• Title/Summary/Keyword: reverse-transcription-PCR

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Identification of Ornithogalum mosaic virus isolated from ornithogalum.

  • Chang, Yun-Young;Lee, Hae-Eun;Lee, Jae-Bong;Lee, Key-Woon
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.139.2-140
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    • 2003
  • Ornithogalum showing mosaic symptoms were collected from the isolated field of National Plant Quarantine Service in Sengrimmyon of Kyungnam province. Electron microscopic examination of negatively strained preparation was filamentous particle of 740nm in lenght. Indirect-ELISA determined that the virus was serologically related to potyvirus. A single major protein band of Mr 30,000 was observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Indicator plant test showed masaic, necrotic local lesion and sunken areas in leaves of Nicotiana clevelandii and Tetragonia expansa, while the others of indicator plants did not infect. An enzyme-aided purification protocal was used, which eliminated a highly viscous mucilage from extracts of the Omithogalum. Total RNA extracted from infected Omithogalum leaves were amplified of 411b.p fragment in reverse transcription (RT)-PCR when primers specilic for the coat protein gene. An isolate of Omithogalum mosaic virus (OrMV) of the genus Potyvirus was identified as the casual agent of the disease on the basis of electron microscopic, biological and serological reaction.

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Heme Oxygenase-1(HO-1) induction by UVB(290-320nm) radiation in ICR mice

  • Choi, Wook-Hee;Kim, Tae-Hwan;Ahn, Ryoung-Me
    • Proceedings of the Korean Environmental Health Society Conference
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    • 2005.11a
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    • pp.166-168
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    • 2005
  • The induction of heme oxygenase-1(HO-1) by ultraviolet(UV) radiation provides a protective defense against oxidative stress, and has been well demonstrated in UVA-irradiated skin, but not UVB. In this study in mice, we show that the UVB(290-320nm) radiation can be attributed to the induction of cutaneous heme oxygenase-1. The expression of HO-1 mRNA was assessed in vivo by the reverse transcription-polymerase chain reaction (RT-PCR) analysis, and HO-1 enzyme activity was measured in microsomal preparation from irradiated mice. The mRNA level of HO-1 increases in liver and skin from 24h to 72h after UVB($3KJ/m^3$) radiation. The results of gene expression were same pattern of HO enzyme activity in skin, but not in liver. HO-1 mRNA in liver resulted in a progressive increase to 96h after UVB radiation, but HO activity in liver increased to 48h. This finding indicates that UVB radiation is an important inducer of HO-1 and increases in HO activity may protect tissues directly or indirectly from oxidative stress.

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Heme Oxygenase-1 (HO-1) Induction by UVB (290-320nm) Radiation in ICR Mice (ICR mouse의 피부와 간에서 자외선 B파에 의한 Heme Oxygenase-1의 유도)

  • Choi, Wook-Hee;Kim, Tae-Hwan;Ahn, Ryoung-Me
    • Journal of Environmental Health Sciences
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    • v.34 no.1
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    • pp.49-54
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    • 2008
  • The induction of heme oxygenase-1(HO-1) by UV radiation provides a protective defence against oxidative stress, and has been well demonstrated in skin irradiated with UVA, but not UVB. In this study, we show that the induction of cutaneous HO-l can be attributed to UVB radiation. The expression of HO-1 mRNA was assessed in vivo by reverse transcription-polymerase chain reaction (RT-PCR) analysis, and HO-1 enzyme activity was measured in microsomal preparation from irradiated mice. The mRNA level of HO-1 increases in liver and skin from 1d to 3d after UVB $(3KJ/m^2)$ exposure. The results of gene expression were same pattern of HO-1 enzyme activity in skin, but not in liver. HO-1 mRNA in liver resulted in a progressive increase to 4d after UVB exposure, but HO-1 activity in liver increased to 2d. This finding indicates that UVB radiation is an important inducer of HO-1 and increases in HO activity may protect tissue directly or indirectly from oxidative stress.

