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

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Estradiol Valerate Exerts Neuroprotective Effects in Ischemic Rat Brain when Administered after Middle Cerebral Artery Occlusion

  • Yoo, Seong-Jin;Yu, Jeong-Min;Youm, Mi-Young;Kim, Do-Rim;Kim, Jee-Yun;Kang, Sung-Goo
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.111-111
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    • 2002
  • Stroke occurs when local thrombosis, embolic particle or the rupture of blood vessele interrupts the blood floe to the brain. $\beta$-estradiol 17-valerate has been reported to exert neuroprotective effects when administered before an ischemic insult. Recently, the pathophysiology of cerebral ischemia has been studied extensively in rat with various methods. In the present study, we investigates whether $\beta$-estrodiol 17-valerate can protect against brain injury. RNA sample were extracted from the hippocampus of female rat, reverse-transcription in the presence of [$\alpha$32p] dATP. Differential gene express-ion profiles were revealed (Bone morphogenetic protein type 1A receptor, Protein disulphide isomerase, Leukemia inhibitor factor receptor, cytochrome bc- 1 complex-x core P, thiol-specific antioxidant protein). RT-PCR was used to validate the relative expression pattern obtained by the cDNA array. The precise relationship between the early expression of recovery genes and stroke is a matter of luther investigation. This Study was supported by the Korea Science and Engineering Foundation(KOSEF) through the Biohealth Products Research Center(BPRC), Inje University, Korea.

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Effect of temperature on the Concentration of Chrysanthemum stunt viroid in CSVd-infected Chrysanthemum

  • Chung, Bong-Nam;Huh, Eun-Joo;Kim, Jeong-Soo
    • The Plant Pathology Journal
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    • v.22 no.2
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    • pp.152-154
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    • 2006
  • CSVd-infected chrysanthemum plants grown under $10^{\circ}C\;or\;15^{\circ}C$ growth chamber for 2 months resulted a higher dilution endpoint of template RNA for Reverse transcription and polymerase chain reaction (RT-PCR) than those grown for 1 month: $10^{-4}(1.35{\times}10^{-2}ug/ml)$ for 1 month, and $10^{-3}(1.35{\times}10^{-1}ug/ml)$ for 2 months. Independent experiment, shoots cut from CSVd (Chrysanthemum stunt viroid)-in footed chrysanthemum plants grown under $10^{\circ}C\;or\;20^{\circ}C$ growth chamber for 2 months showed the same CSVd concentration as control($30^{\circ}C$) at 8 weeks after moving them to normal green-house condition($30^{\circ}C$). From those results, it was concluded that even though the concentration of CSVd was reduced in plants grown at low temperatures, when they were moved to normal glass-house temperature CSVd concentration increased to that of untreated plants in 8 weeks. This conclusion was supported by the rapid replication of CSVd in chrysanthemum after infection.

A Subpopulation of RNA3 of Cucumber mosaic virus Quasispecies

  • Park, Seung-Kook;Park, Sun-Hee;Yoon, Ju-Yeon;Park, Jang-Kyung;Ryu, Ki-Hyun
    • The Plant Pathology Journal
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    • v.19 no.4
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    • pp.210-216
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    • 2003
  • This study examined the existence of genetically diverse population of Cucumber mosaic virus (CMV), known as quasispecies, from lily, Nicotiana benthamiana and from purified virions. Based on the conserved sequences of CMV lily isolates in intergenic region (IR) on RNA3, the genetic variation of IR from three different sources was investigated by a specific restriction endonuclease hydrolysis of amplified reverse transcription-polymerase chain reaction (RT-PCR) products using virus-specific primers, and was compared with IR sequences. The IR nucleotide sequences of CMV lily isolates were highly conserved, however, quasispecies was detected from all three sources in low level, containing sub-populations of RNA3. These subpopulations of RNA3 were inoculated onto zucchini squash by in vitro transcripts from corresponding full-length cDNA clones together with Eny RNA1 and 2 transcripts. The systemic symptom of zucchini plants infected by these quasispecies was chlorotic spotting, which was milder than severe mosaic and stunt symptom caused by Eny-CMV. The severity of symptom was correlated with RNA accumulation of viruses. These results suggest that the genome of CMV lily isolates consists of quasispecies populations.

