• Journal of Korean Neurosurgical Society
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• v.55 no.3
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• pp.131-135
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• 2014

Objective : With the growing interests of bacteria as a targeting vector for cancer treatment, diverse genetically engineered Salmonella has been reported to be capable of targeting primary or metastatic tumor regions after intravenous injection into mouse tumor models. The purpose of this study was to investigate the capability of the genetically engineered Salmonella typhimurium (S. typhimurium) to access the glioma xenograft, which was monitored in mouse brain tumor models using optical bioluminescence imaging technique. Methods : U87 malignant glioma cells (U87-MG) stably transfected with firefly luciferase (Fluc) were implanted into BALB/cAnN nude mice by stereotactic injection into the striatum. After tumor formation, attenuated S. typhimurium expressing bacterial luciferase (Lux) was injected into the tail vein. Bioluminescence signals from transfected cells or bacteria were monitored using a cooled charge-coupled device camera to identify the tumor location or to trace the bacterial migration. Immunofluorescence staining was also performed in frozen sections of mouse glioma xenograft. Results : The injected S. typhimurium exclusively localized in the glioma xenograft region of U87-MG-bearing mouse. Immunofluorescence staining also demonstrated the accumulation of S. typhimurium in the brain tumors. Conclusion : The present study demonstrated that S. typhimurium can target glioma xenograft, and may provide a potentially therapeutic probe for glioma.

• Journal of Microbiology
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• v.38 no.3
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• pp.183-186
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• 2000

Several types of bioluminescent reporter strains have been developed for the detection and monitoring of pollutant aromatics contaminating the environment. In this study, a bioluminescent reporter strain, E. coli SHP3, was constructed by fusing the luc gene of firefly luciferase with the promoter of pcbC responsible for the meta-cleavage of aromatic hydrocarbons. the bioluminescence expressed by the luc gene in the reporter was well triggered by the promoter when it was exposed to 2,3-dihydroxybiphenyI (2,3-DHBP) at 0.5 to 1 mM concentrations. The bioluminescent response was more extensive when the reporter strain was exposed to 5 mM catechol and 2 mM 4-chlorocatechol. These different types of bioluminescent responses by E. coli SHP3 appeared to be characterized by the nature of the aromatics to stress. Since E. coli SHP3 responded to 2,3-DHBP quite sensitively, this reporter strain could be applied for detecting some catecholic pollutants.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.207-211
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• 2015

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken ${\beta}-actin$ promoter-driven firefly improved luciferase cDNA (${\beta}-act/luc^+$) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (${\beta}-act/luc^+$) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice.

• The Journal of Korean Society of Virology
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• v.29 no.2
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• pp.129-136
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• 1999

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CD14 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with $10\;{\mu}M$, $50\;{\mu}M$ or $100\;{\mu}M$ of TPA. The cell morphology change was observed and the expression of the CD11b and CD14 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 hand 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.

• The Journal of Natural Sciences
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• v.5 no.2
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• pp.3-11
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• 1992

As concentration of ADP was increased, the rate of phosphocreatine formation by respiring heart mitochondria was increased. The value of apparent Km of the phosphocreatine-forming mitochondria for ADP was estimated to be 0.0185 mM. This value was much lower than that of Km for ATP (0.31 mM) which was determined from the reaction of the soluble form of mitochondrial creatine kinase. The concentration of ATP remained constant during the respiring in the presence of ADP. The rate of accumulation of oxidative-phosphorylated ATP in the mitochondrial respiration medium was continuously monitored as a function of ADP concentration with the firefly luciferase-coupled assay. In that case, exogenous creatine did not affect the rate of accumulation of ATP, indicating that phosphocreatine-forming (i.e.,respiring) mitochondria in the presence of ADP did not use the ATP in the medium as a substrate.These results suggest that the heart mitochondrial creatine kinase bound to the inner membrane functionally tight-coupled to the oxidative phosphorylating system with respect to the respired ATP.

