• Title/Summary/Keyword: Differential display

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AMP-activated protein kinase determines apoptotic sensitivity of cancer cells to ginsenoside-Rh2

  • Kim, Min-Jung;Yun, Hee;Kim, Dong-Hyun;Kang, Insug;Choe, Wonchae;Kim, Sung-Soo;Ha, Joohun
    • Journal of Ginseng Research
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    • v.38 no.1
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    • pp.16-21
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    • 2014
  • Ginseng saponins exert various important pharmacological effects with regard to the control of many diseases, including cancer. In this study, the anticancer effect of ginsenosides on human cancer cells was investigated and compared. Among the tested compounds, ginsenoside-Rh2 displays the highest inhibitory effect on cell viability in HepG2 cells. Ginsenoside-Rh2, a ginseng saponin isolated from the root of Panax ginseng, has been suggested to have potential as an anticancer agent, but the underlying mechanisms remain elusive. In the present study, we have shown that cancer cells have differential sensitivity to ginsenoside-Rh2-induced apoptosis, raising questions regarding the specific mechanisms responsible for the discrepant sensitivity to ginsenoside-Rh2. In this study, we demonstrate that AMP-activated protein kinase (AMPK) is a survival factor under ginsenoside-Rh2 treatment in cancer cells. Cancer cells with acute responsiveness of AMPK display a relative resistance to ginsenoside-Rh2, but cotreatment with AMPK inhibitor resulted in a marked increase of ginsenoside-Rh2-induced apoptosis. We also observed that p38 MAPK (mitogen-activated protein kinase) acts as another survival factor under ginsenoside-Rh2 treatment, but there was no signaling crosstalk between AMPK and p38 MAPK, suggesting that combination with inhibitor of AMPK or p38 MAPK can augment the anticancer potential of ginsenoside Rh2.

Genes involved in leaf senescence and regulation of their expression

  • Watanabe, Akira;Fujiki, Yuki;Yoshikawa, Yoko;Biswall, Basanti;Ito, Masaki
    • Proceedings of the Botanical Society of Korea Conference
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    • 1999.07a
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    • pp.63-67
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    • 1999
  • We have isolated more than a dozen cDNA clones corresponding to genes that were expressed in Arabidopsis leaves when they were kept in the dark. The nucleotide sequence analysis showed that some of the clones encoded proteins with significant homology to $\beta$-glucosidase (din2), branched-chain $\alpha$-keto acid dehydrogenase subunit E1$\beta$(din3), and another subunit E2 (din4), yeast RAD23 (din5), asparagine synthetase (din6), pre-mRNA splicing factor SRp35 (din7), phosphomannose isomerase (din9), seed imbibition protein (din10), and 2-oxoacid-dependent oxidase (din11). Accumulation of transcripts from din3,4,6 and 10 occurred rapidly after the plants were transferred to darkness. Transcripts from din2,9, and 11 could be detected only after 24 h of dark treatment. Inhibition of photo-synthesis by DCMU strongly induced the accumulation of transcripts from those genes, and application of sucrose to detached leaves suppressed the accumulation both in the dark and by DCMU. These observations indicate that expression of the genes is caused by sugar starvation resulted from the cessation of photosynthesis. We further showed that din2-encoded protein also accumulated in senescing leaves. Given these results, possible roles of din genes in leaves in the dark and senescing leaves are discussed.

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The Phase Space Analysis of 3D Vector Fields (3차원 벡터 필드의 위상 공간 분석)

  • Jung, Il-Hong;Kim, Yong Soo
    • Journal of Digital Contents Society
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    • v.16 no.6
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    • pp.909-916
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    • 2015
  • This paper presents a method to display the 3D vector fields by analyzing phase space. This method is based on the connections between ordinary differential equations and the topology of vector fields. The phase space analysis should be geometric interpolation of an autonomous system of equation in the form of the phase space. Every solution of it system of equations corresponds not to a curve in a space, but the motion of a point along the curve. This analysis is the basis of this paper. This new method is required to decompose the hexahedral cell into five or six tetrahedral cells for 3D vector fields. The critical points can be easily found by solving a simple linear system for each tetrahedron. The tangent curves can be integrated by finding the intersection points of an integral curve traced out by the general solution of each tetrahedron and plane containing a face of the tetrahedron.

