• Journal of the Society of Cosmetic Scientists of Korea
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• v.27 no.1
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• pp.17-27
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• 2001

A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

• BMB Reports
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• v.38 no.4
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• pp.386-390
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• 2005

Based on the reported cDNA sequences of $BmK{\alpha}Txs$, the genes encoding toxin $BmK{\alpha}Tx11$ and $BmK{\alpha}Tx15$ were amplified by PCR from the Chinese scorpion Buthus martensii Karsch genomic DNA employing synthetic oligonucleotides. Sequences analysis of nucleotide showed that an intron about 500 bp length interrupts signal peptide coding regions of $BmK{\alpha}Tx11$ and $BmK{\alpha}Tx15$. Using cDNA sequence of $BmK{\alpha}Tx11$ as probe, southern hybridization of BmK genome total DNA was performed. The result indicates that $BmK{\alpha}Tx11$ is multicopy genes or belongs to multiple gene family with high homology genes. The similarity of $BmK{\alpha}$-toxin gene sequences and southern hybridization revealed the evolution trace of $BmK{\alpha}$-toxins: $BmK{\alpha}$-toxin genes evolve from a common progenitor, and the genes diversity is associated with a process of locus duplication and gene divergence.

• The Journal of Korean Society of Virology
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• v.29 no.4
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• pp.221-233
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• 1999

Prospect Hill Virus (PHV) is the well known serotype of hantavirus, a newly established genus in family Bunyaviridae. Extensive studies have upheld the original view of PHV genetics with three genes such as nucleocapsid (N) protein, envelope proteins (G1, G2) and RNA dependent RNA polymerase. In this study, we report the existence of additional gene that is encoded in an overlapping reading frame of the N protein gene within S genome segment of PHV. This gene is expected to encode a nonstructural small (NSs) protein and it seems to be only found in PHV infected cell. The presence and synthesis of NSs protein could be demonstrated in the cell infected with PHV using anti-peptide sera specific to the predicted amino acid sequence deduced from the second open reading frame. Ribosomal synthesis of this protein appears to occur at AUG codon at the 83rd base of S genome segment, downstream of N protein initiation codon. This protein is small in size (10.4 KDa) and highly basic in nature. The expression strategy of NSs protein appears that a signal mRNA is used to translate both N and NSs protein in PHV infected cell. 10 KDa protein in virus infected cell lysates can bind to mimic dsRNA. This fact strongly suggests that NSs protein may be involved in virus replication on late phase of viral life cycle.

• Fisheries and Aquatic Sciences
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• v.10 no.1
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• pp.1-7
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• 2007

The induction of cellular and humoral immunity and cytokine gene expression by synthetic CpG oligodexoynucleotides (CpG-ODNs) has not been investigated systematically in olive flounder Paralichthys olivaceus in vivo. We optimized the proper concentration of CpG-ODNs using an in vitro assay for the superoxide anion $(O_2^-)$. CpG-ODNs induced $O_2^-$ and nitric oxide (NO) production, lysozyme activity, and the proinflammatory cytokine gene expression of $IL-1{\beta}$ and $TNF-{\alpha}$ in olive flounder significantly in vivo, whereas non-CpG-ODNs did not produce these effects or produced them to a lesser extent. This implied that CpG-ODNs could stimulate cellular and humoral immunity and cytokine gene expression in olive flounder. This is the first evidence of NO production and the first study on the mRNA expression of the proinflammatory cytokine genes $IL-1{\beta}$ and $TNF-{\alpha}$ in olive flounder in response to CpG-ODNs. Comparison of the variation in NO production and lysozyme activity to that of other studies led us to postulate that a group-specific difference exists in the immune responses of olive flounder against CpG-ODNs. Furthermore, the detailed immunostimulatory spectrum of CpG-ODNs in olive flounder could be a useful index with which to analyze the effect of CpG-ODNs against the challenge test prior to field applications.

• BMB Reports
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• v.52 no.2
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• pp.133-138
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• 2019

Upon viral infection, the 2', 5'-oligoadenylate synthetase (OAS)-ribonuclease L (RNaseL) system works to cleave viral RNA, thereby blocking viral replication. However, it is unclear whether OAS proteins have a role in regulating gene expression. Here, we show that OAS1 and OAS3 act as negative regulators of the expression of chemokines and interferon-responsive genes in human macrophages. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology was used to engineer human myeloid cell lines in which the OAS1 or OAS3 gene was deleted. Neither OAS1 nor OAS3 was exclusively responsible for the degradation of rRNA in macrophages stimulated with poly(I:C), a synthetic surrogate for viral double-stranded (ds)RNA. An mRNA sequencing analysis revealed that genes related to type I interferon signaling and chemokine activity were increased in $OAS1^{-/-}$ and $OAS3^{-/-}$ macrophages treated with intracellular poly(I:C). Indeed, retinoic-acid-inducible gene (RIG)-I- and interferon-induced helicase C domain-containing protein (IFIH1 or MDA5)-mediated induction of chemokines and interferon-stimulated genes was regulated by OAS3, but Toll-like receptor 3 (TLR3)- and TLR4-mediated induction of those genes was modulated by OAS1 in macrophages. However, stimulation of these cells with type I interferons had no effect on OAS1- or OAS3-mediated chemokine secretion. These data suggest that OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages.

