• Biomedical Science Letters
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• v.17 no.4
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• pp.297-304
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• 2011

Influenza A virus of the Orthomyxoviridae family is a contagious respiratory pathogen that continues to evolve and burden in the human public health. It is able to spread efficiently from human to human and have the potential to cause pandemics with significant morbidity and mortality. It has been estimated that every year about 500 million people are infected with this virus, causing about approximately 0.25 to 0.5 million people deaths worldwide. Influenza A viruses are classified into different subtypes by antigenicity based on their hemagglutinin (HA) and neuraminidase (NA) proteins. The sudden emergence of influenza A virus subtypes and access for epidemiological analysis of this subtypes demanded a rapid development of specific diagnostic tools. Also, rapid identification of the subtypes can help to determine the antiviral treatment, because the different subtypes have a different antiviral drug resistance patterns. In this study, our aim is to detect influenza A virus subtypes by using real-time nucleic acid sequence based amplification (NASBA) which has high sensitivity and specificity through molecular beacon. Real-time NASBA is a method that able to shorten the time compare to other molecular diagnostic tools and is performed by isothermal condition. We selected major pandemic influenza A virus subtypes, H3N2 and H5N1. Three influenza A virus gene fragments such as HA, NA and matrix protein (M) gene were targeted. M gene is distinguished influenza A virus from other influenza virus. We designed specific primers and molecular beacons for HA, NA and M gene, respectively. In brief, the results showed that the specificity of the real-time NASBA was higher than reverse transcription polymerase chain reaction (RT-PCR). In addition, time to positivity (TTP) of this method was shorter than real-time PCR. This study suggests that the rapid detection of neo-appearance pandemic influenza A virus using real-time NASBA has the potential to determine the subtypes.

• Journal of Aquaculture
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• v.19 no.4
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• pp.315-322
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• 2006

The objective of the present study was to investigate the expression of heat shock protein 90 (HSP90) and 70 (HSP70) mRNA as cellular stress responses, the levels of plasma cortisol with glucose as neuro-endocrine stress responses during water temperature rising in freshwater adapted black porgy, Acanthopagrus schlegeli. A cDNA fragment of 891 (HSP90) and 465 (HSP70) bp was cloned from black porgy testis by Reverse transcription-polymerase chain reaction (RT-PCR) with primers designed from the conserved regions of other teleost. The PCR product of HSP90 showed very high homology to red seabream (99%), rainbow trout (95%), Atlantic salmon (94%), zebrafish (94%) HSP90, HSP70 of black porgy was also highly similar to those of rainbow trout (96%), silver seabream (95%), zebrafish (95%) HSP70. Water temperature rising ($20{\sim}30^{\circ}C$) induced elevation of HSP90 mRNA in black porgy gonad, liver, brain, intestine and kidney, whereas it resulted in an induction of the HSP70 mRNA expression in gonad only. Plasma cortisol levels increased significantly at $30^{\circ}C$ in the fish compared to those at $20^{\circ}C$. Glucose levels of the fish showed a tendency of co-increase with cortisol during water temperature rising. These results suggest that increased HSP90 mRNA in liver with plasma cortisol following heat shock may be related to increasing glucose for homeostasis in this species.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.1
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• pp.365-370
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• 2015

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic growth factor and immune modulator. The aim of this study was to evaluate the effects of GM-CSF on the development and cell number of porcine parthenotes, as well as on their expression of implantation-related genes. In the present study, porcine parthenogenatic activated embryos were cultured in a protein-free culture medium in the absence or presence of 5, 10 and 20 ng/ml GM-CSF for 7 days. The percentage of blastocyst formation, total cell number and gene expressions were evaluated. The results showed that the addition of 20 ng/ml GM-CSF to protein-free culture medium significantly increased the blastocoel formation ($26.14{\pm}2.03%$ vs. $3.55{\pm}0.51%$, p < 0.05). In addition, the cell number also increased when they were cultured in the presence of 20 ng/ml GM-CSF ($43.51{\pm}3.6%$ vs. $30.68{\pm}5.51%$, p < 0.05). A real time reverse transcripts polymerase chain reaction (RT-PCR) showed that GM-CSF enhances mRNA expression of the interleukin-6, but does not influence the leukemia inhibitory factor (LIF) receptor mRNA expression in blastocyst stage parthenotes. These results suggest that GM-CSF may enhance the viability of porcine embryos developing in vitro in a defined culture medium.

