• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7825-7829
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• 2014

Objective: To investigate the effect of high expression of XAF1 in vivo or in vitro on lung cancer cell growth and apoptosis. Methods: 1. The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/F35 - Null at the same multiplicity of infection (MOI); (hereinafter referred to as transient transfected cell strain); XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. 2. Methyl thiazolyl tetrazolium (MTT) and annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis before and after infection of Ad5/F35 - XAF1 with Western blotting for apoptosis related proteins, caspase 3, caspase - 8 and PARP. 3. After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1 (hereinafter referred to as stable expression cell strain). Twenty nude mice were randomly divided into groups A and B, 10 in each group: A549/XAF1 stable expression cell strain was subcutaneously injected in group A, and A549/Ctrl stable cell line stable expression cell strain in group B (control group), to observe transplanted tumor growth in nude mice. Results: The mRNA and protein expression of XAF1 in A549 cells transfected by Ad5/F35 - XAF1 was significantly higher than in the control group. XAF1 mediated by adenovirus vector demonstrated a dose dependent inhibition of lung cancer cell proliferation and induction of apoptosis. This was accompanied by cleavage of caspase -3, -8, -9 and PARP, suggesting activation of intrinsic or extrinsic apoptotic pathways. A cell strain of lung cancer highly expressing XAF1 was established, and this demonstrated delayed tumor growth after transplantation in vivo. Conclusion: Adenovirus mediated XAF1 gene expression could inhibit proliferation and induce apoptosis in lung cancer cells in vitro; highly stable expression of XAF1 could also significantly inhibit the growth of transplanted tumors in nude mouse, with no obvious adverse reactions observed. Therefore, the XAF1 gene could become a new target for lung cancer treatment.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2004.11a
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• pp.77-85
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• 2004

A significant portion (about 8% in human genome) of mammalian mRNA sequences contains AU(Adenine and Uracil) rich elements or AREs at their 3' untranslated regions (UTR). These mRNA sequences are usually stable. ARE motifs are assorted into three classes. The importance of AREs in biology is that they make certain mRNA unstable. We analyzed the occurrences of AREs and Alu, and propose a possible mechanism on how human mRNA could acquire and keep A REs at its 3' UTR originated from Alu repeats. Interspersed in the human genome, Alu repeats occupy 5% of the 3' UTR of mRNA sequences. Alu has poly-adenine (poly-A) regions at the end that lead to poly -thymine (poly-T) regions at the end of its complementary Alu. It has been discovered that AREs are present at the poly -T regions. In the all ARE's classes, 27-40% of ARE repeats were found in the poly -T region of Alu with mismatch allowed within 10% of ARE's length from the 3' UTRs of the NCBI's reference m RNA sequence database. We report that Alu, which has been reported as a junk DNA element, is a source of AREs. We found that one third of AREs were derived from the poly -T regions of the complementary Alu.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.211-217
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• 1999

Effects of subsh-ate RNA configuration on the tians-splicing reactcon by group I intron ribozyme of Tetralzynzena thern\ulcornerophila were analyzed with substrate RNAs which have been generated to have very stable structures with stem-loop. RNAinapping strategy was perfo~med in vivo as well as in virro to search the mosl accessible siles to the ~irms-splicing ribozymes in the substrate RNAs. Sequences present in the loop of the target RNAs have shown to be well recognized by and reacted with group I inlron ribozymes while sequences present in the stein do not. Thesc results were confirmed with the experiments of trans-cleavage and rmnssplicing reactmn with ihe specific ribozyines recognizing those sequences. Moreover, sequence analysis of the trans-splicing products have shown that irons-splicing reaction can proceed with high fidelity. In conclusion, the secondary structure of substrate RNAs is one of the most important factors to detemine the ribozyme activity.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• Proceedings of the Microbiological Society of Korea Conference
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• 2003.05a
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• pp.83-86
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• 2003

Major advances in positive-sense RNA virus research have been facilitated by the development of reverse genetics systems. These systems consist of an infectious cDNA clone that encompasses the genome of the virus in question. This clone is then used as a template for the subsequent synthesis of infectious RNA for the generation of synthetic viruses. However, the construction of infectious cDNA for the Japanese encephalitis virus (JEV) has been repeatedly thwarted by the instability of its cDNA. As JEV is an important human pathogen that causes permanent neuropsychiatric sequelae and even fatal disease, a reliable reverse genetics system for this virus is highly desirable. The availability of this tool would greatly and the development of effective vaccines as well as facilitate studies into the basic biology of the virus, including the molecular mechanisms of viral replication, neurovirulence, and pathogenesis. We have successfully constructed a genetically stable infectious JEV cDNA containing full-length viral RNA genome. Synthetic RNA transcripts generated in vitro from the cDNA were highly infectious upon transfection into susceptible cells, and the cDNA remained stable after it had been propagated in E. coli for 180 generations. Using this infectious JEV cDNA, we have successfully expressed a variety of reporter genes from the full-length genomic and various subgenomic RNAs in vitro transcribed from functional JEV cDNAS. In summary, we have developed a reverse genetics system for JEV that will greatly facilitate the research on this virus in a variety of different fields. It will also be useful as a heterologous gene expression vector and aid the development of a vaccine against JEV.

