• Title/Summary/Keyword: Reverse transcriptase polymerase chain reaction

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Effect of Taurine on Sperm Characteristics and Gene Expressions in Fresh Boar Semen during In Vitro Storage

  • 장현용;정혜윤;조명현;정희태;박춘곤;김정익;양부근
    • Proceedings of the KSAR Conference
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    • 2004.06a
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    • pp.227-227
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    • 2004
  • The purpose of this study was to investigate the effect of taurine on sperm characteristics and gene expressions(bax and Gpx) in fresh boar semen during in vitro storage. The motility of spermatozoa in Modena, Modana plus taurine 25 mM, Modana plus taurine 50 mM, Modana plus taurine 75 mM and Modana plus taurine 100 mM were 63.1%, 65.1%, 65.3%, 82.5% and 80.8%, respectively. (omitted)

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Cloning of a matrix metalloproteinase cDNA from Scylliorhinus torazame (두툽상어 matrix metalloproteinase 유전자 cDNA의 클로닝)

  • Kim, Jon Won;Cho, Won Jin;Chun, Kwang Ho;Kim, Kyu-Won;Kim, Yung-Jin;Lee, Sang-Jun;Shin, Hae-Ja;Lim, Woon Ki
    • Journal of Life Science
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    • v.8 no.3
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    • pp.235-240
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    • 1998
  • Matrix metalloproteinases(MMPs) are a group of zinc enzymes responsible for degradation of the matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling and in many disease processes such as arthritis, cancer, periodontitis, and osteprocess. Genetically distince MMPs have been characterized and their genes have been cloned thus far from a variaty of species but not from fishes. In this stydy, a mmp cDNA was cloned by using RT-PCR(reverse transcriptase dependent polymerase chain reaction) from Scylliorhinus toraxzame(shark), agroup of cartilaginous fish, abundant in the coast of Pusan, Korea. It has 74% base homologue with membrane type matrix matalloproteinase-3 genes(mt3-mmps) from human, rat and chick, and also shows more than 90% residue homologue with them. In addition, it has cysteine switch domain, zinc binding domain(HExGH motif), propeptide cleavage site, and RRKR motif, which are present in MMPs. This result indicates that cDNA fragment cloned here may be mt3-mmp or its analogous gejne cDNA fragment of Scylliorhinus torzame.

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Studies on Inhibitory Effect of Inflammatory Cytokines Secretion from Brain Astrocytes by Polygala Tenuifolia (원지(遠志)에 의한 뇌(腦) 성장세포(星狀細胞)로부터 염증성(炎症性) 세포활성물질(細胞活性物質) 분필(分泌)의 억제(抑制) 효과(效果)에 관(關)한 연구(硏究))

  • Hwang Si-Young;Gang Hyeong-Won;Lyu Yeong-Su
    • Journal of Oriental Neuropsychiatry
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    • v.10 no.1
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    • pp.95-108
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    • 1999
  • We investigated whether an aqueous extract of Polygala tenuifolia root (PTAE) inhibits secretion of inflammatory cytokines from primary cultures of mouse astrocytes. PTAE dose-dependently inhibited the Tumor necrosis $factor-{\alpha}$ $(TNF-{\alpha})$ secretion by astrocytes stimulated with substance P (SP) and lipopolysaccharide (LPS). Interleukin-1 (IL-1) has been shown to elevate $TNF-{\alpha}$ secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore also investigated whether IL-1 mediated inhibition of $TNF-{\alpha}$ secretion from primary astrocytes by PTAE. Treatment of PTAE to astrocytes stimulated with both LPS and SP decreased IL-1 secretion to the level observed with LPS alone. Moreover, incubation of astrocytes with IL-1 antibody abolished the synergistic cooperative effect of LPS and SP. Reverse transcriptase-polymerase chain reaction analysis demonstrated the significantly reduced level of the $TNF-{\alpha}$ mRNA was expressed in astrocytes treated with PTAE. These results suggest that PTAE has an antiinflammatory activity on the central nervous system curing some pathological disease states.