• Korean Journal of Poultry Science
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• v.41 no.3
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• pp.191-196
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• 2014

The identification and utilization of potential candidate genes with significant effects on economically important traits have become increasingly important in poultry breeding programs. The ovocalyxin-32 (OCX-32) gene is located chromosome 9 in chicken, plays an important role in eggshell formation. This study was performed to assess the association between single nucleotide polymorphisms (SNPs) of OCX-32 gene and egg production traits in the Korean native chicken. Four Korean native chicken population (n = 181; including 46 females of Ogol, 46 females of white, 43 females of gray and 46 females of black) were used to analyze two SNPs (c.494A>C and c.267T>G) in the OCX-32 gene by PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism). We measured egg production traits of age at first egg, first egg weight, egg production ratio and egg weight. The SNP c.494A>C was significantly associated with egg production ratio in Korean Ogol chickens (p<0.001) and egg weight in Korean white chickens (p<0.05). SNP c.267T>C was significantly associated with egg weight in Korean Ogol chickens (p<0.05). But there was no significant association in Korean gray and black chickens. Results suggest the possibility of using molecular markers in OCX-32 gene as a tool for performance and egg production traits in Korean native chicken breeding program.

• Journal of Preventive Medicine and Public Health
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• v.31 no.1 s.60
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• pp.1-14
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• 1998

The genetically determined CYP2D6 activity as considered to be associated with cancer susceptibility with inter-individual variation. Genetic polymorphism of CYP2D6(B) and CYP2D6(T) was determined by the two polymerase chain reaction(PCR) and BstN1 and EcoN1 restriction fragment length polymorphisms(RFLP) for 67 lung cancer cases and 95 healthy volunteer controls. The cases were composed of 26 squamous cell carcinoma, 14 small cell carcinoma, 10 adenocarcinoma, 3 large cell undifferentiated carcinoma, and 14 not histologically diagnosed. The results were gained from the 142 subjects (57 cases and 85 controls) who observed successfully in two PCR and BstNl/EcoN1 RELP. Only one and no mutant allele of the CYP2D6(B) and CYP2D6(T) gene was detected, that is, the frequency of mutant allele was very low; 0.7%(1/142) and 0%(0/142), respectively. Detected mutant allele of the CYP2D6(B) was beterozygous type(WM). The odds ratios for lung cancer susceptibility with CYP2D6(B) and CYP2D6(T) genotype were not calculated. These results are similar to the previous understanding that the mutant allele is very rare in Orientals compared to Caucasians, therefore, it considered that CYP2D6(B) and CYP2D6(T) genotypes have maybe no association with lung cancer susceptibility in Koreans. This is the basic data of CYP2D6(B) and CYP2D6(T) genotypes for Koreans. It would be hepful for further study to determine lung cancer susceptibility of Koreans with the data about CYP1A1, CYP2E1, GSTM1 from future study.

• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.381-388
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• 1996

The hemodynamic changes in septic patients produced by inhalational anesthetics are sufficient to threaten the anesthesiologists. The effect of hydroxocobalamin, a vitamin $B_{12a}$, on contractile responses to phenylephrine during administration of inhalational anesthetics were evaluated in aortic ring preparations obtained from LPS-treated rats. The sepsis was developed by intraperitoneal injection of LPS (1.5 mg/kg for l8h) and confirmed by iNOS expression using RT-PCR. Statistical significances (P<0.05) were analyzed by Student's t-test or paired t-test according to data characteristics. The blood pressure, but not heart rate, was decreased in LPS-treated rats as compared to control rats. The contractile response to phenylephrine were dose-dependently increased from the doses of $10^{-8}\;M$ to that of $10^{-5}$ and were attenuated in LPS-treated rings. Both halothane and enflurane, at the doses of 1 MAC, decreased the contractile responses to phenylephrine while isoflurane did not significantly affect the contractile responses. Hydroxocobalamin ($10^{-5}$ M) significantly potentiated the contractile responses in the LPS-treated aortic ring preparations during administration of each inhalational anesthetic or not. From these results, it is suggested that hydroxocobalamin may improve the hemodynamics of septic patients during inhalational anesthesia. Abbreviations: LPS, lipopolysaccharide; RT-PCR, reverse transcription-polymerase chain reaction; MAC, minimum alveolar concentration; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.109-122
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• 2001

Gingival fibroblasts(GF) and periodontal ligament fibroblasts(PDLF) are the major cellular components of periodontal soft connective tissues, but the precise molecular biological differences between these cells are not yet known. In the present study, we investigated the expression of S100A4, S100A2 calcium-binding protein and osteoblast-specific factor 2(OSF-2, Periostin) mRNA in GF and PDLF in vitro through the process of reverse transcription-polymerase chain reaction(RT-PCR) and Northern blot analysis in each. Human GF and PDLF were isolated from the gingival connective tissue and the middle third of freshly extracted healthy third molars. They were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 10% fetal bovine serum and cells in the third passage were used in the experiments. After extracting total RNA from cultured cells, RT-PCR and Northern analysis were performed using S100A4-, S100A2- and Periostin-specific oligonucleotide primers and subcloned cDNA probes in each. In PT-PCR and Northern analysis, the expression of S100A4 and Periostin mRNA in GF was slightly detectable. Interestingly, the expression of S100A4 and periostin mRNA in PDLF was much higher than that in GF. On the other hand, S100A2 mPNA was highly expressed in both GF and PDLF. Since there was a marked difference of S100A4 and Periostin expression between GF and PDLF in vitro, these data suggest that S100A4 and periostin could be used as a useful marker for distinguishing cultured gingival fibroblasts and periodontal ligament cells.