• Genomics & Informatics
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• v.16 no.2
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• pp.22-29
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• 2018

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (~28%) and well above the predicted error of Sanger sequencing (~2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

• Korean Chemical Engineering Research
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• v.43 no.2
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• pp.313-317
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• 2005

Synthetic gene carriers such as poly-cationic polymers easily form complexes with plasmid DNA which contains negative charge. Chitosan is a polysaccharide that demonstrates much potential as a gene delivery system. The ability of depolymerized chitosan to condense DNA was determined using electrophoresis. Dynamic laser scattering and scanning electron microscopy were used to examine the size and the morphology of the chitosan/DNA complex. Parameters such as chitosan molecular weight and charge density influenced the complex size and the DNA amount condensed with chitosan. The cell viabilities in the presence of chitosan ranged between 84-108% of the control in all experiments. Gene expression efficacy using chitosan/DNA complex was enhanced in 293 cells relative to that using naked DNA, although it was lower than that using lipofecamine. Transfection efficacy using low molecular weight chitosan (Mw=8,517) was higher than those of the control and the other chitosan (MW=4,078). The low molecular weight chitosan (MW=8,517) with a high charge density (18.32 mV) fulfilled the requirements for a suitable model gene delivery system with respect to the condensing ability of DNA, complex formation, and transfection efficacy.

• Interdisciplinary Bio Central
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• v.4 no.3
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• pp.10.1-10.12
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• 2012

Introduction: We investigated frameshift suppressor tRNAs previously reported to use five-base anticodon-codon interactions in order to provide a collection of frameshift suppressor tRNAs to the synthetic biology community and to develop modular frameshift suppressor logic devices for use in synthetic biology applications. Results and Discussion: We adapted eleven previously described frameshift suppressor tRNAs to the BioBrick cloning format, and built three genetic logic circuits to detect frameshift suppression. The three circuits employed three different mechanisms: direct frameshift suppression of reporter gene mutations, frameshift suppression leading to positive feedback via quorum sensing, and enzymatic amplification of frameshift suppression signals. In the course of testing frameshift suppressor logic, we uncovered unexpected behavior in the frameshift suppressor tRNAs. The results led us to posit a four-base binding hypothesis for the frameshift suppressor tRNA interactions with mRNA as an alternative to the published five-base binding model. Conclusion and Prospects: The published five-base anticodon/codon rule explained only 17 of the 58 frameshift suppression experiments we conducted. Our deduced four-base binding rule successfully explained 56 out of our 58 frameshift suppression results. In the process of applying biological knowledge about frameshift suppressor tRNAs to the engineering application of frameshift suppressor logic, we discovered new biological knowledge. This knowledge leads to a redesign of the original engineering application and encourages new ones. Our study reinforces the concept that synthetic biology is often a winding path from science to engineering and back again; scientific investigations spark engineering applications, the implementation of which suggests new scientific investigations.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.736-738
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• 2003

Ornithine decarboxylase (ODC) is the key enzyme in the synthetic pathway of polyamines. This enzyme is a homodimeric and a pyridoxal 5-phosphate (PLP) dependent enzyme. We have isolated, a cDNA clone encoding ODC from brain cDNA library constructed from flounder (Paralichthys olivaceus). The ODC cDNA contained a complete ORF consisting of 460 amino acids and one stop codon with comparison to nucleotide sequences of the flounder, zebrafish and rat ODC genes, the ODC genes were highly conserved. The transcription of ODC was analyzed with reverse transcription-polymerase chain reaction (RT-PCR) species in brain, kidney, liver, and embryo.

• Genomics & Informatics
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• v.11 no.1
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• pp.55-57
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• 2013

Growing numbers of studies employ cell line-based systematic short interfering RNA (siRNA) screens to study gene functions and to identify drug targets. As multiple sources of variations that are unique to siRNA screens exist, there is a growing demand for a computational tool that generates normalized values and standardized scores. However, only a few tools have been available so far with limited usability. Here, we present siMacro, a fast and easy-to-use Microsoft Office Excel-based tool with a graphic user interface, designed to process single-condition or two-condition synthetic screen datasets. siMacro normalizes position and batch effects, censors outlier samples, and calculates Z-scores and robust Z-scores, with a spreadsheet output of >120,000 samples in under 1 minute.