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Molecular cloning and characterization of metallothionein cDNA gene in channel catfish (챠넬메기의 metallothionein cDNA 유전자의 cloning 및 그 특성에 관한 연구)

  • Lee, In-Jung;Song, Young-Hwan
    • Journal of fish pathology
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    • v.5 no.2
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    • pp.143-152
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    • 1992
  • Metallothionein is an essential and common protein to regulate the intracellular concentration of heavy metals, which exist in most organisms from bacteria to vertebrates. Although the detailed function of metallothianein has not been fully identified until yet, it may be involoved in the cellular protection against the heavy metal toxicity and in the global regulation of several other genes and the expression of metalloproteins. We have cloned the full cDNA clone of metallothionein gene in Channel Catfish by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) starting from poly(A)-containing mRNAs. All PCR fragments have been subcloned into EcoRV site of pBluescript SK+ and dT-tailed at Smal site of pUC19, then PCR products are recovered by the double digestion of recombinant plasmids wiht EcoRI and HindIII, which are adjacent to EcoRV site in multicloning sites or by rapid PCR screening. The nucleotide sequence analysis of pMT150(one of the PCR clones) showed high homology with several other piscine metallothionein cDNA genes.

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Cloning of Bovine Macrophage Colony-stimulating Factor

  • Kim, Tae-Yung;Kim, Cheol-Ho;Lee, Sang-Gil;Kang, Chung-Boo
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.6
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    • pp.892-897
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    • 2005
  • Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.

Acute viral gastroenteritis in children hospitalized in Iksan, Korea during December 2010-June 2011

  • So, Cheol Whoan;Kim, Dong Sup;Yu, Seung Taek;Cho, Ji-Hyun;Kim, Jong Duck
    • Clinical and Experimental Pediatrics
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    • v.56 no.9
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    • pp.383-388
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    • 2013
  • Purpose: Viral etiology is common in cases of children with acute diarrhea, and antibiotic therapy is usually not required. Therefore, it is important to determine the distribution of common viruses among children hospitalized with acute diarrhea. Methods: We included 186 children who suffered from acute diarrhea and were hospitalized at the Wonkwang University Hospital Pediatric ward from December 1, 2010 to June 30, 2011 in this study. Stool samples were collected and multiplex reverse transcriptase polymerase chain reaction (multiplex RT-PCR) was used to simultaneously determine the viral etiology such as rotavirus, norovirus, astrovirus, or adenovirus. Results: Causative viruses were detected in 72 of the 186 cases (38.7%). The mean age of the virus-positive cases was 1 year and 9 months (range, 1 month to 11 years). Rotavirus was detected in 50/186 (26.9%); norovirus, in 18/186 (9.7%); and astrovirus, in 3/186 cases (1.6%). Adenovirus was not detected in any of the cases. Proportions of norovirus genogroups I and II were 21.1% and 78.9%, respectively. Four of the 51 rotavirus-positive cases (7.8%) had received rotavirus vaccination at least once. The mean duration of diarrhea was 2.8 days (range, 1 to 10 days) and vomiting occurred in 39 of the 72 cases (54.2%). Conclusion: Viral etiology was confirmed in about one-third of the children with acute diarrhea, and the most common viral agent was rotavirus, followed by norovirus.

