scholarly journals mkp-1 Encoding Mitogen-Activated Protein Kinase Phosphatase 1, a Verotoxin 1 Responsive Gene, Detected by Differential Display Reverse Transcription-PCR in Caco-2 Cells

2000 ◽  
Vol 68 (5) ◽  
pp. 2791-2796 ◽  
Author(s):  
Shihoko Kojima ◽  
Itaru Yanagihara ◽  
Gengo Kono ◽  
Tomomi Sugahara ◽  
Hatsumi Nasu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Differential Display ◽  
Reverse Transcription ◽  
Mitogen Activated Protein Kinase ◽  
28S Rrna ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mitogen Activated Protein ◽  
A Subunit ◽  
Differential Display Reverse Transcription

ABSTRACT The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing Escherichia coli is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release. Four differentially expressed genes, including mkp-1 (encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells. Northern blot analysis showed the induction ofmkp-1 mRNA 6 h after VT1 stimulation. Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced mkp-1 mRNA, but mkp-1mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor. Therefore, we concluded that the A subunit of VT1 was essential for mkp-1 induction. Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin. Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1. This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins.

Mitogen-activated Protein Kinase 7 Promotes Cell Proliferation, Migration and Invasion in SOSP-M Human Osteosarcoma Cell Line

2015 ◽  
Vol 103 (5) ◽  
pp. 483-488 ◽  
Author(s):  
Yan Huang ◽  
Jianhua Yao ◽  
Bing Zhu ◽  
Jianzheng Zhang ◽  
Tiansheng Sun
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Expression Level ◽  
Migration And Invasion ◽  
M Cells ◽  
Rt Pcr ◽  
Blank Group ◽  
Mitogen Activated Protein

Purpose Osteosarcoma (OS) is the most common primary bone tumor and has low cure rates. Our study aimed to evaluate the roles of mitogen-activated protein kinase 7 (MAPK7) in cell proliferation, migration and invasion using the SOSP-M human OS cell line as an in vitro model. Methods SOSP-M cells were transfected with PCDNA3.1-MAPK7 and siRNA-MAPK7 plasmids using Lipofectamine 2000. Quantitative real-time polymerase chain reaction (RT-PCR) was performed to determine the relative expression level of MAPK7 and Western blot analysis was carried out to determine the expression level of ERK5 protein. Then MTT, scratch wound healing and Matrigel transwell assays were used to investigate the roles of MAPK7 expression in the proliferation, migration and invasion, respectively, of SOSP-M cells in vitro. Results RT-PCR analysis showed that the expression level of MAPK7 increased significantly after transfection with PCDNA3.1-MAPK7 plasmid compared with the blank group, while it decreased significantly after transfection with siRNA-MAPK7 plasmid. Similar results for ERK5 expression were obtained by Western blot analysis. In addition, the cell proliferation rate, cell migration rate and invasive cell number in the PCDNA3.1-MAPK7 transfection group increased significantly compared with the blank group, while they decreased significantly in the siRNA-MAPK7 transfection group. Conclusions Our results indicate that overexpression of MAPK7 in human OS cells could promote cell proliferation, migration and invasion, whereas knockdown of MAPK7 expression had the opposite effect. All the results suggest that MAPK7 may serve as a potent target for drug development.

scholarly journals Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

2000 ◽  
Vol 13 (3) ◽  
pp. 408-427 ◽  
Author(s):  
Joy Sturtevant
Keyword(s):  
Gene Expression ◽  
Differential Display ◽  
Reverse Transcription ◽  
Drug Targets ◽  
Cell Activation ◽  
Histoplasma Capsulatum ◽  
Medical Mycology ◽  
Reverse Transcription Pcr ◽  
Differential Display Reverse Transcription ◽  
Plant Pathogenesis

SUMMARY The host-fungus interaction is characterized by changes in gene expression in both host and pathogen. Differential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysis of gene expression among several cell populations. Several limitations and drawbacks to this procedure have now been addressed, including the large number of false-positive results and the difficulty in confirming differential expression. Modifications that simplify the reaction time, allow the use of minute quantities of RNA, or address unusual species- or gene-specific sequences have been reported. DDRT-PCR has been used to address biological questions in mammalian systems, including cell differentiation, cell activation, cell stress, and identification of drug targets. In microbial pathogenesis and plant pathogenesis, DDRT-PCR has allowed the identification of virulence factors, genes involved in cell death, and signaling genes. In Candida albicans, DDRT-PCR studies identified TIF-2, which may play a role in the upregulation of phospholipases, and the stress-related genes, CIP1 and CIP2. In Histoplasma capsulatum and C. albicans, genes involved in the host-pathogen interaction, including a member of the 100-kDa family in Histoplasma and an ALS and 14-3-3 gene in Candida, were potentially identified by DDRT-PCR. Although very few reports have been published in medical mycology, studies in mammalian, nonfungal microbial, and plant pathogen systems are easily applied to basic questions in fungal pathogenesis and antifungal therapeutics.

scholarly journals Identification of Salt-Stress-Induced Genes from the RNA-Seq Data ofReaumuria trigynaUsing Differential-Display Reverse Transcription PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Zhen-hua Dang ◽  
Qi Qi ◽  
Hui-rong Zhang ◽  
Hao-yu Li ◽  
Shu-Biao Wu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Differential Display ◽  
Reverse Transcription ◽  
Expression Patterns ◽  
Digital Gene Expression ◽  
Rna Seq ◽  
Sequencing Data ◽  
Reverse Transcription Pcr ◽  
Wide Range ◽  
Differential Display Reverse Transcription

Next generation sequencing (NGS) technologies have been used to generate huge amounts of sequencing data from many organisms. However, the correct choice of candidate genes and prevention of false-positive results computed from digital gene expression (DGE) of RNA-seq data are vital when using these genetic resources. We indirectly identified 18 salt-stress-inducedReaumuria trigynatranscripts from the transcriptome sequencing data using differential-display reverse transcription PCR (DDRT-PCR) combined with local BLAST searches. Highly consistent with the DGE results, the quantitative real-time PCR expression patterns of these transcripts showed strong upregulation by salt stress, suggesting that these genes may play important roles inR. trigyna’s survival under high-salt environments. The method presented here successfully identified responsive genes from the massive amount of RNA-seq data. Thus, we suggest that DDRT-PCR could be employed to mine NGS data in a wide range of applications in transcriptomic studies. In addition, the genes identified in the present study are promising candidates for further elucidation of the salt tolerance mechanisms inR. trigyna.

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