• Bulletin of the Korean Chemical Society
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• 제31권4호
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• pp.829-834
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• 2010

Ligand variation was carried out on a cobalt(III) complex of Salen-type ligand comprised of 1,2-cyclohexenediamine and salicylaldehyde bearing a methyl substituent on 3-position and -[$CMe(CH_2CH_2CH_2N^+Bu_3)_2$] on 5-position, which is a highly active catalyst for $CO_2$/propylene oxide copolymerization. Replacement of the methyl substituent with bulky isopropyl group resulted in alteration of the binding mode, consequently lowering turnover frequency significantly. Replacement with an ethyl group preserved binding mode and activity. Replacement of the tributylammonium unit with trihexylammonium or trioctylammonium, or replacement of 1,2-cyclohexenediamino unit with -$NC(Me)_2CH_2N$- decreased activity, even though the binding mode was unaltered.

• BMB Reports
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• 제29권1호
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• pp.38-44
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• 1996

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method, which is being used for amplifying DNA. Not only does Taq DNA polymerase have high commercial value commercial value for the polymerase chain reaction application, but it is also important in studying DNA replication, because it is apparently an homologue to E. coli DNA polymerase I, which has long been used for DNA replication study (Lawyer et ai., 1993). The crystal structure determination of Taq DNA polymerase was initiated. An X-ray diffraction pattern breaks down a crystal structure into discrete sine waves in a Fourier series. The original shape of a crystal object in terms of electron density may be represented as the sum of those sine waves with varying amplitudes and phases in three dimensions. The molecular replacement method was initially employed to provide phase information for the structure of Taq DNA polymerase. The rotation search using the program MERLOT resulted in a solution peak with 5.4 r.m.s. PC-refinement of the X-PLOR program verified the result and also optimized the orientation angles. Next, the translation search using the X-PLOR program resulted in a unique solution peak with 7.35 r.m.s. In addition, the translation search indicated $P3_121$ to be the true space group out of two possible ones. The phase information from the molecular replacement was useful in the MIR phasing experiment.

• 한국세라믹학회지
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• 제51권2호
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• pp.127-131
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• 2014

Precipitated calcium carbonate(PCC) inorganic fillers for plastic offera higher replacement ratio with improved mechanical properties than any other inorganic fillers. Due to its secure economic feasibility, its fields of application areexpanding. For optimized PCC grain size and polymer replacement ratio, it is good to maintain at least $0.035{\mu}m$ grains and keep double the grain size of distance between particles, depending on the molecular weight and volume replacement rate of the polymer. PCC has unique characteristics, ie, with smaller grain size, dispersibility decreases, and if grain size is not homogenous, polymer cracking occurs. The maximum replacement ratio of PCC is approximately 30%, but in the range of 10 - 15% it produces the highest mechanical strength. When mixed with a biodegradable plastic like starch, it also improves initial environmental degradability.

• Journal of Microbiology
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• 제41권4호
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• pp.340-344
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• 2003

The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권5호
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• pp.311-315
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• 2002

By the in vitro combinatorial mutagenesis, which is a sequential reaction of PCR mutagenesis and in vitro coupled transcription/translation with Escherichia coli S30 extract, S65 and E222 of green fluorescent protein of Aequarea victoria were comprehensively changed to all possible combinations of amino acids, thus totally 400 mutant (including a wild type) proteins were simultaneously produced and their fluorescent properties were analyzed. Although a few mutations had been reported so far at the 222nd position, replacement E222 to all other19 amino acids gave fluorescent signal to the mutants by changing Ser 65 to Ala together. Among the mutants, replacement to G, A, S, Q, H and C gave relatively high fluorescence. The in vitro combinatorial mutagenesis, therefore, has been proved valuable for comprehensive structure-function studies of proteins.

• Genomics & Informatics
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• 제7권4호
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• pp.181-186
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• 2009

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans -splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3'-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a trans-splicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

• Genomics & Informatics
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• 제5권1호
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• pp.32-35
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• 2007

Telomerase reverse transcriptase (TERT) is an enzymatic ribonucleoprotein that prolongs the replicative life span of cells by maintaining protective structures at the ends of eukaryotic chromosomes. Telomerase activity is highly up-regulated in 85-90% of human cancers, and is predominately regulated by hTERT expression. In contrast, most normal somatic tissues in humans express low or undetectable levels of telomerase activity. This expression profile identifies TERT as a potential anticancer target. By using an RNA mapping strategy based on a trans-splicing ribozyme library, we identified the regions of mouse TERT (mTERT) RNA that were accessible to ribozymes. We found that particularly accessible sites were present downstream of the AUG start codon. This mTERTspecific ribozyme will be useful for validation of the RNA replacement as cancer gene therapy approach in mouse model with syngeneic tumors.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.844-848
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• 2002

In more than half of human tumors, the p53 tumor suppressor gene is mutated. Thus, restoration of wild-type p53 activity by repair of mutant RNA could be a potentially promissing approach to cancer treatment. To explore the potential use of RNA repair for cancer therapy, trans-splicing group I ribozymes were developed that could replace mutant p53 RNA with RNA sequence attached to the 3'end of ribozymes. By employing a mapping library of ribozymes, we first determined which regions of the p53 RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, trans-splicing ribozymes were generated that specifically recognized the sequences around these accessible regions. Subsequently, the ribozymes reacted with and altered the p53 transcripts by transferring a 3'exon tag sequence onto the targeted p53 RNA with high fidelity. Thus, these ribozymes could be utilized to repair mutant p53 in tumors, which would revert the neoplastic phenotype.

• Genomics & Informatics
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• 제3권4호
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• pp.172-174
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• 2005

Recent anti-cancer approaches have been based to target tumor-specifically associated and/or causative molecules such as RNAs or proteins. As this specifically targeted anti-cancer modulator, we have previously described a novel human cancer gene therapeutic agent that is Tetrahymena group I intron-based trans-splicing ribozyme which can reprogram and replace human telomerase reverse transcriptase (hTERT) RNA to selectively induce tumor-specific cytotoxicity in cancer cells expressing the target RNA. Moreover, the specific ribozyme has been shown to efficiently retard tumor tissues in xenograft mice which had been inoculated with hTERT-expressing human cancer cells. In this study, we assessed specificity of trans-splicing reaction in cells to evaluate the therapeutic feasibility of the specific ribozyme. In order to analyze the trans-spliced products by the specific ribozyme in hTERT-positive cells, RT, 5'-end RACE-PCR, and sequencing reactions of the spliced RNAs were employed. Then, whole analyzed products resulted from reactions only with the hTERT RNA. This study suggested that the developed ribozyme perform highly specific RNA replacement of the target RNA in cells, hence trans-splicing ribozyme will be one of specific agents for genetic approach to revert cancer.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.1070-1076
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• 2009

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.