• BMB Reports
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• v.44 no.12
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• pp.816-820
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• 2011

There is a broad range of different small heat shock proteins (sHSPs) that have diverse structural and functional characteristics. To better understand the functional role of mitochondrial sHSP, NtHSP24.6 was expressed in Escherichia coli with a hexahistidine tag and purified. The protein was analyzed by non-denaturing PAGE, chemical cross-linking and size exclusion chromatography and the $H_6NtHSP24.6$ protein was found to form a dimer in solution. The in vitro functional analysis of $H_6NtHSP24.6$ using firefly luciferase and citrate synthase demonstrated that this protein displays typical molecular chaperone activity. When cell lysates of E. coli were heated after the addition of $H_6NtHSP24.6$, a broad range of proteins from 10 to 160 kD in size remained in the soluble state. These results suggest that NtHSP24.6 forms a dimer and can function as a molecular chaperone to protect a diverse range of proteins from thermal aggregation.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.2
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• pp.81-85
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• 2007

Glucocorticoid hormone regulates numerous physiological processes, such as regulation of metabolism, and anti-inflammatory and immunosuppressive actions via the activation and repression of gene expression. Here we described a lentivirus-based reporter vector system expressing red fluorescent protein (mRFP) or firefly luciferase (Luc) under the control of a glucocorticoid-responsive element that allows observation of the temporospatial pattern of glucocorticoid induced GR-mediated signaling on a cellular level. Moreover, usage of the chromatin insulator of the chicken ${\beta}$-globin locus induced a marked increase of sensitivity of glucocorticoid inducible promoter of a reporter gene. Use of this method will be applicable of screening for agonist and antagonist of GR in vitro, and also a reporter gene assay for the in vivo determination of the GR-mediated gene activation.

• Genomics & Informatics
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• v.2 no.1
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• pp.45-52
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• 2004

The self-splicing group I intron from Tetrahymena thermophila has been demonstrated to perform splicing reaction with its substrate RNA in the trans configuration. In this study, we explored the potential use of the trans-splicing group I ribozymes to replace a specific RNA with a new RNA that exerts any new function we want to introduce. We have chosen thymidine phosphorylase (TP) RNA as a target RNA that is known as a valid cancer prognostic factor. Cancer-specific expression of TP RNA was first evaluated with RT-PCR analysis of RNA from patients with gastric cancer. We determined next which regions of the TP RNA are accessible to ribozymes by employing an RNA mapping strategy, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. A specific ribozyme recognizing the most accessible sequence in the TP RNA with firefly luciferase transcript as a 3' exon was then developed. The specific trans-splicing ribozyme transferred an intended 3' exon tag sequence onto the targeted TP transcripts, resulting in a more than two fold induction of the reporter activity in the presence of TP RNA in mammalian cells, compared to the absence of the target RNA. These results suggest that the Tetrahymena ribozyme can be a potent anti-cancer agent to modify TP RNAs in tumors with a new RNA harboring anti-cancer activity.

• Biomolecules & Therapeutics
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• v.23 no.5
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• pp.486-492
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• 2015

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their interassay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

• Journal of Life Science
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• v.33 no.1
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• pp.34-42
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• 2023

In a signal transduction network, from the perception of stress signals to stress-responsive gene ex- pression, binding of various transcription factors to cis-acting elements in stress-responsive promoters coordinate the adaptation of plants to abiotic stresses. Among the AP2/ERF transcription factor family genes, group VII ERF genes, such as RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, and AtERF71/ HRE2, are known to be involved in the response to hypoxia stress in Arabidopsis. In this study, we dissected the HRE1 promoter to identify hypoxia-responsive region(s). The 1,000 bp upstream promoter region of HRE1 showed increased promoter activity in Arabidopsis protoplasts and transgenic plants under hypoxia conditions. Analysis of the promoter deletion series of HRE1, including 1,000 bp, 800 bp, 600 bp, 400 bp, 200 bp, 100 bp, and 50 bp upstream promoter regions, using firefly luciferase and GUS as reporter genes indicated that the -200 to -100 region of the HRE1 promoter is responsible for the transcriptional activation of HRE1 in response to hypoxia. In addition, we identified two putative hypoxia-responsive cis-acting elements, the ERF-binding site and DOF-binding site, in the -200 to -100 region of the HRE1 promoter, suggesting that the expression of HRE1 might be regulated via the ERF transcription factor(s) and/or DOF transcription factor(s). Collectively, our results suggest that HRE1 contains hypoxia-responsive cis-acting elements in the -200 to -100 region of its promoter.