Biomedical subjects

Seung Pil Pack

Publications and source records attributed to Seung Pil Pack.

16 recordsLinked to original sources

PubMed2006

Reactive oxygen species generation through NADH oxidation by 6-formylpterin derivatives in the dark.

6-formylpterin (6FP) has been reported to produce reactive oxygen species (ROS) such as *O2- and H2O2 from O2 in the presence of NADH under light condition. In the present study, we prepared a variety of 6FP derivatives and found that 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridin-4-one and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-methylpteridin-4-one, in which the 2-amino groups are modified by a dimethylaminomethylene group and the 3-positions by pivaloyl and methyl groups and 2-amino-6-formyl-3-methylpteridin-4-one in which the amino group at the 2-position is free and the 3-position is modified by a methyl group generated H2O2 from O2 on oxidation of NADH to NAD+ in the dark. However, 6FP and 2-(N,N-dimethylaminomethyleneamino)-6-formylpteridin-4-one, in which the 3-position is free did not yield H2O2. These results indicate that modification of the 3-position is essential to make the activities of 6FP available in the dark and would be suggestive for designing pharmaceutical compounds that generate appropriate and controllable amounts of ROS in vivo.

Darkness↗

PubMed2006

Development of an efficient amine-functionalized glass platform by additional silanization treatment with alkylsilane.

Aminosilane-treated molecular layers on glass surfaces are frequently used as functional platforms for biosensor preparation. All the amino groups present on the surface are not available in reactive forms, because surface amino groups interact with remaining unreacted surface silanol groups. Such nonspecific interactions might reduce the efficiency of chemical immobilization of biomolecules such as DNA, enzymes, antibodies, etc., in biosensor fabrication. To improve immobilization efficiency we have used additional surface silanization with alkylsilane (capping) to convert the remaining silanol groups into Si-O-Si linkages, thereby liberating the amino groups from nonspecific interaction with the silanol groups. We prepared different types of capped amine surface and evaluated the effect of capping on immobilization efficiency by investigating the fluorescence intensity of Cy3-NHS (N-hydroxysuccinimide) dye that reacted with amino groups. The results indicate that most of the capped amine surfaces resulted in enhanced efficiency of immobilization of Cy3-NHS compared with the untreated control amine surface. We found a trend that trialkoxysilanes had greater capping effects on immobilization efficiency than monoalkoxysilanes. It was also found that the aliphatic chain of alkylsilane, which does not participate in the capping of the silanol, had an important function in enhancing immobilization efficiency. These results would be useful for preparation of an amine-modified surface platform, with enhanced immobilization efficiency, which is essential for developing many kinds of biosensors on a silica matrix.

Alkylation↗

PubMed2006

Novel 6-formylpterin derivatives: chemical synthesis and O2 to ROS conversion activities.

6-Formylpterin (6FP) has been demonstrated to have strong neuroprotective effects against transient ischemia-reperfusion injury in gerbils. Also it has been shown that in rats, 6FP protected retinal neurons even when it was administered after the ischemic insult. Since there is a significant need for such a compound that effectively suppresses the events caused by the lack of oxygen supply, 6FP has attracted further investigation. Unfortunately, however, 6FP is hardly soluble in water at neutral pH and in organic solvents because of its self-assembling ability. Although a several mM solution of 6FP is available in alkaline water, it is unstable. In the present study, a novel chemical derivatization of 6FP has been developed which maintains the formyl group on the 6-position of 6FP, which is essential for the physiological activities of 6FP, and increases solubility in water and organic solvents. In the method, the 2- and 3-positions of 6FP were modified by a three component coupling reaction: 6FP was subjected to the reaction with acid chloride and N,N-dimethylformamide. The derivatives synthesized here, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one 1, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-isobutyrylpteridine-4-one 2, and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-o-toluoylpteridine-4-one 3, showed high solubility in water (1.0-5.6 mM) and organic solvents. The O(2) conversion property has also been determined for the derivative 1. Using an oxygen electrode, it has been found that O(2) is consumed in the presence of 1 and NADH at around pH 7.4 and that the rate of O(2) consumption is enhanced by UV-A irradiation. Electron paramagnetic resonance (EPR) analysis coupled with DMPO spin trapping has also revealed that in the presence of NADH, 1 converts O(2) to O(2)(-), which is further reduced to OH. By UV-A illumination in the analogous systems, (1)O(2) formation was observed. These results are similar to those reported previously for 6FP.

Electrodes↗

PubMed2006

Structural property and enzymatic response of oxanine in DNA strands.