• BMB Reports
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• v.55 no.2
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• pp.65-71
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• 2022

Regenerative medicine is a research field that develops methods to restore damaged cell or tissue function by regeneration, repair or replacement. Stem cells are the raw material of the body that is ultimately used from the point of view of regenerative medicine, and stem cell therapy uses cells themselves or their derivatives to promote responses to diseases and dysfunctions, the ultimate goal of regenerative medicine. Stem cell-derived extracellular vesicles (EVs) are recognized as an attractive source because they can enrich exogenous microRNAs (miRNAs) by targeting pathological recipient cells for disease therapy and can overcome the obstacles faced by current cell therapy agents. However, there are some limitations that need to be addressed before using miRNA-enriched EVs derived from stem cells for multiplexed therapeutic targeting in many diseases. Here, we review various roles on miRNA-based stem cell EVs that can induce effective and stable functional improvement of stem cell-derived EVs. In addition, we introduce and review the implications of several miRNA-enriched EV therapies improved by multiplexed targeting in diseases involving the circulatory system and nervous system. This systemic review may offer potential roles for stem cell-derived therapeutics with multiplexed targeting.

• Journal of Microbiology
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• v.44 no.1
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• pp.1-10
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• 2006

Adaptation to any undesirable change in the environment dictates the survivability of many microorganisms, with such changes generating a quick and suitable response, which guides the physiology of bacteria. During nutritional deprivation, bacteria show a stringent response, as characterized by the accumulation of (p)ppGpp, resulting in the repression of stable RNA species, such as rRNA and tRNA, with a concomitant change in colony morphology. However, genes involved in amino acid biosynthesis become over-expressed to help bacteria survive under such conditions. The survivability of pathogenic bacteria inside a host cell also depends upon the stringent response demonstrated. Therefore, an understanding of the physiology of stringent conditions becomes very interesting in regulation of the growth and persistence of such invading pathogens.

• Korean Journal of Microbiology
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• v.25 no.2
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• pp.94-102
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• 1987

Rat liver LDH A-cDNA has been isolated from a .lambda.gt11-rat lover cDNA library and partially characterized. The size of the isolated rat liver LDH A-cDNA if shown to be 1.6Kb and restriction enzyme sites for the rat liver LDH A-cDNA are also mapped. 682-nucleotide sequence coding for 3'-end of rat liver LDH A-cDNA has been analyzed and compared to the nucleotide sequence of the same region of rat $C_6$-glioma cell LDH A-cDNA which has been cloned from the hormonally stimulated cell cultures. The result shows that 177 nucleotide sequences coding for the C-terminal 59-amino acids are identical but 505 nucleotide sequences of 3'-nontranslated region of the two LSH A-cDNA exhibit characteristic differences in thier nucleotide sequences. Computer analysis for the folding structures for 3'-end 400 nucleotide sequences of the two LDH A-cDNA shows a possibility implying that the two LDH A-mRNAs isolated from different tissues of rats may have different half life and therefore their translational efficiency may be different. It has been previously demonstrated that isoproterenol stimulated rat $C_6$ -glioma cell cultures produce LDH A-mRNA showing 2 to 3-fold longer half life in comparison to that of noninduced LHD A-mRNA. The result therefore support for the idea that hormonally stimulated rat $C_6$-glioma cells may produce LDH A-mRNA containing different nucleotide sequences at the 3'-end nontranslated region by which the hormonally induced LDH A-mRNA could have more stable secondary mRAN structure in comparison to that of noninduced LDH A-mRNA.

• Journal of Life Science
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• v.19 no.3
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• pp.305-310
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• 2009

Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the $M13{\Delta}lac182/229$ phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.239-245
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• 2009

Translation initiation factor eIF1A performs essential functions in various initiation steps including 43S preinitiation complex formation in eukaryotes, and contains a highly conserved oligonucleotide-binding (OB) fold. In our previous study, we discovered that eIF1A possesses RNA annealing activity and forms a stable complex with double-stranded RNA. In this study, we initiated site-directed mutations in eIF1A to find the active sites for these biochemical activities and to investigate whether they are essential functions for yeast cell growth. A truncated protein, eIF1A($\Delta$T), devoid of both N- and C-terminal domains but containing an intact OB-fold exhibited RNA annealing activity. In contrast, all point mutations in OB-fold domain, except R57D, impaired both RNA annealing and dsRNA binding activities, indicating that the intact OB-fold domain is required for both activities. Viabilities of the mutant yeast cells were not correlated with RNA annealing activity but with the in vivo protein stabilities in the case of R57D and K94D.