O_2$-induced apoptosis, we have performed morphological and biochemical analyses for the detection of apoptotic phenomena in the pineal tumor cell line $PGT-{\beta}$ We have also peformed cytochemical and immunocytochemical analyses for the detection of changes in nitric oxide synthase (NOS) activity and estimated the expression . of apoptotic genes using reverse transcription-polymerase chain reaction (RT-PCR) Methods. $PGT-{\beta}\;cells$ were pretreated with Hominis Placenta extracts $(0,\;10^{-2}\;{\mu}g/ml)$ for 2 hours and then exposed to $H_2O_2\;(0,\;50\;{\mu}M)$ for 3 hours. Appearance of apoptotic characteristics were monitored using 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining assay, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and flow cytometric analysis. NOS activity was measured by NADPH-diaphorase cytochemistry. Expression of inducible NOS (iNOS) and nuclear factor kappa B (NF k B) was assessed via immunocytochemistry. The expression of apoptotic genes was examined by RT-PCR. Results. After 3 flours of exposure to $H_2O_2$, it was shown that $PGT-{\beta}\;cells$ treated with $H_2O_2(50\;{\mu}M)$ exhibit classical apoptotic features and increases in NOS activity and caspase-3 expression. Treatment with Hominis Placenta extract resulted in a reduced occurrence of apoptotic features. DAPI staining, TUNEL and flow cytometric assays revealed decreases in the occurrence of nuclear fragmentation and in the sub-Gl fraction in the $PGT-{\beta}\;cells$ treated with Hominis Placenta extract. Cells treated with Hominis Placenta extract also showed lower activity of NADPH-diaphorase and immunoreactivities of both iNOS and NF k B than those of $H_2O_2$-treated cells which were not treated with Hominis Placenta extract. By RT-PCR, it was shown that the level of caspase-3 mRNA was derreased In the cells treated with Hominis Placenta . extract. Conclusions. This study shows that Hominis Placenta extract prevents $H_2O_2$-induced apoptosis in $PGT-{\beta}\;cells$; inhibitions of iNOS and caspnse-3 are possible mechanisms of the protection against apoptosis.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1366-1372
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• 2018

Objective: A disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to toll-like receptor 3 (TLR3) stimulation. Methods: The ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly[I:C]; a TLR3 ligand). Results: The chADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very dif­ferent. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly(I:C) stimulation (p<0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling. Conclusion: In this report, the genetic information of chADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important to examine the molecular mechanisms during different regulation, depending on innate immune receptor activation.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.2
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• pp.97-104
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• 2006

Objective: To investigate whether polymorphism of Catechol-O-methyltransferase(COMT) gene is associated with the risk of polycystic ovary syndrome (PCOS) in Korean women. Methods: One hundred and thirty-six PCOS patients and eighty four controls were enrolled. Blood samples were collected from the patients diagnosed according to the 2003 revised criteria of the Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group. Age matched women with regular menstruation from same geographic region were recruited as control subject. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of PCR products were done to determine all individuals' genotype. Results: In women with $COMT^{LL}$ genotype, there was decreased PCOS risk and this difference was statistically significant (OR 0.24, 95% CI 0.11~0.51). Conclusion: The results suggest that the $COMT^{LL}$ genetic polymorphism might be associated with PCOS risk in Korean women.

• Journal of dental hygiene science
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• v.13 no.3
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• pp.296-303
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• 2013

Lysyl oxidase (LOX), extracellular matrix enzyme, is catalyzing lysine-derived crosslinks in collagen and elastin. Recently, several LOX-like proteins (LOXL, LOXL2, LOXL3 and LOXL4) have been identified in human but their specific functions are still largely unknown. The purpose of this study was to evaluate the function of the LOX family genes during odontoblastic differentiation of human dental pulp (HDP) cells induced with odontogenic supplement (OS). The messenger RNA (mRNA) expression of LOX family genes and differentiation markers was assessed by reverse transcriptase polymerase chain reaction analysis (RT-PCR). The formation of mineralization nodules was evaluated by alrizarin red S staining. Amine oxidase activity of HDP cells was measured by peroxidase-coupled fluormetric assay. The expressions of differentiation markers, such as alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), dentin matrix protein1 (DMP1), dentin sialophosphoprotein (DSPP) in HDP cells were increased after treatment with OS media. The LOX and LOXL mRNA expression were gradually increased in OS media, whereas LOX enzyme activities were markedly detected on day 7. The mRNA expression and LOX enzyme activity of collagen type I was very similar to the pattern of LOX gene. In this study, the expression of LOX and its isoforms, and activity of LOX were highly regulated during odontoblastic differentiation. Thus, these results suggest that LOX plays a key role in odontoblastic differentiation of HDP cells.

• Journal of Veterinary Clinics
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• v.27 no.5
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• pp.622-625
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• 2010

Inflammatory polyps in feline ear are nonneoplastic, inflammatory growths that arise from the middle ear or the eustachian tube and extended into the pharynx or external ear canal. Two 2-year-old female Russian blue cats showed 2-3 weeks history of aural discharge, crust formation in external ear, and head or ear shaking. Two masses were surgically excised from ear canal, and submitted for diagnosis. Histopathologically, these masses were covered with hyperplastic ciliated epithelium or nonkeratinizing squamous epithelium with partial erosion and ulceration. The core of masses was consisted of proliferated connective tissue and massive infiltration of mononuclear cells. Immunohistochemically, about 90% of infiltrated mononuclear cells demonstrated CD3 positive T cell. According to both polymerase chain reaction (PCR) and reverse transcriptase-PCR, tissues samples were negative for feline viral pathogens. Based on the clinical, gross, histopathologic findings, these two cases were diagnosed as inflammatory polyps originated from the middle ear in cats.