Differentially Expressed Genes in Porcine Parthenogenetic 2-cell and Blastocyst Embryos

  • Lee, Hwa-young;Kim, Bong-Ki;Cui, Xiang-Chun;Hwang, Kyu-Chan;Chung, Hak-Jae;Ko, Yeoung-Gyu;Jung, Jin-Kwan;Chang, Won-Kyong;Kim, N. H.
    • Proceedings of the KSAR Conference
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    • 2004.06a
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    • pp.198-198
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    • 2004
  • The identification of embryo-specific genes would provide insights into early embryonic development. However, the current methods employed to identify the genes that are expressed at a specific developmental stage are labor intensive and suffer from high rates of false positives. Here we employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) technology that involves annealing control primers(ACPs) to identify the genes that are specifically expressed in porcine blastocysts compared to 2-cell stage embryos. (omitted)

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In Vitro Expression of the Recombinant hFSH Gene using Retrovirus Vector System (In Vitro에서 Retrovirus Vector System을 이용한 재조합 hFSH 유전자의 발현)

  • Min, Gyeong-Heon;Kwon, Mo-Sun;Kim, Teoan;Koo, Bon-Chul
    • Reproductive and Developmental Biology
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    • v.35 no.1
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    • pp.115-121
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    • 2011
  • hFSH is a glycoprotein secreted from anterior pituitary and consists of ${\alpha}$ and ${\beta}$ subunits. Because of its major biological functions including sperm formation in the male and for follicular growth, FSH is used to cure woman's sterility. In this study we tried to produce recombinant hFSH in vitro using a retrovirus expression vector. Two major components of the vector we constructed are: ( i ) a DNA fragment containing ${\alpha}$ and ${\beta}$ genes fused by a DNA sequence coding carboxyl terminal peptide (CTP) of human chorionic gonadotropin, (ii) a DNA fragment corresponding woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Evaluation of expression profile of the recombinant FSH using reverse transcription PCR and enzyme-linked immunosorbent assay (ELISA). Among three cell lines tested, HeLa cells were the best for hFSH expression (5,395 mIU/ml), then followed by chicken embryonic fibroblast (CEF) cells and Chinese hamster ovary (CHO) cells in the order of hFSH production. In addition to the amount, the FSH produced from HeLa cells was highest in terms of biological activity which was determined by measuring cAMP.

Photoprotective Effect of Topical EGb 761 and Korean Red Ginseng in C57BL/6 Mice

  • Choi, Wook-Hee;Han, Seon-Kyu;Yi, Seh-Yoon;Ann, Hyoung-Soo;Ahn, Ryoung-Me
    • Proceedings of the Korean Environmental Health Society Conference
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    • 2005.06a
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    • pp.306-310
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    • 2005
  • Exposure to UVB radiation can cause diverse biological photodamage to skin. Eeb 761 and Korean red ginseng are the major and most effective natural drug against a variety of oxidative damage. But, the protective effects against UVB radiation have not been clearly identified. In this study, we investigated the protective effect of topical EGb 761 and Korean red ginseng on pigmentation by UVB radiation. Pro-inflammatory cytokines($IL-l{\beta}$, IL-6 and $TNF-{\alpha}$) and melanogenesis proteins(tyrosinase, TRP-1 and TRP-2) mRNA were measured by reverse transcription-polymerase chain reaction(RT-PCR) analysis. The in vivo protection against pigmentation was calculated using chromameter. The mRNA level of IL-lf and TNF-a were increased by UVB irradiation in treated and non-treated group, while no significant changes were observed in IL-6 level. Topical treatment with EGb 761 and Korean red ginseng remarkably reduced expression of tyrosinase, TRP-1 and TRP-2 in the non-irradiated and irradiated skin. Application of EGb 761 and Korean red ginseng significantly protected the WB-induced skin pigmentation and Korean red ginseng was more effective. Our study suggests that topical ECb761 and Korean red ginseng can regulate melanogenic proteins and protect UVB radiation on skin pigmentation.

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Effects of geagibokrounghwan on superoxide dismutase gene expression in experimental mouse fed cholesterol (계지복령환이 Cholesterol 식이 생쥐의 SOD 유전자발현능에 미치는 영향)

  • Hong Yun-Pyo;Ro Sung-Hyun
    • Herbal Formula Science
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    • v.6 no.1
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    • pp.215-226
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    • 1998
  • Geagibokrounghwan (桂技茯笭湯) has long been used to cure human diseases such as vascular and blood disorders. However, it is still unkown on its action mechanism, physiolosical and biochemical meaning. Thus, many attempts were tried to show the scientific background covering the above mentioned mechanism. The effect of Geagibokrounghwan, which was known to date, as follow; effective circulation of body blood system, proliferation of leucocytes and antioxidative action. In this study, we have applied the Geagibokrounghwan administration and feed to mouse, to see effects on the expression of superoxide dismutase(SOD) mRNA as antioxidative agent and oxygen radical scavenger. Total RNAs includingmRNA have been isolated from liver and white blood cells after mice were fed with cholesterol in high dose. Also, in a separate group, the cholesterol-administrated mice were fed with Geagibokrounghwan to see the effects on SOD transcription. and then reverse transcriptase-polymerase chain reaction (RT-PCR) usion each primer set (SOD-F;GATGAAAGCGGTGT-3'; SOD-R; 5'-CCTGTGGAGTGATT-3') were performed to trace theamounts of mRNA. SOD mRNA was specifically expressed in Geagibokrounghwan-fed mice at 2 weeks after treatment, however, gradually reduced after 4 weeks. These results indicate that Geagibokrounghwan is highly applicable in treatment of the above mentioned human diseases.

