• Genomics & Informatics
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• v.5 no.4
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• pp.174-178
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• 2007

The earliest stages of mammalian embryogenesis are governed by the activity of maternally inherited transcripts and proteins. Cytoplasmic polyadenylation of selected maternal mRNA has been reported to be a major control mechanism of delayed translation during preimplantation embryogenesis in mice. The presence of cis-elements required for cytoplasmic polyadenylation (e.g., CPE) can serve as a useful tag in the screening of maternal genes partaking in key functions in the transcriptionally dormant egg and early embryo. However, due to its relative simplicity, UA-rich sequences satisfying the canonical rule of known CPE consensus sequences are often found in the 3'-UTR of maternal transcripts that do not actually undergo cytoplasmic polyadenylation. In this study, we developed a method to confirm the validity of candidate CPE sequences in a given gene by a multiplex comparison of 3'-UTR sequences between mammalian homologs. We found that genes undergoing cytoplasmic polyadenylation tend to create a conserved block around the CPE, while CPE-like sequences in the 3'-UTR of genes lacking cytoplasmic polyadenylation do not exhibit such conservation between species. Through this cross-species comparison, we also identified an alternative CPE in the 3'-UTR of tissue-type plasminogen activator (tPA), which is more likely to serve as a functional element. We suggest that verification of CPEs based on sequence conservation can provide a convenient tool for mass screening of factors governing the earliest processes of mammalian embryogenesis.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2003.05a
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• pp.183-184
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• 2003

Sophoricoside was isolated as the inhibitor of IL-5 bioactivity from Sophora japonica (Leguminosae). It has been reported to have an anti-inflammatory effect on rat paw edema model. To develop as an anti-allergic drug, genotoxicity of sophoricoside was investigated in bacterial and mammalian cell system such as Ames bacterial test, chromosomal aberration assay, Comet assay and MOLY assay. In Ames test, sophoricoside of 5000 ∼ 313 $\mu\textrm{g}$/plate concentrations was not shown significant mutagenic effect in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains. The cytotoxicity (IC$\_$50/ and IC$\_$20/) of sophoricoside was determined above the concentration of 5000 $\mu\textrm{g}$/ml in Chinese hamster lung (CHL) fibroblast cell and L5178Y mouse lymphoma cell line. At concentrations of 5000, 2500 and 1250 $\mu\textrm{g}$/ml, this compound was not induced chromosomal aberration in CHL fibroblast cell in the absence and presence of S-9 metabolic activation system. Also in comet assay, DNA damage was not observed in L5178Y cell line. Also in MOLY assay, sophoricoside of 5000 ∼ 313 $\mu\textrm{g}$/ml concentrations was not shown significant mutagenic effect in absence of S-9 metabolic activation system. However, the higher concentration of 5000 and 2500 $\mu\textrm{g}$/ml of sophoricoside induced the increased mutation frequency (MF) in the presence of S-9 metabolic activation system. From these results, no genotoxic effects of sophoricoside observed in bacterial systems whereas, genotoxic effects observed in mammalian cell systems in the presence of metabolic activation system. These results suggested that the metabolite(s) of sophoricoside can cause some genotoxic effects in mammalian cells.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.234-241
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• 2001

The C-terminal 393 bp region of the human interleukin-16 (IL-16) gene was cloned and expressed in E. coli along with mammalian cell lines. Recombinant IL-16 expressed from E. coli was 22 kDa on SDS-PAGE and showed 260% of chemoattractant activity at a concentration of $0.1\;{\mu}g/ml$. HeLa, COS, and Neuro-2a cells were transduced by recombinant retrovirus vector pLNC/IL-16/IRES/TK and the intracellular and secreted amounts of IL-16 produced by HeLa/IL-16/TK, COS/IL-16/TK, and Neuro-2a/IL-16/TK cells were determined by enzyme-linked immunosorbent assay (ELISA). HeLa/IL-16/TK $(1{\times}10^5)$ and COS/IL-16/TK $(1{\times}10^5)$ cells secreted 36.1 and 13.3 ng of IL-16 for 48 h, respectively. Forty-nine ng and 86.4 ng of IL-16 remained in the cell lysates of HeLa/IL-16/TK and COS/IL-16/TK. Intracellular and secreted amounts of IL-16 from Neuro-2a/IL-16/TK $(5{\times}10^5)$ cells during 24 h cultivation were 50 ng and 3.3 ng, respectively. Also, HeLa and COS cells wee stably transfected with mammalian expression vector pCRIII/IL-16. Both culture media and cell lysates prepared from HeLa/IL-16 cells and COS/IL-16 cells showed chemoattractant activity ranging from 190% to 460% as compared to the control experiment. Expression of the herpes simplex virus thymidine kinase (HSV0tk) gene in pLNC/IL-16/ IRES/TK bicistronic retroviral expression vector was verified by performing a genciclovir (GCV) sensitivity assay. Finally, IL-16 repressed Tat-transactivated human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter activity.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.3
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• pp.421-426
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• 2002

The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.409-413
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• 2012

Indolicidin (ID) is a 13-residue Trp-rich antimicrobial peptide (AMP) isolated from bovine neutrophils. In addition to having a high antimicrobial potency, it is also toxic to mammalian cells. To develop novel ID-derived AMPs with shorter lengths and enhanced prokaryotic selectivities (meaning potent antimicrobial activity against bacterial cells without toxicity against mammalian cells) over the parental ID, several ID analogs were designed and synthesized. Finally, 10-residue ID analogs (SI, SI-PA, SI-WF and SI-WL) with much higher prokaryotic selectivity than the parental ID were developed. Our results suggest that the hydrophobic and aromatic amino acids at the central position of the analog SI with the highest prokaryotic selectivity are important for potent antimicrobial activity, but two Pro residues do not affect antimicrobial activity. The order of prokaryotic selectivity for ID and its designed analogs was SI > SI-PA > SI-WF > SI-WL > ID > SI-WA. Taken together, our designed short ID analogs could be developed as therapeutic agents for treating bacterial infections.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.257-260
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• 2002

