• Molecules and Cells
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• v.27 no.3
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• pp.337-343
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• 2009

We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutaminase activity in mammalian cells. Transglutaminases are a family of $Ca^{2+}$-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N'-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the $[^3H]putrescine$-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transglutaminase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases.

• Bulletin of the Korean Chemical Society
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• v.27 no.10
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• pp.1537-1541
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• 2006

The $^1H$ and $^{13}C$ chemical shifts of eleven flavone derivatives were completely determined by basic 1D and 2D NMR experiments. Nineteen flavone derivatives including the above eleven derivatives were examined for anti-oxidative effects using the 1,1-diphenyl-2-picryl-hydrazyl assay and Caenorhabditis elegans. In order to understand the relationships between the structures of flavone derivatives and their anti-oxidative activities, a Comparative Molecular Field Analysis was performed.

• BMB Reports
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• v.30 no.3
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• pp.205-210
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• 1997

p53 tumor suppressor plays an important role in the regulation of cellular proliferation. To identify proteins regulating the expression of p53 in rat liver, we analyzed p53 promoter by electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay. We found that a protein binds the sequence CACGTG, bHLH consensus sequence in rat p53 promoter. Southwestern blotting analysis with oligonucleotides containing this sequence shows that the molecular weight of the protein is 100 kDa. This size is not compatible with the bHLH family such as USF or c-Myc/Max which is known to regulate the expression of the human and mouse p53 gene. Therefore this 100 kDa protein may be a new protein regulating basal transcription of rat p53. We purified this 100 kDa protein through sequence-specific DNA affinity chromatogaphy.

• BMB Reports
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• v.37 no.4
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• pp.460-465
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• 2004

A 16-residue polypeptide model with the sequence acetyl-YALSLAATLLKEAASL-OH was derived by rational de novo peptide design. The designed sequence consists of amino acid residues with high propensity to adopt an alpha helical conformation, and sequential order was arranged to produce an amphipathic surface. The designed sequence was chemically synthesized using a solid-phase method and the polypeptide was purified by reverse-phase liquid chromatography. Molecular mass analysis by electro-spray ionization mass spectroscopy confirmed the correct designed sequence. Structural characterization by circular dichroism spectroscopy demonstrated that the peptide adopts the expected alpha helical conformation in 50% acetonitrile solution. Liposome binding assay using Small Unilamellar Vesicle (SUV) showed a marked release of entrapped glucose by interaction between the lipid membrane and the tested peptide. The channel-forming activity of the peptide was revealed by a planar lipid bilayer experiment. An analysis of the conducting current at various applied potentials suggested that the peptide forms a cationic ion channel with an intrinsic conductance of 188 pS. These results demonstrate that a simple rational de novo design can be successfully employed to create short peptides with desired structures and functions.

• Molecules and Cells
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• v.42 no.2
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• pp.104-112
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• 2019

Tracking the fate of individual cells and their progeny through lineage tracing has been widely used to investigate various biological processes including embryonic development, homeostatic tissue turnover, and stem cell function in regeneration and disease. Conventional lineage tracing involves the marking of cells either with dyes or nucleoside analogues or genetic marking with fluorescent and/or colorimetric protein reporters. Both are imaging-based approaches that have played a crucial role in the field of developmental biology as well as adult stem cell biology. However, imaging-based lineage tracing approaches are limited by their scalability and the lack of molecular information underlying fate transitions. Recently, computational biology approaches have been combined with diverse tracing methods to overcome these limitations and so provide high-order scalability and a wealth of molecular information. In this review, we will introduce such novel computational methods, starting from single-cell RNA sequencing-based lineage analysis to DNA barcoding or genetic scar analysis. These novel approaches are complementary to conventional imaging-based approaches and enable us to study the lineage relationships of numerous cell types during vertebrate, and in particular human, development and disease.

• Bulletin of the Korean Chemical Society
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• v.27 no.5
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• pp.699-704
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• 2006

Escherichia coli RNase P is a ribonucleoprotein composed of M1 RNA and C5 protein. Previously, analysis of RNA aptamers selected for C5 protein from a synthetic RNA library showed that C5 protein could bind various RNA molecules as an RNA binding protein. In this study, we searched cellular RNA motifs that could be recognized by C5 protein by a genomic SELEX approach. We found various C5 protein-binding RNA motifs derived from E. coli genomic sequences. Our results suggest that C5 protein interacts with various cellular RNA species in addition to M1 RNA.

• Molecular & Cellular Toxicology
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• v.1 no.3
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• pp.164-171
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• 2005

Toxicological studies have an object of detecting adverse effects of a chemical on an organism based on observed toxicity marker (i.e., serum biochemical markers and chemical-specific gene expression) or phenotypic outcome. To date, most toxicogenomic studies concentrated on hepatic toxicity. cDNA microarray analysis enable discrimination of the responses in animals exposed to different classes of hepatotoxicants. In an effort to further characterize the mechanisms of 2, 3, 7, 8,-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin)-mediated toxicity, comprehensive temporal-responsive microarray analyses were performed on hepatic tissue from Sprague-Dawley rats treated with TCDD. Hepatic gene expression profiles were monitored using custom DNA chip containing 490 cDNA clones related with toxicology. Gene expression analysis identified 26 features which exhibited a significant change. In this study, we observed that the genes related with oxidative stress in rats exposed to Dioxin, such as CYPIIA3 and glutathione S-transferase, were up-regulated at 24hr after exposure. In this study, we carried out to discover novel evidence for previously unknown gene expression patterns related to mechanism of hepatic toxicity in rats exposed to dioxin, and to elucidate the effects of dioxin on the gene expression after exposure to dioxin.

• International Journal of Oral Biology
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• v.37 no.4
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• pp.181-188
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• 2012

As the demand for large-scale analysis of gene expression using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We compared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM) of mercaptohexanol or hepthanethiol, which create hydrophilic or hydrophobic surface properties, respectively. A hydrophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA molecules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The optimal DNA concentration for immobilization was found to be 0.5 ${\mu}M$. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized conditions, molecular biology techniques including DNA hybridization, ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We demonstrated from our present analysis the importance of surface polarity in solid-phase molecular biological applications. A hydrophilic SAM generated a far more favorable environment than hydrophobic SAM for solid-state molecular techniques. Our findings suggest that the conditions and methods identified here could be used for DNA-DNA hybridization applications such as DNA chips and for the further development of solid-phase genetic engineering applications that involve DNA-enzyme interactions.

• BMB Reports
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• v.39 no.5
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• pp.595-606
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• 2006

Metallothioneins are a group of low molecular mass and cysteine-rich metal-binding proteins, ubiquitously found in most living organisms. They play an important role in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting against intracellular oxidative damages. Analysis of complete rice genome sequences revealed eleven genes encoding putative metallothionein (OsMT), indicating that OsMTs constitute a small gene family in rice. Expression profiling revealed that each member of the OsMT gene family differs not only in sequence but also in their tissue expression patterns, suggesting that these isoforms may have different functions they perform in specific tissues. On the basis of OsMT structural and phylogenetic analysis, the OsMT family was classified as two classes and class I was subdivided into four types. Additionally, in this paper we also present a complete overview of this family, describing the gene structure, genome localization, upstream regulatory element, and exon/intron organization of each member in order to provide valuable insight into this OsMT gene family.

• Molecules and Cells
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• v.19 no.3
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• pp.328-333
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• 2005

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as $70^{\circ}C$. Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.