Comparative Study of Colour Recognition According to Background Lightness and Stimulus Size (배경의 밝기와 자극의 크기에 따른 색채 인지 비교 연구)

  • Hong, Ji-Young;Park, Yun-Sun
    • The Journal of the Institute of Internet, Broadcasting and Communication
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    • v.15 no.6
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    • pp.61-70
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    • 2015
  • This study, under the assumption that there may be a difference in colours recognized depending on background lightness and colour stimulus size, applied background lightness in a differential way and conducted an experiment by comparing the sizes of stimuli equivalent to $2^{\circ}$ and $10^{\circ}$. Based on the results, by reflecting the results of this experiment as a difference in colour recognition, which may occur when the input image is converted from a large-sized screen to a mobile-sized screen, power efficiency, one of the biggest issues, can be reflected in mobile devices and may contribute to effective three-dimensional image reproduction and image quality in 3-D or hologram as well as 2-D images.

Molecular Cloning and Characterization of Attacin from the Swallowtail Butterfly, Papilio xuthus

  • Kim, Seong-Ryul;Hwang, Jae-Sam;Park, Seung-Won;Goo, Tae-Won;Kim, Ik-Soo;Kang, Seok-Woo
    • International Journal of Industrial Entomology and Biomaterials
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    • v.23 no.2
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    • pp.231-238
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    • 2011
  • Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, $Papilio$ $xuthus$. A full length cDNA of $P.$ $xuthus$ attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete $P.$ $xuthus$ attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of $P.$ $xuthus$ attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of $P.$ $xuthus$ attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature $P.$ $xuthus$ attacin into the expression vector, highly expressed in $E.$ $coli$ BL21 cells, and its antibacterial activity was analyzed. Recombinant $P.$ $xuthus$ attacin evidenced considerably antibacterial activity against Gram-negative bacteria, $E.$ $coli$ ML 35 and $Klebsiella$ $pneumonia$.

Cloning of Superoxide Dismutase (SOD) Gene of Lily 'Marcopolo' and Expression in Transgenic Potatoes

  • Park, Ji-Young;Kim, Hyun-Soon;Youm, Jung-Won;Kim, Mi-Sun;Kim, Ki-Sun;Joung, Hyouk;Jeon, Jae-Heung
    • Journal of Applied Biological Chemistry
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    • v.49 no.1
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    • pp.1-7
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    • 2006
  • Differential display reverse transcription PCR (DDRT-PCR) analysis was performed on lily 'Marcopolo' bulb scale for isolation of expressed genes during bulblet formation. Cu/Zn lily-superoxide dismutase (LSOD) of 872 bp gene, with ability to scavenge reactive oxygen in stress environment, was isolated. Northern blot analysis showed expression levels of LSOD maximized 12 days after bulblet formation. Ti plasmid vectors were constructed with sense and antisense expressions of LSOD gene and transformed into potato. Southern blot analysis of transgenic potatoes revealed different copies of T-DNA were incorporated into potato genome. In transgenic potatoes, lily SOD gene was overexpressed in sense lines and not in antisense lines. In native polyacrylamide gel electrophoresis analysis, additional engineered LSOD was detected in sense overexpressed transgenic line only. Transgenic potatoes were subjected to oxidative stress, such as herbicide methyl viologen (MV). Transgenic potato lines with sense orientation exhibited increased tolerance to MV, whereas in antisense lines exhibited decreased tolerance. In vitro tuberization of transgenic potato with sense orientation was promoted, but was inhibited in transgenic potato with antisense orientation.