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Amygdalin Extract from Armeniacae semen Induces Apoptosis in Human COLO 201 Colon Cancer Cells

  • Kim, Kyung-Nam;Song, Yun-Kyung;Lim, Hyung-Ho
    • The Journal of Korean Medicine
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    • v.26 no.4
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    • pp.108-121
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    • 2005
  • Backgrounds: Amygdalin (D-mandelonitrile B-gentiobioside), a cynogenic compound, is found in sweet and bitter almond, Persicae semen, and Armeniacae semen. Aqueous extract of amygdalin was made from Armeniacae semen and used in this study. Objectives: Apoptosis is a very important mechanism in cancer treatment. In the present study, it was investigated whether amygdalin induces apoptotic cell death in human COLO 201 colon cancer cells. Materials and Methods: For this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, flow cytometric analysis, reverse transcription-polymerase chain reaction(PR-PCR), western blot analysis, and caspase-3 enzyme assay were performed on COLO 201 cells. Cells treated with amygdalin exhibited several characteristics of apoptosis. Results: Amygdalin treatment enhanced Bax expression and suppressed Bcl-2 expression in COLO 201 cells. Amygdalin also was shown to increase the caspase-3 activity. Conclusions: Amygdalin induces apoptotic cell death via Bax-dependent caspase-3 activation in COLO 201 cells.

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The Protective Effect of Paeoniae Radix Extract against 1-methyl-4-Phenylpyridium-induced Apoptosis on SK-N-MC Neuroblastoma Cells

  • Chae, Ki-Heon;Song, Yun-Kyung;Lim, Hyung-Ho
    • The Journal of Korean Medicine
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    • v.26 no.4
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    • pp.74-86
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    • 2005
  • Background: There are increasing neuro-degenerative disorders with aging. Paeoniae Radix(PR) possesses various pharmacological effects such as sedative, analgesic, anti-inflammatory, anti-stress and neuro-protective actions. Also antiaging and anti-cancer effects of PR were reported. Our purpose was to investigate whether PR is useful on the treatment of Parkinson's disease, one of the neuro-degenerative disorders. Objective: We investigated whether Paeonia Radix possesses a protective effect against 1-methyl-4 phenylpyridine(MPP+)-induced cytotoxicity in neuronal cells. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, flow cytometry, DNA fragmentation assay, reverse transcription-polymerase chain reaction(RT-PCR), and Western blotting were performed on SK-N-MC neuroblastoma cells. Results: Cells treated with MPP+ exhibited several apoptotic features, while cells pre-treated with Paeonia Radix prior to MPP+ exposure showed s decrease in the occurrence of apoptotic features. Conclusions: These results suggest that Paeonia Radix may exert a protective effect against MPP+-induced apoptosis in SK-N-MC neuroblastoma cells.

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Effect of Berberine on MUC5AC Mucin Gene Expression and Mucin Production from Human Airway Epithelial Cells

  • Sikder, Md. Asaduzzaman;Lee, Hyun-Jae;Lee, Su-Yel;Bae, Heung-Seog;Kim, Jang-Hyun;Chang, Gyu-Tae;Lee, Choong-Jae
    • Biomolecules & Therapeutics
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    • v.19 no.3
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    • pp.320-323
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    • 2011
  • We conducted this study to investigate whether berberine signifi cantly affects MUC5AC mucin gene expression and mucin production induced by epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) from human airway epithelial cells. Confl uent NCI-H292 cells were pretreated with varying concentrations of berberine for 30 min and then stimulated with EGF, PMA or TNF-${\alpha}$ for 24 h. MUC5AC mucin gene expression and mucin production were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Berberine was found to inhibit the expression of MUC5AC mucin gene induced by EGF, PMA or TNF-${\alpha}$. Berberine also inhibited the production of MUC5AC mucin protein stimulated by the same inducers. This result suggests that berberine can regulate the expression of mucin gene and production of mucin protein, by directly acting on human airway epithelial cells.