Determination of Complete Genome Sequence of Korean Isolate of Potato virus X

  • Choi, Sun-Hee;Ryu, Ki-Hyun
    • The Plant Pathology Journal
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    • v.24 no.3
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    • pp.361-364
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    • 2008
  • The complete nucleotide sequences of a Korean isolate of Potato virus X(PVX-Kr) has been determined. Full-length cDNA of PVX-Kr has been directly amplified by long template reverse transcription and polymerase chain reaction(RT-PCR) using virus specific 5'-end primer and 3'-end primer, and then constructed in a plasmid vector. Consecutive subclones of a full-length cDNA clone were constructed to identify whole genome sequence of the virus. Total nucleotide sequences of genome of PVX-Kr were 6,435 excluding one adenine at poly A tail, and genome organization was identical with that of typical PVX species. Comparison of whole genome sequence of PVX-Kr with those of European and South American isolates showed 95.4-96.8% and 77.4-77.9%, in nucleotide similarity, respectively. Sequenced PVX-Kr in this study and twelve isolates already reported could be divided into two subgroups in phylogeny based on their complete nucleotide sequences. Phylogenetic tree analysis demonstrated that PVX-Kr was clustered with European and Asian isolates(Taiwan, os, bs, Kr, S, X3, UK3, ROTH1, Tula) in the same subgroup and South American isolates(CP, CP2, CP4, HB) were clustered in the other subgroup.

Curcumin Effect on MMPs and TIMPs Genes in a Breast Cancer Cell Line

  • Hassan, Zeinab Korany;Daghestani, Maha Hassan
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.7
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    • pp.3259-3264
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    • 2012
  • Curcumin (CM) possesses anti-cancer activity against a variety of tumors. Matrix metalloproteinases (MMPs) play an important role in remodeling the extracellular matrix and their activities are regulated by tissue inhibitor of metalloproteinases (TIMPs) family. Control of MMP and TIMP activity are now of great significance. In this study, the effect of CM is investigated on metastatic MMPs and anti-metastatic TIMPs genes on MDA breast cancer cells cultured in a mixture of DMEM and Ham's F12 medium and treated with different concentrations of CM (10, 20 and $40{\mu}M$ for various lengths of time. Reverse transcription followed by quantitative real time PCR was used to detect the gene expression levels of MMPs and TIMPs in CM-treated versus untreated cases and the data were analyzed by one-way ANOVA. At high concentrations of curcumin, TIMP-1, -2, -3 and -4 genes were up-regulated after 48 hours of treatment, their over-expression being accompanied by down-regulation of MMP-2 and MMP-9 gene expression levels in a concentration- and time-dependent manner. These results suggest that curcumin plays a role in regulating cell metastasis by inhibiting MMP-2 and MMP-9 and up-regulating TIMP1 and TIMP4 gene expression in breast cancer cells.

SLC35B2 Expression is Associated with a Poor Prognosis of Invasive Ductal Breast Carcinoma

  • Chim-ong, Anongruk;Thawornkuno, Charin;Chavalitshewinkoon-Petmitr, Porntip;Punyarit, Phaibul;Petmitr, Songsak
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.15
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    • pp.6065-6070
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    • 2014
  • Background: Breast cancer is the most common malignancy in women worldwide, including Thailand, and is a major cause of mortality and morbidity, despite advances in diagnosis and treatment. Novel gene expression in breast cancer is a focus in searches for prognostic biomarkers and new therapeutic targets. Materials and Methods: The mRNA expression of novel B4GALT4, SLC35B2, and WDHD1 genes in breast cancer were examined in invasive ductal breast carcinoma (IDC) patients using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). Results: Among these genes, increased expression of SLC35B2 mRNA was significantly associated with TNM stage III + IV of IDC (p<0.001). Hence, up-regulation of SLC35B2 may serve as a prognostic biomarker for poor prognosis, and is also a potential therapeutic target in breast cancer.

Effects of substance P on mineralization markers and heme oxygenase-1 Expression in human immortalized periodontal ligament cells

  • Cho, You-Min;Suh, Chung-Hwan;Chun, Sang-Woo;Kim, Eun-Cheol;Kang, Kyung-Hwa
    • International Journal of Oral Biology
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    • v.33 no.4
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    • pp.131-135
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    • 2008
  • Substance P (SP) is known to be expressed in the nerve fibers of dental pulp and periodontal tissues. It was recently reported that SP expression increased in response to orthodontic force. In the present study, we investigated the effect of SP on expression of mineralization markers and heme oxygenase-1 (HO-1) in human immortalized periodontal ligament (IPDL) cells. Cell viability was measured using a 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of mineralization markers, including alkaline phosphatase (ALP), osteonectin (ON) and bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. SP did not significantly change human IPDL cell viability, with the exception of the 24 hour treatment group. Treatment of human IPDL cells with $10^{-10}$ to $10^{-4}M$ SP upregulated mineralization marker and HO-1 expression in a time- and concentration-dependent manner. Our results suggest that SP may modulate osteoblastic cell differentiation of human IPDL cells through a mechanism involving HO-1 expression.