Identification of novel susceptibility genes associated with bone density and osteoporosis in Korean women

  • Bo-Young Kim;Do-Wan Kim;Eunkuk Park;Jeonghyun Kim;Chang-Gun Lee;Hyun-Seok Jin;Seon-Yong Jeong
    • Journal of Genetic Medicine
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    • v.19 no.2
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    • pp.63-75
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    • 2022
  • Purpose: Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility. In this study, we aimed to identify novel genes for susceptibility to osteoporosis and/or bone density. Materials and Methods: To identify differentially expressed genes (DEGs) between control and osteoporosis-induced cells, annealing control primer-based differential display reverse-transcription polymerase chain reaction (RT-PCR) was carried out in pre-osteoblast MC3T3-E1 cells. Expression levels of the identified DEGs were evaluated by quantitative RT-PCR. Association studies for the quantitative bone density analysis and osteoporosis case-control analysis of single nucleotide polymorphism (SNPs) were performed in Korean women (3,570 subjects) from the Korean Association REsource (KARE) study cohort. Results: Comparison analysis of expression levels of the identified DEGs by quantitative RT-PCR found seven genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib, and Scara5, that showed significantly different expression between the dexamethason-treated and untreated MC3T3-E1 cells and between the ovariectomized osteoporosis-induced mice and sham mice. Association studies revealed that there was a significant association between the SNPs in the five genes, ANXA6, COL5A1, ENO1, MYOF, and SCARA5, and bone density and/or osteoporosis. Conclusion: Using a whole-genome comparative expression analysis, gene expression evaluation analysis, and association analysis, we found five genes that were significantly associated with bone density and/or osteoporosis. Notably, the association P-values of the SNPs in the ANXA6 and COL5A1 genes were below the Bonferroni-corrected significance level.

Isolation and Characterization of Defense Genes Mediated by a Pathogen-Responsive MAPK Cascade in Tobacco (담배에서 병원균에 반응하는 MAPK 신호전달체계에 의해 매개되는 방어 유전자들의 분리 및 특성화)

  • Jang, Eun-Kyoung;Kang, Eun-Young;Kim, Young-Cheol;Cho, Baik-Ho;Yang, Kwang-Yeol
    • Journal of Life Science
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    • v.18 no.8
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    • pp.1023-1030
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    • 2008
  • NtMEK2, which is the tobacco MAPK kinase that is upstream of SIPK and WIPK, was identified using the dexamethasone (DEX)-inducible gain-of-function transgenic system. Expression of $NtNEK2^{DD}$, a constitutively active mutant of NtNEK2, leads to HR-like cell death, which indicates that the NtMEK2-SIPK/WIPK cascade controls defense responses in tobacco. However, little is known about the downstream target substrates or defense-related genes that are regulated by the NtMEK2-SIPK/ WIPK cascade. In this study, ACP-based differential display RT-PCR was used to isolate the downstream effectors mediated by the NtMEK2-SIPK/WIPK cascade in $NtNEK2^{DD}$ transgenic plants. The results identified 6 novel differentially expressed genes (DEGs). These included pathogen induced protein 2-4 (pI2-4), monoterpene synthase 2 (MTS2), seven in absentia protein (SINA), cell death marker protein 1 (CDM1), hydroxyproline-rich glycoprotein (HRGP) and unknown genes (DEG45). The induction of these genes was confirmed by RT-PCR of samples obtained from $NtNEK2^{DD}$ plants. Additionally, when compared with other isolated DEGs, the pI2-4, CDM1 and HRGP genes were significantly up-regulated in response to treatment with salicylic acid and tobacco mosaic virus. Taken together, these results suggest that three novel DEGs were regulated by the NtMEK2-SIPK/WIPK cascade involved in disease resistance in tobacco.

BCR (Brown Color Repressor) gene isolation related to mycelial browning of Lentinus edodes (표고균사 갈변과 관련된 BCR (Brown Color Repressor) 유전자 분리)