A Case of Vertical Transmission of Hepatitis C Virus in an Infant of a Mother Who had Hepatitis C during Pregnancy (C형 간염 산모로부터 출생한 영아에서 C형 간염 Virus의 수직 전파 1례)

  • Oh, Sang-Hyun;Kim, Kuk-Hwan;Yang, Eun-Seok;Park, Sang-Kee;Moon, Kyung-Rye
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.2 no.1
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    • pp.109-115
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    • 1999
  • Hepatitis C virus (HCV) has been identified as an important cause of posttransfusion hepatitis, but vertical transmission of chronic infected HCV RNA positive mothers has been documented in some cases. The reports of the risk of perinatal infection have been widely varied in the literature. The authors experienced one case of vertical transmission of HCV in an infant of a mother who had hepatitis C during pregnancy. At admission, HCV RNA (+), Ig G anti HCV (+) and Ig M anti HCV (+) were found in the mother. Also at admission, HCV RNA (+), Ig G anti HCV (+), Ig M anti HCV (+), elevation of liver aminotransferase level and hepatosplenomegaly on ultrasonography were found in the baby on day 31. HCV RNA (-), Ig M anti HCV (-) and normal of liver aminotransferase level were noted on day 250 in the serum of the infant. We used reverse transcriptase polymerase chain reaction (RT-PCR) technique to find a very small amount of HCV RNA in the serum. All the findings suggest vertical transmission of HCV RNA from mother to infant during 3rd trimester of pregnancy.

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Detection rate and clinical impact of respiratory viruses in children with Kawasaki disease

  • Kim, Ja Hye;Yu, Jeong Jin;Lee, Jina;Kim, Mi-Na;Ko, Hong Ki;Choi, Hyung Soon;Kim, Young-Hwue;Ko, Jae-Kon
    • Clinical and Experimental Pediatrics
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    • v.55 no.12
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    • pp.470-473
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    • 2012
  • Purpose: The purpose of this prospective case-control study was to survey the detection rate of respiratory viruses in children with Kawasaki disease (KD) by using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR), and to investigate the clinical implications of the prevalence of respiratory viruses during the acute phase of KD. Methods: RT-PCR assays were carried out to screen for the presence of respiratory syncytial virus A and B, adenovirus, rhinovirus, parainfluenza viruses 1 to 4, influenza virus A and B, metapneumovirus, bocavirus, coronavirus OC43/229E and NL63, and enterovirus in nasopharyngeal secretions of 55 KD patients and 78 control subjects. Results: Virus detection rates in KD patients and control subjects were 32.7% and 30.8%, respectively (P=0.811). However, there was no significant association between the presence of any of the 15 viruses and the incidence of KD. Comparisons between the 18 patients with positive RT-PCR results and the other 37 KD patients revealed no significant differences in terms of clinical findings (including the prevalence of incomplete presentation of the disease) and coronary artery diameter. Conclusion: A positive RT-PCR for currently epidemic respiratory viruses should not be used as an evidence against the diagnosis of KD. These viruses were not associated with the incomplete presentation of KD and coronary artery dilatation.

Development of Reverse Transcriptase-Polymerase Chain Reaction of fimA Gene to Detect Viable Salmonella in Milk (우유 내 활력있는 Salmonella를 검출하기 위한 fimA 유전자의 역전사중합효소 연쇄반응의 개발)

  • Choi, S.H.;Lee, S.B.
    • Journal of Animal Science and Technology
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    • v.46 no.5
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    • pp.841-848
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    • 2004
  • Rapid detection of viable Salmonella in pasteurized milk is important to protect public health from food poisoning. Reverse transcriptase-polymerase chain reaction(RT-PCR) is recognized as a molecular genetical method to differentiate between live and dead bacteria The RT-PCR in this study was designed to detect specifically viable Salmonella in milk by using the primers whose nucleotide sequences were determined based on fimA gene which encodes the submit of type 1 fimbriae. Treatment of RNA preparation with RNase-free DNase was adequate enough to destroy DNA, which may otherwise be amplified in the RT PCR Seven strains of Salmonella were detected in the RT-PCR but Escherichia coli, Shigella sonnei, Citrobacter freundii, and Klebsiella pneumoniae were not. $10^7/ml$ and $10^6/ml$ of dead Salmonella which were heat-treated in milk were detectable by using the RT-PCR but $10^5{\sim}10/ml$ of the dead bacteria were not. The sensitivity of the RT-PCR in detecting viable Salmonella was 100 cells/ml.