• Bulletin of the Korean Chemical Society
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• v.26 no.12
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• pp.2033-2037
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• 2005

In vitro selection was used to isolate $Mg^{2+}$-dependent self-cleaving ribozymes with cis-cleavage activity from a pre-tRNA library having 40-mer random sequences attached to 5'-end of E. coli $tRNA^{Phe}$. After 8 rounds of SELEX (Systematic Evolution of Ligands by Exponential Enrichment), RNA molecules which can self-cleave at the high concentration of $Mg^{2+}$ were isolated. The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM $Mg^{2+}$ but not in 10 mM $Mg^{2+}$. The cleavage sites of the ribozymes are located at +3 and +4 of $tRNA^{Phe}$, compared with +1 position of 5'-end cleavage site of pre-tRNA by RNase P. New RNA constructs deprived of its D stem-loop, anticodon stem-loop, variable loop and T stem-loop, respectively showed the cleavage specificity identical to a ribozyme having the intact tRNA structure. Also, the new ribozyme fused with both a ribozyme and $tRNA^{Leu}$ showed the cleavage activities at the various sites within its sequences, different from two sites of position +3 and +4 observed in the ribozyme with $tRNA^{Phe}$. Our results suggest that the selected ribozyme is not structural-specific for tRNA.

• Bulletin of the Korean Chemical Society
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• v.26 no.8
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• pp.1251-1254
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• 2005

It has been proposed that oxidation of L-3,4-dihydroxyphenylalanine (DOPA) may contribute to the pathogenesis of neurodegenerative disease. In this study, L-DOPA-Fe(III)-mediated DNA cleavage and the protection by carnosine and homocarnosine against this reaction were investigated. When plasmid DNA was incubated with L-DOPA in the presence of Fe(III), DNA strand was cleaved. Radical scavengers and catalase significantly inhibited the DNA breakage. These results suggest that $H_2O_2$ may be generated from the oxidation of DOPA and then $Fe^{3+}$ likely participates in a Fenton’s type reaction to produce hydroxyl radicals, which may cause DNA cleavage. Carnosine and homocarnosine have been proposed to act as anti-oxidants in vivo. The protective effects of carnosine and homocarnosine against L-DOPA-Fe(III)-mediated DNA cleavage have been studied. Carnosine and homocarnosine significantly inhibited DNA cleavage. These compounds also inhibited the production of hydroxyl radicals in L-DOPA/$Fe^{3+}$ system. The results suggest that carnosine and homocarnosine act as hydroxyl radical scavenger to protect DNA cleavage. It is proposed that carnosine and homocarnosine might be explored as potential therapeutic agents for pathologies that involve damage of DNA by oxidation of DOPA.

• BMB Reports
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• v.33 no.5
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• pp.379-384
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• 2000

The native conformation of serpins (serine protease inhibitors) is strained. Upon cleavage of the reactive center loop of serpins by a protease, the amino terminal portion of the cleaved loop is inserted into the central ${\beta}-sheet$, A sheet, as the fourth strand, with the concomitant release of the native strain. We questioned the role of protease in this conformational switch from the strained native form into a stable relaxed state. Chemical cleavage of the reactive center loop of ${\alpha}_1-antitrypsin$, a prototype serpin, using hydroxylamine dramatically increased the stability of the serpin. A circular dichroism spectrum and peptide binding study suggests that the amino terminal portion of the reactive center loop is inserted into the A sheet in the chemically-cleaved ${\alpha}_1-antitrypsin$, as in the enzymatically-cleaved molecule. These results indicate that the structural transformation of a serpin molecule does not require interaction with a protease. The results suggest that the serpin conformational switch that occurred during the complex formation with a target protease is induced by the cleavage of the reactive center loop per se.

• Bulletin of the Korean Chemical Society
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• v.23 no.9
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• pp.1185-1186
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• 2002

• Bulletin of the Korean Chemical Society
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• v.25 no.3
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• pp.403-406
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• 2004

• Bulletin of the Korean Chemical Society
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• v.29 no.2
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• pp.301-302
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• 2008

• Bulletin of the Korean Chemical Society
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• v.31 no.6
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• pp.1467-1468
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• 2010

• Bulletin of the Korean Chemical Society
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• v.25 no.11
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• pp.1623-1624
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• 2004

• Bulletin of the Korean Chemical Society
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• v.29 no.6
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• pp.1137-1140
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• 2008

M1 RNA, the catalytic subunit of Escherichia coli RNase P, is an essential ribozyme that processes the 5' leader sequence of tRNA precursors (ptRNAs). Using KS2003, an E. coli strain generating only low levels of M1 RNA, which showed growth defects, we examined whether M1 RNA is involved in polycistronic mRNA processing or degradation. Microarray analysis of total RNA from KS2003 revealed six polycistronic operon mRNAs (acpP-fabF, cysDNC, flgAMN, lepAB, phoPQ, and puuCBE) showing large differences in expression between the adjacent genes in the same mRNA transcript compared with the KS2001 wild type strain. Model substrates spanning an adjacent pair of genes for each polycistronic mRNA were tested for RNase P cleavage in vitro. Five model RNAs (cysNC, flgMN, lepAB, phoPQ, and puuBE) were cleaved by RNase P holoenzyme but not by M1 RNA alone. However, the cleavages occurred at non-ptRNA-like cleavage sites, with much less efficiency than the cleavage of ptRNA. Since cleavage products generated by RNase P from a polycistronic mRNA can have different in vivo stabilities, our results suggest that RNase P cleavage may lead to differential expression of each cistron.

• Bulletin of the Korean Chemical Society
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• v.32 no.3
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• pp.964-972
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• 2011

This work investigated the difference between $Fe^{2+}$ autoxidation-induced and Fenton-type cleavage of pBR322 plasmid DNA. $^{\cdot}OH$ generation reactions in the absence and presence of $H_2O_2$ under various conditions were also investigated. Although both the $Fe^{2+}$ autoxidation and Fenton-type reactions showed DNA cleavage and $^{\cdot}OH$ generation, there were significant differences in their efficiencies and reaction rates. The rate and efficiency of the cleavage reaction were higher in the absence of 1.0 mM of $H_2O_2$ than in its presence in 20 mM phosphate buffer. In contrast, the $^{\cdot}OH$ generation reaction was more prominent in the presence of $H_2O_2$ and showed a pH-independent, fast initial reaction rate, but the rate was decreased in the absence of $H_2O_2$ at across the entire tested pH range. Studies using radical scavengers on DNA cleavage and $^{\cdot}OH$ generation reactions in both the absence and presence of $H_2O_2$ confirmed that both reactions spontaneously involved the active oxygen species $^{\cdot}OH$, ${O_2}^{\cdot-}$, $^1O_2$ and $H_2O_2$, indicating that a similar process may participate in both reactions. Based on the above observations, a new mechanism for the $Fe^{2+}$ autoxidation-induced DNA cleavage reaction is proposed.