Antiaging Effects of Musa sapientum L. (Banana) Leaf Extract (바나나 잎 추출물의 주름개선 효과)

  • Yoo, Dae Sung;Jang, Su Jin;Park, Yun Jeong;Kim, Seung Hoi;Hwang, Hyung-seo
    • KSBB Journal
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    • v.31 no.2
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    • pp.126-134
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    • 2016
  • To examine the possibility of using Musa sapientum L. (Banana) leaf extract as a cosmetic raw material, banana leaves grown in Jeju Island were extracted with 70% ethanol. Polysaccharides present in banana leaf extract were discarded by precipitation with cold ethanol. Polysaccharide-discarded banana leaf extract promoted procollagen and COL1A1 gene expression, but inhibited matrix metalloproteinase (MMP)-1 and MMP-2 gene expression in human skin fibroblasts when examined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The active compound in banana leaves was identified by fractionation with various solvents. The chloroform fraction showed the highest anti-wrinkle efficacy and the active compound of chloroform fraction was identified as corosolic acid by NMR, FT-IR, EA, and HPLC-MS. In addition, banana leaf extract showed anti-oxidative efficacy with an IC50 value of 67.91 ppm, as determined by DPPH free radical scavenging assay. Finally, the anti-wrinkle efficacy of banana leaf extract-containing cream was confirmed by clinical tests. Based on these results, banana leaves could have an application as a cosmetic raw material with anti-wrinkle efficacy.

Proinflammatory Effects of Bacterial Lipopolysaccharide (LPS) in Rainbow Trout (Oncorhynchus mykiss) Macrophage Cells

  • Hong Suhee;Jeong Hyun Do
    • Fisheries and Aquatic Sciences
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    • v.6 no.3
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    • pp.130-134
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    • 2003
  • Proinflammatory effects of bacterial lipopolysaccharide (LPS) have been assessed by analysing the induction of two inflammatory genes, $interleukin-1\beta$ $(IL-1\beta)$ and cyclooxygenase-2 (COX-2), in rainbow trout (Oncorhynchus mykiss) macrophage cells. Production of a metabolite of arachidonic acid by COX-2, prostaglandin $E_2\;(PGE_2)$, was also analysed in macrophage cells after LPS stimulation. Northern blot analysis revealed that LPS $(5{\mu}g/mL)$ significantly upregulated $IL-1\beta$ (54 times) and COX-2 (40.7 times) gene expression in macrophage cells after 4 h stimulation. According to RT-PCR (Reverse Transcription Polymerase Chain Reaction) analysis, $IL-1\beta$ gene induction in LPS stimulated macrophage cells was started within 1h and significantly increased thereafter until 4h. Meanwhile, COX-2 gene induction by LPS was delayed in comparison with $IL-1\beta$ gene induction as a faint band was observed after 4h stimulation in head kidney macrophage cells. LPS also significantly increased $PGE_2$ production in head kidney leucocytes, presumably via activating COX-2 expression that metabolites arachidonic acid to $PGE_2$. In conclusion, it was demonstrated that LPS could induce two main inflammatory and immune related genes, $IL-1\beta$ and COX-2, and increase $PGE_2$ production in trout head kidney macrophage cells, representing a strong inflammatory activity.

Expressions of Semenogelin Gene in Male Syrian Hamsters according to Photoperiod

  • Kim, Tae Hong;Kim, Hyeon Jeong;Lee, Sung-Ho;Cheon, Yong-Pil;Choi, Donchan
    • Development and Reproduction
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    • v.23 no.4
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    • pp.355-365
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    • 2019
  • The morphogenetically matured spermatozoa (sperm) are generated in the testes by the spermatogenesis. They travel male reproductive tract with many substances secreted from the accessory reproductive organs. One of the substances is the semenogelin (SEMG) released from the seminal vesicles that is involved in the post-testicular maturation. The expression of SEMG gene was investigated in seminal vesicle tissues of sexually matured and regressed male Syrian hamsters by reverse transcription polymerase chain reaction (RT-PCR). The SEMG gene was uniquely identified in the seminal vesicles of the matured Syrian hamsters and compared to the genes reported previously. But the expression of SEMG gene was not observed in reproductively and completely regressed testes of Syrian hamsters. These results indicate that the expressions of the SEMG gene are related to the reproductive capability in the male Syrian hamsters.