Dissolved oxygen level of cell culture media has a critical effect on cellular metabolism, which governs specific productivity of recombinant proteins and mammalian cell growth However, in the cores of cell aggregates or cell-immobilized beads, oxygen level frequently goes below a critical level. Mammalian cells have a number of genes induced in the lower level of oxygen, and the genes contain a common cis-acting element (-RCGTG-), hypoxia response element (HRE). By binding of hypoxia inducible factor-l (HIF-I) to the HRE, promoters of hypoxia inducible genes are activated, which is a survival mechanism. In this work, to develop a CHO cell capable of producing recombinant proteins in immobilization and high density cell culture efficiently, mammalian expression vectors containing human tissue-type plasminogen activator (t-PA) gene controlled by HRE were constructed and stably transfected into the CHO cells. In $Ba^{2+}$ -alginate immobilization culture, CHO/pCl/dhfr/2HRE-t-PA cells produced 2 folds higher recombinant t-PA activity than CHO/pCl/dhfrlt-PA cells without $CoCl_2$ treatment. Furthermore, in repeated fed batch culture, productivity of t-PA in immobilized CHO/pCI/dhfr/2HRE-t-PA cells was 121 ng/ml/day, total production of 0.968 mg/day at 11 days culture while CHO/pCIIdhfrlt-PA cells was 22.8 ng/ml/day. All these results indicate that HRE is very useful for the enhancement of protein productivity in mammalian cell cultures.

• Animal cells and systems
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• v.6 no.4
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• pp.317-322
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• 2002

To improve conventional yeast one-hybrid screening, we have developed an efficient mammalian one-hybrid system that allows rapid isolation of com-plementary DNAs which are able to induce human p14$^{ARF}$. tumor suppressor gene. A 1.5 kb promoter region of p14$^{ARF}$ was fused to EGFP to generate ARF promoter-EGFP reporter vector. This reporter plasmid was stably trans-fected into NIH3T3 cells for generation of reporter cell line. When the reporter cell line was infected with E2F-1 together with excess amounts of empty vector, the cells that received the positive modulator were readily identifiable by green fluorescence using FACS. The GFP-positive cells were cloned directly from the cultured cells and expanded in bulk culture. The genomic DNAs from GFP-positive cells were prepared and the CDNA insert in integrated retroviral genome was recovered by PCR using primers annealing to the retroviral vector sequences flanking the insert-cloning site. This system should be useful for efficient screening of expression CDNA libraries in mammalian cells to identify novel upstream regulators for spe-cific genes by one-hybrid interaction.ion.

• Journal of Ecology and Environment
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• v.45 no.2
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• pp.88-96
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• 2021

Background: Sika deer, Cervus nippon, were originally introduced to South Korea from Japan and Taiwan for commercial farming purposes. Unfortunately, they were released into the wild during religious events and have since begun to impact the native ecosystem and species endemic to South Korea. The study of activity patterns can improve our understanding of the environmental impact of non-native species and their association with sympatric species. Using camera traps, we studied the diel and seasonal activity patterns of non-native sika deer and quantified the temporal overlap with sympatric mammalian species in the Muljangori-oreum wetlands of Hallasan National Park, South Korea. Results: A total of 970 trap events were recorded for five mammalian species from nine locations during the camera-trap survey. Siberian roe deer (Capreolus pygargus tianschanicus) had the highest number of recorded events (72.0%), followed by sika deer (Cervus nippon) (16.2%), wild boar (Sus scrofa) (5.0%), Asian badger (Meles leucurus) (4.5%), and the Jeju weasel (Mustela sibirica quelpartis) (2.0%). Sika deer had bimodal activity patterns throughout the year, with peaks throughout the spring-autumn twilight, and day and night time throughout the winter. Relating the daily activity of sika deer with other mammalian species, roe deer expressed the highest degree of overlap (Δ₄ = 0.80) while the Asian badger demonstrated the lowest overlap (Δ₄ = 0.37). Conclusions: Our data show that sika deer are a crepuscular species with seasonal variations in daily activity patterns. Additionally, we identified the temporal differences in activity peaks between different mammals in the Muljangori-oreum wetlands and found higher degree of overlap between sika deer and roe deer during twilight hours.

• Journal of Veterinary Science
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• v.24 no.4
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• pp.53.1-53.12
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• 2023

Background: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. Objectives: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. Methods: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. Results: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. Conclusions: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

• BMB Reports
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• v.42 no.2
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• pp.113-118
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• 2009

Despite the growing body of evidence suggesting a role for MsrA in antioxidant defense, little is currently known regarding the function of MsrB in cellular protection against oxidative stress. In this study, we overexpressed the mammalian MsrB and MsrA genes in Saccharomyces cerevisiae and assessed their subcellular localization and antioxidant functions. We found that the mitochondrial MsrB3 protein (MsrB3B) was localized to the cytosol, but not to the mitochondria, of the yeast cells. The mitochondrial MsrB2 protein was detected in the mitochondria and, to a lesser extent, the cytosol of the yeast cells. In this study, we report the first evidence that MsrB3 overexpression in yeast cells protected them against $H_2O_2$-mediated cell death. Additionally, MsrB2 overexpression also provided yeast cells with resistance to oxidative stress, as did MsrA overexpression. Our results show that mammalian MsrB and MsrA proteins perform crucial functions in protection against oxidative stress in lower eukaryotic yeast cells.