  • Kim, Young-Ho;Park, Soo-Cheol;Jhune, Chang-Sung;You, Chang-Hyun;Sung, Jae-Mo;Kong, Won-Sik
    • Journal of Mushroom
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    • v.10 no.3
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    • pp.120-128
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    • 2012
  • Recently sawdust cultivation of Shiitake mushroom (Lentinula edodes) is increasing. It is important to make mycelia to be brown on the substrate surface. This browned surface in sawdust cultivation plays an important role like as artificial bark of the oak log, which protects the other pests and suppresses water evaporation in the substrate. In order to isolate genes which related to brown color formation, differential display method was used. Two cDNA fragments obtained by DD-PCR were 1.2 and 1.6kb and these were expressed in white colored mycelia from L. edodes, but not brown colored mycelia. Partial sequencing of these cDNA fragments showed that the 1.6kb cDNA had 100% identity with the microsatellites gene from Dugenia polichroa. However, the other 1.2kb cDNA fragment had poly T tail on 3' region of partial open reading frame on 5' region. The new primer designed based on the sequence of 1.2kb cDNA was constructed. RT-PCR analysis using the newly designed 0.12kb cDNA specific primer showed that the gene was only expressed in white color mycelia, but not in brown color mycelia. Sequence analysis of 5' region of this 1.2kb cDNA revealed that this gene contained partial open reading frame consisted of 110 amino acid. Homology search using DNASIS database showed that this gene had high sequence homology of 66.7% in DNA level and 69.2 % in amino acid level with dTDP-glucose 4,6-dehydratases gene from Arabidopsis thaliata. The dTDP-glucose 4,6-dehydratases gene was known to be function to have tolerance with oxidation stress. These results strongly suggest that this gene isolated from white mycelia of L. edodes might have a function of repressor against mycelia browning. Therefore I designated this gene as BCR (Brown Color Repressor) gene.

Expression Pattern of Skeletal-Muscle Protein Genes and Cloning of Parvalbumin mRNA in Dark-banded Rockfish (Sebastes inermis) (볼락(Sebastes inermis) 근육단백질 유전자의 성장단계별 발현 양상과 parvalbumin 유전자 클로닝)

  • Jang, Yo-Soon
    • Korean Journal of Ichthyology
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    • v.23 no.1
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    • pp.1-9
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    • 2011
  • Differentially Expressed Gene (DEG) was obtained from Differential Display Reverse Transcription (DDRT)-PCR using Annealing Control Primer (ACP) to search and clone genes related to developmental stages of Sebastes inermis. By using 120 ACPs, the nucleotide sequences obtained from 16 DEGs showing higher expression in 6-month-old skeletal muscle than 18-month-old ones and from 22 DEGs displaying stronger expression in 18-month-old than 6-month-old were analyzed and BLAST was conducted. The results identified that DEGs shared 69~95% homology with genes of parvalbumin (PVALB), nucleoside diphosphate kinase (NDK) B, tropomyosin (TPM), troponin I (TnI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), muscle-type creatine kinase (CKM2), small EDRK-rich factor 2 (SERF2), adenosine monophosphate deaminase (AMPD), Trimeric intracellular cation channel type A (TRICA), Rho GTPase-activating protein 15 (ARHGAP15), S-formylglutathione hydrolase (Esterase D; ESD), heat shock protein 70 (hsp70), type 1 collagen alpha 2 (COL1A2), glutathione S-transferase, Mid1-interacting protein 1 (Mid1lip1), myosin light chain 1 (MYL1), sarcoplasmic/endoplasmic reticulum calcium ATPase 1B (SERCA1B), and ferritin heavy subunit (FTH1). Expression pattern by developmental stage of DEG14 and PVALB exhibiting strong expression in 6-month-old skeletal muscle was investigated using real time PCR. Expression was reduced as Sebastes inermis grew. Expression of PVALB gene was extremely low after 6 months of age. Expression of CKM2 showed higher expression in 18-month-old skeletal muscle than in 6-month-old muscles, and increased continuously until 4 years old, after which CKM2 expression became gradually reduced. By analysis of tissue-specific expression patterns of DEG, DEG14 was expressed mainly in skeletal muscle, liver, kidney and spleen tissues, whereas PVALB expression was expressed in skeletal muscle and kidney, but not in liver and spleen tissues. CKM2 was expressed in skeletal muscle, kidney, and spleen tissues, but not in liver tissues. PVALB gene was composed of 110 amino acids, which constituted 659 bp nucleotides. The results reported here demonstrate that the expression patterns of parvalbumin and CKM2 could be used as molecular markers for selecting fishes exhibiting fast growth.