Oxanine (Oxa, O), one of the major products generated from guanine (Gua) by nitrosative oxidation has been expected as mutagenic lesion involved in NO- or HNO(2) -induced genotoxicity. Here, to elucidate the biological meaning of Oxa in DNA strands, several kinds of Oxa-containing oligodeoxynucleotides (Oxa-ODNs) were synthesized and applied to biophysical and biochemical characterization. CD and NMR analyses revealed that the conformations of all the Oxa-containing duplexes are basically B-type without causing any severe distortion in the whole DNA structure. It was also determined that restriction endonucleases recognize and cleave the specific base-sequence even when Gua was substituted by Oxa in the sequence. When Oxa-ODN was testified as substrates for other DNA-relevant enzymes, the enzymatic functions were not largely affected by Oxa.

Alkaline Phosphatase↗

PubMed2006

Synthesis and application of new amine-modified oligonucleotides using H-phosphonate chemistry.

Oligodeoxynucleotide probes tethered with amine linkers are commonly used for development of DNA based biosensor tools for studying gene expression and biological assays. However, the current preparation methods for synthesizing amine tethered probes often show defects during acid deprotection and during hydrophobic purification with the amine linker protecting groups. Here, we developed a prospective preparation method of using modified H-phosphonate chemistry to prepare amine tethered oligonucleotide probes. The present developed method will be helpful for preparing efficient amine tethered oligodeoxynucleotide probes that can be used in DNA biosensor applications.

Amines↗

PubMed2006

Development of DNA-arrayed column for sensitive and selective analysis of DNA.

Quantitative separation of target DNA molecules was performed by DNA-arrayed silica capillary column on the basis of base pairing interaction of nucleic acids. We prepared DNA-arrayed silica capillary column by conjugating 5'-amniohexyl oligonucleotide (probe) on inner surface of the capillary column pre-treated with 3-aminopropyltriethoxysilane (APS) and disuccinimidyl glutarate (DSG) cross-linker. Sufficient resolution was observed by controlling of target-DNAs concentration, salt gradients, and temperature gradients. Finally, we succeeded to separate two different DNA targets even with same melting temperature according to their own concentrations. These results would be useful for developing quantitative analysis of cDNA, which is related to mRNA levels in biological sample.

Chromatography, Affinity↗

PubMed2006

Chemical natures and application of 6-formylpterin derivatives.

Pterin, an analog of guanine, is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological and pharmacological activities. In vitro, 6FP converts molecular oxygen to reactive oxygen species (ROS) in the presence of NADH or NADPH under light illumination, with the oxidation of NADH is to NAD(+). In the present study, it has been elucidated that some of 6FP derivatives in which the 3-position of 6FP is modified have such unique activities even in the dark where the most of in vivo biological events occur.

Darkness↗

PubMed2005

Chemical synthesis and thermodynamic characterization of oxanine-containing oligodeoxynucleotides.

Oxanine (Oxa, O), one of the major damaged bases from guanine generated by NO- or HNO2-induced nitrosative deamination, has been considered as a mutagen-potent lesion. For exploring more detailed properties of Oxa, large-scale preparation of Oxa-containing oligodeoxynucleotide (Oxa-ODN) with the desired base sequence is a prerequisite. In the present study, we have developed a chemical synthesis procedure of Oxa-ODNs and characterized thermodynamic properties of Oxa in DNA strands. First, 2'-deoxynucleoside of Oxa (dOxo) obtained from 2'-deoxyguanosine by HNO2-nitrosation was subjected to 5'-O-selective tritylation to give 5'-O-(4,4'-dimethoxytrityl)-dOxo (DMT-dOxo) with a maximum yield of 70%. Subsequently, DMT-dOxo was treated with conventional phosphoramidation, which resulted in DMT-dOxo-amidite monomer with a maximum yield of 72.5%. The amidite obtained was used for synthesizing Oxa-ODNs: the coupling yields for Oxa incorporation were over 93%. The prepared Oxa-ODNs were employed for analyzing the thermodynamic properties of DNA duplexes containing base-matches of O:N [N; C (cytosine), T (thymine), G (guanine) or A (adenine)]. Melting temperatures (Tm) and thermodynamic stability (DeltaG37(0)) were found to be lower by 6.83 approximately 13.41 degrees C and 2.643 approximately 6.047 kcal mol(-1), respectively, compared with those of oligodeoxynucleotides, which had the same base sequence except that O:N was replaced by G:C (wild type). It has also been found that Oxa-pairing with cytosine shows relatively high stability in DNA duplex compared with other base combinations. The orders of DeltaDeltaG37(0) were O:C > O:T > O:A > O:G. The chemical synthesis procedure and thermodynamic characteristics of Oxa-ODNs established here will be helpful for elucidating the biological significance of Oxa in relation to genotoxic and repair mechanisms.

Base Pairing↗

PubMed2005

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface.

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.