• Journal of Aquaculture
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• v.9 no.4
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• pp.445-452
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• 1996

Endogeneous activities of ${\beta}-galactosidase$-like enzyme in various tissues from several finfishes and shellfishes were examined by histochemical analysis based on X-gal staining and by fluorimetric measurement using 4-methylumbelliferyl-${\beta}$-D-galactoside (4-MUG). Species used in this study were 3 freshwater fishes, mud loach (Misgurnus mizolepis), common carp (Cyprinus carpio) and tilapia (Oreochromis niloticus) ; 3 marine fishes, olive flounder (Paralichthys olivaceus), stone flounder (Kareius bicoloratus) and marbled sole (Limanda yokohamae) ; and 4 shellfishes, abalone (Haliotis discus hannai), Pacific oyster (Crassoskra gigas), pearl oyster (Pinctada fucata martensii) and ark shell (Anadara broughtonii). The activities of ${\beta}-galactosidase$-like enzyme in all finfishes examined were significantly different among species, with the wide variations between tissues in a species. In general, the tissues such as kidney, intestine and liver were ones which showed the significantly higher values in 4-MUG fluorimetry and deeper staining patterns in X-gal analysis compared to other tissues. On the other hand, serum and muscle revealed the significantly lower activities than others did, regardless of species. Shellfishes were also found to have endogenous activities of ${\beta}-galactosidase$-like enzyme which were significantly varied depending on both species and organs in a species. Hepatopancreas from all shellfishes examined showed the deepest pattern in X-gal staining and also the highest value in 4-MUG analysis, while activities of ${\beta}-galactosidase$-like enzyme in adductor muscles and mantle muscles from all shellfish species in this study except Pacific oyster were negligible : Pacific oyster had the significant activity of this enzyme in muscle tissues. Putative endogenous lacZ fragment was amplified from both finfishes and shellfishes by polymerase chain reaction (PCR). The molecular size of PCR products was about 510 bp, and there was no difference in size among species examined.

• Korean journal of applied entomology
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• v.36 no.3
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• pp.206-214
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• 1997

The genetic relationships among the twenty parental silkworm, Bombyx mori strains authorized in Korea were evaluated using RAPDs-PCR(Random Amplified Polymorphic DNAs-Polymerase Chain Reaction). Twenty-six different 10-mer oligonucleotide primers were used to screen genetic characteristics of parental twenty silkworm strains by RAPD-PCR analysis. 24 primers showed different banding patterns among the strains. Based on these RAPD patterns, the genetic relationships among the silkworm strains were analyzed by UPGMA(Unweighed Pair-Group Method with Arithmetic average) method. The phylogenetic relationships in the twenty silkworm strains were classified into two major sub-groups at the genetic similarity coefficient of 0.60. The first sub-group included Jaml13, Jaml 19, Jaml20, Jam123, Jam1 25 and Jam 127. Jamll4, Jam1 2 I, Jam 122, Jam 124, Jam1 26, Jam 128, Jam129, Jam 130, Jam 131, Jam1 32, Jam133, Jam134, Jam301 and Jam302 were included in the 2nd group. The genetic distance values among Jam1 14, Jam120 and Jam127 were lower than those among the other strains, while Jam129 is very closely related to Jam131 as showing coefficient value of 1 .O.

• Parasites, Hosts and Diseases
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• v.34 no.2
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• pp.135-142
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• 1996

Antigenic domain of jai or surface protein (p30) of Toxoplosmc Sondii was analyzed after polymerase chain reaction (PCR) of its gene fragments. Hydrophilic or hydrophobic moiety of amino acid sequences were expressed as glutathione S-transferase (G57) fusion proteins. Fragments of p30 gene were as follows: 737, total p30 open reading frame (ORF) ; S28, total ORF excluding N-terminal signal sequence and C-terminal hydrophobic sequence; Al9, N-terminal 2/3 parts of A28; A19, N-terminal 2/3 of S28; P9, C-terminal 2/3 part of S28; Z9. middle 1/3 of S28; and 29, C-terminal 1/3 of S28. respectively. Primer of each fragment was synthesized to include clamp sequence of EcoR I restriction site. PCR amplified DNA was inserted info GST (26 kDa) expression vector, PGEX-47-1 to transform into Escheri,hia coei (.JM105 strain). G57 fusion proteins were expressed with IPTG induction as 63. 54, 45, 45, 35, 36. and 35 kDa proteins measured by SDS-PAGE. Each fusion protein was confirmed with G57 detection kit. Western blot analysis with the serum of a toxoplasmosis patient revealed antigenicity in proteins expressed by T37. S28, and Al9 but not those by Pl8. X9, Y10, and Z9. Antigenicity of p30 seems to be located either in N-terminal 115 part in the presence of middle 1/3 part or in the oligopeptides between margins of the first and second 1/3 parts.