• Journal of Life Science
• /
• v.16 no.7 s.80
• /
• pp.1229-1234
• /
• 2006

This study investigated whether matrix metalloproteinases (MMPs) can be modified in apoptotic smooth muscle cell (SMC) using the SMC that undergoes apoptotic death by expressing Fas-associated death domain containing protein (FADD) when they are grown without tetracycline in culture medium. In the absence of tetracycline, FADD-SMC lost adherence and showed the fragmentation of the nuclei. In proportion to duration of tetracycline removal, phosphorylated form of p38 MAPK and of ERK increased, whereas phosphorylation of protein kinase B (PKB) was not changed very much in response to tetracycline The levels of cyclin A and cyclin D were also decreased in a time dependent manner. Up-regulation of MMP-9 expression and activity was observed when the SMC were grown without tetracycline. Immunoreactivity of MMP-9 was detected from both attached and floating FADD-SMCs grown without tetracycline. An inhibitor of MAPK kinase, PD098059, and an inhibitor of p38 MAPK, SB203580, inhibited the up-regulation of MMP-9. Treatment of the SMC with a synthetic MMP inhibitor, BB94, attenuated death occurring in the absence of tetracycline. These results indicate that SMC undergoing death is able to up-regulate MMP-9 and that the enzyme can affect cell viability.

• BMB Reports
• /
• v.43 no.2
• /
• pp.110-114
• /
• 2010

The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.

• Journal of Life Science
• /
• v.17 no.1 s.81
• /
• pp.45-50
• /
• 2007

This study investigated biological activity of tumor necrosis factor $(TNF)-\alpha$ secreted from smooth muscle cell (SMC) destined for death by expressing Fas associated death domain containing protein (FADD) (FADD-SMC) when the cells are grown without tetracycline in culture medium. In the absence of tetracycline the FADD-SMC secreted approximately 1000 pg/ml $TNF-\alpha$, whereas hardly detectable amount of the cytokine existed in the presence of tetracycline. The culture medium collected from the FADD-SMC grown in the absence of tetracycline increased phosphorylated form of p38 MAPK and up-regulated nuclear factor kappa B (NF-kB). The medium collected without tetracycline also caused death of L929 cells. Depletion of $TNF-\alpha$ with the soluble TNF receptor (sTNFR) inhibited the phosphorylation of p38 MAPK, the up-regulation of NF-kB activity and the death activity of the medium collected from FADD-SMC in the absence of tetracycline. These results indicate that $TNF-\alpha$ secreted from SMC undergoing death is biologically active and can affect cellular function.

• Journal of Fisheries and Marine Sciences Education
• /
• v.25 no.4
• /
• pp.828-835
• /
• 2013

Products from aquaculture have sometimes been focused on the problems caused by the contamination of chemical agents as the use of chemical agents in aquaculture has been annually increased. The risk of contamination of products by chemical agents is greater in freshwater than in seawater. In order to evaluate the food safety of a fish grown in freshwater, we investigated the residues of antibiotics (tetracycline, oxolinic acid and ciplofloxacin) and malachite green in cultured rainbow trout, Oncorhynchus mykiss. Malachite green, which was prohibited in the application of aquaculture, was not detected in samples tested in this study. The residual content of tetracycline was determined to be less than the permissible amount, <0.2 mg/kg. The contents of ciplofloxacin was also less than the permissible amount, <0.1 mg/kg. However, in case of oxolinic acid, one of samples was only exhibited higher content than the permissible amount (<0.1 mg/kg). The results obtained in this study suggested that the control and regulation of chemical agents such as antibiotics was important to maintain a safe and worry-free seafood supply.

• Reproductive and Developmental Biology
• /
• v.30 no.3
• /
• pp.157-162
• /
• 2006

Endogenous 84 amino acid parathyroid hormone(PTH) is synthesized as a pre-pro hormone by the chief cells of the parathyroid glands. Physiological actions of PTH include regulation of bone metabolism, renal tubular reabsorption of calcium and phosphate, and intestinal calcium absorption. In addition, PTH stimulates new bone formation by extraordinary stimulation of osteoblastic activity and decreasing calcium excretion by the kidney. In this study, we constructed and tested retrovirus vectors designed to express the human parathyroid hormone(hPTH) gene under the control of the tetracycline-inducible promoters. To increase the hPTH gene expression at turn-on state, woodchuck hepatitis virus posttranscriptional regulatory element(WPRE) sequence was also introduced into retrovirus vector at downstream region of either the hPTH gene or the sequence encoding reverse tetracycline-controlled transactivator(rtTA). Transformed primary culture cells(porcine fetal fibroblast, PFF, chicken embryonic fibroblast, CEF) were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the hPTH gene expression level using two step RT-PCR and ELISA Higher hPTH expression($3{\tims}10^4\;pg/ml,\;5.3{\times}10^4\;pg/ml$) and tighter expression control(up to 8 fold) were observed from the vector in which the WPRE sequence was placed at downstream of the hPTH gene. The resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Reproductive and Developmental Biology
• /
• v.29 no.1
• /
• pp.49-55
• /
• 2005

We cloned cDNA of the PGH(porcine growth hormone) gene and constructed retrovirus vector designed to express PGH gene under the regulation of CMV (cytomegalovirus) promoter. To maximize the expression, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was placed at the downstream of the PGH gene. After infection with recombinant viruses, approximately 1×10/sup 6/ PFF(porcine fetal fibroblast) cells released PGH protein into the media as much as 1,400 ng. In a subsequent experiment, a modifications of the retrovirus vector was made to express the PGH gene in a teracycline-inducible manner. In PFF cells carrying these viral vector sequences, addition of doxycycline to the media resulted in 2∼6 fold increase in PGH synthesis. In the modified retrovirus vectors, the WPRE sequence also played a role in boosting the effect of the tetracycline induction. This result indicates that our tetracycline-inducible expression system might be a promising candidate in alleviating the complicate physiological problems caused by constitutive expression of the exogenous genes in the transgenic animals.

• Reproductive and Developmental Biology
• /
• v.33 no.1
• /
• pp.63-69
• /
• 2009

In this study we tried to construct a more efficient tetracycline-inducible gene expression system by replacing previous key elements with more advance ones. At the beginning, we substituted PGK (phophoglycerate kinase) promoter for CMV (cytomegalovirus) promoter to control "$rtTA2^sM2$" which has been known for high induction efficiency in response to tetracycline. With this modification, expression of the EGFP marker gene under the induction condition was significantly increased. Next, we replaced "TRE" fragment with a modified version named "TRE-tighf" which has been reported to have higher affinity and specificity to the transactivator by minor base change of the "TRE" DNA fragment sequence. Use of "TRE-tighf" instead of "TRE" resulted in more than 10 fold increment in terms of induction efficiency and significant decrement of background expression in non-inducible condition. By combining PGK promoter and "TRE-tight" fragment, we could upgrade previous tetracycline-inducible system to show more stringent turn on/off gene switch ability and stronger expression of the gene of our interest. Use of this newly developed system must be very helpful to the studies of gene expression, especially to the transgenic animal study in which non-controllable constitutive expression of the transgene has been one of the urgent problems to be solved.

• Reproductive and Developmental Biology
• /
• v.29 no.1
• /
• pp.57-62
• /
• 2005

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2005.10a
• /
• pp.125-125
• /
• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2005.10a
• /
• pp.125-125
• /
• 2005