Base Sequence↗

PubMed2005

Development of a plasmid display system using GAL4 DNA binding domain for the in vitro screening of functional proteins.

A plasmid display system using GAL4 DNA binding domain (GAL4 DBD) was constructed to enrich the molecular diversity and in vitro selection of functional proteins. Model proteins used were enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST). The feasibility of this display system was examined using enrichment experiments of target protein from a model protein mixture and identifying the encoding genes by PCR, in which the model protein mixture includes GAL4 DBD/GST fusion protein, GAL4 DBD/EGFP fusion protein, and xylanase. Target proteins of GAL4 DBD/GST and GAL4 DBD/EGFP from the model protein mixture were efficiently isolated by the plasmid display, respectively. The results show that the display system is sufficiently sensitive to select a target protein from a protein mixture, and that it is possible to discover the functional proteins from large libraries using relatively simple approaches.

Cloning, Molecular↗

PubMed2005

Packing-based difference of structural features between thermophilic and mesophilic proteins.

Twenty pairs of thermophilic and mesophilic proteins were compared in terms of residue packing distribution to obtain structural features related to protein thermostability. Based on residue packing concept, structural features of residues such as residue packing distribution, inner/outer position, secondary structure and water solvation were investigated. The statistical tests revealed that higher frequency in well-packed state of residues, lower frequency in exposed state and higher frequency in well-packed state of inner positioned residues, and higher frequency in well-packed state of 3/10 helix residues could be general structural features thermophilic proteins have.

Archaeal Proteins↗

PubMed2005

Fabrication of efficient DNA microarray by additional surface modification and functional probe design.

DNA microarray, which exploits the preferential binding of complementary single stranded nucleic acids, is a powerful tool for obtaining high-throughput characterization of gene expression. Although this system is evolving rapidly, low-reproducibility of hybridization data is a major drawback to be overcome. Here, we developed additional surface modification step to reduce the hydrolysis of silyl ether bond between glass surface and linker molecule. In addition, we designed functional DNA probe for reducing the chemical treatments of glass surface. These surface/probe modifications will be helpful in fabricating more efficient DNA microarray system.

DNA Probes↗

PubMed2005

Hydrogen bond removal of pterin derivative whose structure is similar to nucleic acid bases.

Pterin, an analog of guanine, is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological and pharmacological activities and it is, therefore, worthwhile to elucidate whole mechanism of its activities. Unfortunately, however, 6FP is hardly soluble in water and organic solvents. Like nucleic acid bases, 6FP makes intermolecular hydrogen bonds and forms stacking structure causing such drawback nature. This has made mechanistic studies on 6FP activities extremely difficult. In this study, we carried out derivatization for 6FP and succeeded in increasing water solubility with maintaining its physiological activities.

Antineoplastic Agents↗

PubMed2005

Biophysical and biochemical properties of oxanine-containing oligodeoxynucleotide.

Since oxanine (Oxa) was found as one of the major products from nitrosative oxidation of guanine, Oxa has been expected as mutagenic lesion related to NO-induced DNA damage. Here, to elucidate the mutagenic significance of Oxa in vitro, several Oxa-containing oligodeoxynucleotides (Oxa-ODNs) were synthesized and subjected to biophysical and biochemical characterization. Oxa-ODNs were analyzed in terms of their duplex stabilities and DNA polymerase responses. These results will be helpful in understanding the genotoxic pattern induced by Oxa.

DNA Polymerase I↗

PubMed2004

Protein thermostability: structure-based difference of amino acid between thermophilic and mesophilic proteins.

Structural distributions of each amino acid were compared between 20 pairs of thermophilic and mesophilic proteins to obtain thermostable factors. Five kinds of residual structure states such as fully-exposed, exposed, partially exposed (or partially buried), buried, well-buried states were considered for analyzing the structural patterns of amino acids. The statistical tests revealed that lower frequency in partially exposed state of SER, lower frequency in exposed state and higher frequency in well-buried state of ALA, higher frequency in buried state of GLU, higher frequency in exposed state of ARG, etc. could be critical factors related with protein thermostability.

Amino Acid Sequence↗

PubMed2004

Chemical synthesis of oxanine-contained oligodeoxynucleotides.

Oxanine, one of major damaged product from deamination of guanine base mediated by NO or HNO2-induced nitrosative reaction, has been considered as a genotoxic lesion. To analyze the biophysical properties of oxanine in context of oligodeoxynucleotide, the chemical incorporation of 2'-deoxyoxanosine (dOxo, nucleoside form of oxanine) into oligodeoxynucleotide is required. Here, we developed the chemical synthesis procedures of dimethoxytritylated dOxo-phosphoramidite (DMT-dOxo-amidite) and oxanine-contained oligonucleotide.

Oligodeoxyribonucleotides↗