• 환경생물
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• 제29권1호
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• pp.74-80
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• 2011

In this study we present a mammalian cell culture model that allows to study the effect of endocrine disruptors (EDCs) on aromatase activity of aquatic amphibian, Bombina orientalis. Bombina orientalis aromatase gene was subcloned into a mammalian expression vector and subsequently transfected to mammalian cells. Although the protein expression level of Bombina orientalis aromatase was low, it had a significant aromatase activity. When EDCs were added to aromatase transfected cells, aromatase activity was significantly decreased. We report here that this system may be used to monitor the effect of EDCs on aromatase activity of aquatic organisms.

• Animal cells and systems
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• 제5권3호
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• pp.225-229
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• 2001

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand cellular processes in which Nek2 participates during mammalian development, we investigated the expression and subcellular localization of Nek2 in vivo. The Nek2 protein was detected in spermatocytes and in a fraction of actively dividing ovarian follicle cells and of embryonic tissues. We also observed that Nek2 was localized in both the nucleus and centrosome in embryonic cells. Such localization pattern supports the proposal that Nek2 is a mitotic regulator that is involved in multiple cell cycle events during mammalian development.

• Molecules and Cells
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• 제24권2호
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• pp.294-300
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• 2007

The mammalian glycosylphosphatidylinositol (GPI) anchor consists of three mannoses attached to acylated GlcN-(acyl)PI to form $Man_3$-GlcN-(acyl)PI. The first of the three mannose groups is attached to an intermediate to generate Man-GlcN-(acyl)PI by the first mannosyltransferase (GPI-MT-I). Mammalian and protozoan GPI-MT-I have different substrate specificities. PIG-M encodes the mammalial GPI-MT-I which has 423 amino acids and multiple transmembrane domains. In this work we cloned PIG-M homologues from humans, Plasmodium falciparum (PfPIG-M), and Saccharomyces cerevisiae (GPI14), to test whether they could complement GPI-MT-I-deficient mammalian cells, since this biosynthetic step is likely to be a good target for selective screening of inhibitors against many pathogenic organisms. PfPIG-M partially restored cell surface expression of the GPI-anchored protein CD59 in PIG-M deficient mammalian cells, and first mannose transfer activity in vitro; however, this was not the case for GPI14.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.62-65
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• 2002

We have previously shown that the addition of silkworm hemolymph to a culture medium increases the longevity of insect and mammalian cells by inhibiting apoptosis. This indicates that the component which inhibits apoptosis is contained in the silkworm hemolymph, The apoptosis-inhibiting component was isolated from silkwonn hemolymph and characterized in our previous study. A database search using the N-terminal amino acid sequence of this component as a template resulted in a 95% homology with a low molecular weight lipoprotein, the so called ’30K protein' of unknown function. In this study, the 30K protein gene was expressed in mammalian and insect cells to confirm the apoptosis-inhibiting effect. The overexpression of 30K protein in mammalian cell inhibited the staurosporin-induced apoptosis by the prevention of the activation of caspase 3. Using an Autographa californicanuclear polyhedrosis virus (AcNPV) system, the 30K protein was overexpressed also in insect cells. The expression of the 30K protein increased the longevity of baculovirus-infected insect cells by inhibiting apoptosis. These results suggest that the 30K protein is a novel anti-apoptotic protein.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1991년도 춘계학술발표대회 논문집
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• pp.82-96
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• 1991

To understand the mechanism of illegitimate recombination in mammalian cells, we have examined the recombination role of DNA topoisomerase II (Topo II ). We found that purified calf thymus Topo II mediates recombination between two phage $\lambda$ DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus Topo II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletion, and most crossovers take place between nonhomologous sequences of $\lambda$ DNA, as judged by the sequences of recombination junctions. In order to study the effects of Topo II on illegitimate recombination in mammalian cells, we have developed a new shuttle vector, pNKl, which contains three bacterial genes, amp(APR), galK and neo($Km^R$). Using this system, we have shown that a Topo II inhibitor, VM26, stimulated deletion formation in pNK1 DNA in monkey COS1 cells. Both in vitro and in vivo results suggest that Topo II participates in illegitimate recombination in mammalian cells.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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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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• 제6권4호
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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.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2011년도 제40회 동계학술대회 초록집
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• pp.7-7
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• 2011

Graphene and graphene derivatives have attracted enormous attention from various research fields for applications in electronic devices, transparent electrodes, biosensors, drug delivery system and surface coatings. In the viewpoint of chemist, the chemical structure of graphene derivatives seems intriguing but detailed structures are being revealed only recently while engineering approaches for various applications are being executed very actively. In addition, cytotoxicity and mammalian cellular responses to graphene have not thoroughly investigated yet in spite of the importance in bio-applications and environment. In this talk, I'll introduce recent studies which report cytotoxicity and behaviors of mammalian cells when the cells are exposed to graphene (as well as some bio-applications of graphene), especially to get closer to answers to these questions, "how we understand and how/why we use graphene in biotechnology".

• 한국가축번식학회지
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• 제18권4호
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• pp.285-297
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• 1995

The mammalian oviduct is a place where ontogeny of an animal begins. Nowadays, however, it is possilbe to manipulate a part of physiological events occurring in the oviduct so that fertilization of gametes and early embryonic development of zygotes could proceed outside oviductal environment. Rabbit zygotes readily develop to blastocysts in a conventional culture condition. Most of the mouse fertilized eggs do so when cultured under a specific environment, e.g., in a medium containing ethylenediamine tetraacetic acid. Similarly, a significant number of zygotes from rat, sheep, pig or cattle can develop to blastocysts if they are cultured in the presence of particular component which appear to be somewhat species-specific. Instead of changing the components of medium, somatic cells including oviductal epithelial cells, have widely been used to improve mammalian embryonic development in vitro. Many investigators have reported that mammalian zygotes, whether fertilized in vivo or in vitro, could develop to blastocysts when they were cultured on a monolayer of various kinds of somatic cells or even in a somatic cell-conditioned medium. While little is known about the nature of embryotrophic factor(s) produced in vitro by somatic cells, the existence fo oviduct-specific protein(s) has consistently been demonstrated in many laboratories. Some of these proteins are reported to be associated with oviductal eggs. However, the physiological role of these proteins has still to be determined. Recently we observed that the perivitelline space of mouse oocytes was fluorescently stained with various fluorochrome-protein conjugates following ovulation into the oviducts or upon their expossure to oviductal extracts. Furthermore, it was also found that cattle or pig oviductal fluid gave similar results when examined using mouse ghost ZP. These observations lead to suggest that mammalian oviduct induces changes of biochemical properties of oocytes. Further studies are needed to clarify the nature of oviductal factor(s) and the physiological meaning of the reaction.