• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.684-687
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• 2000

The super-producing recombinant H.polymorpha mutant is obtained by double membrane screening technique combined with optimum mutation method. The characterization of mutant is carried out to find the change of mutant in m-RNA level, cell wall leakage, protease level and methanol utilization metabolic flux. The change of these properties of mutant was figured out.

• Journal of the Korean Society for Precision Engineering
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• v.31 no.6
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• pp.521-525
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• 2014

Circulating tumor cells (CTCs) in the bloodstream of cancer patients provide an accessible source for detection, characterization, and monitoring of nonhematological cancers. The effectiveness of the CTC-Chip for the isolation of ovarian cancer cells was demonstrated by adapting the herringbone-chip (HB-Chip). The motions of the particles on the HB chip were simulated by a unique combination of buoyant, gravitational forces, and helical flows with a computational modeling. The motions of cells are demonstrated by applying polystylene bead and ovarian cancer cells into the microfabricated HB-Chip. The experimental results from beads and cells are well accordance with the simulated ones, as previously reported by Toner group. Thus, I expect that these modeling and experimental skills will play key roles in the clinical applications on CTC isolation as well as the basic research on characterization of CTCs under flow.

• Bulletin of the Korean Chemical Society
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• v.33 no.12
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• pp.4023-4027
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• 2012

Cytotoxicity assessment of silver nanoparticles (AgNPs) was performed using MTT-based microfluidic image cytometry (${\mu}FIC$). The $LC_{50}$ value of HeLa cells exposed to AgNPs in the microfluidic device was estimated as 46.7 mg/L, which is similar to that estimated by MTT-based IC for cells cultured in a 96 well plate (49.9 mg/L). These results confirm that the ${\mu}FIC$ approach can produce cytotoxicity data that is reasonably well-matched with that of the conventional 96 well plate system with much higher efficiency. This ${\mu}FIC$ method provides many benefits including ease of use and low cost, and is a more rapid in vitro cell based assay for AgNPs. This may aid in speeding up data acquisition in the field of nanosafety and make a significant contribution to the quantitative understanding of nanoproperty-toxicity relationships.

• Journal of Microbiology and Biotechnology
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• v.24 no.8
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• pp.1034-1043
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• 2014

This study determined the effect of light intensity and photoperiod on the dry cell weight and total amount of carotenoids in four isolates of purple non-sulfur bacteria obtained from shaded and exposed microhabitats of a mangrove ecosystem in Kota Kinabalu, Sabah, Malaysia. The initial isolation of the bacteria was carried out using synthetic 112 medium under anaerobic conditions (2.5 klx) at $30{\pm}2^{\circ}C$. On the basis of colony appearance, cell morphology, gram staining, motility test, and 16S rRNA gene sequencing analyses, all four bacteria were identified as Afifella marina. One of the bacterial isolates, designated as Af. marina strain ME, which was extracted from an exposed mud habitat within the mangrove ecosystem, showed the highest yield in dry cell weight ($4.32{\pm}0.03g/l$) as well as total carotenoids ($0.783{\pm}0.002mg/g$ dry cell weight). These values were significantly higher than those for dry cell weight ($3.77{\pm}0.02g/l$) and total carotenoid content ($0.706{\pm}0.008mg/g$) produced by the isolates from shaded habitats. Further analysis of the effect of 10 levels of light intensity on the growth characteristics of Af. marina strain ME showed that the optimum production of dry cell weight and total carotenoids was achieved at different light intensities and incubation periods. The bacterium produced the highest dry cell weight of 4.98 g/l at 3 klx in 72 h incubation, but the carotenoid production of 0.783 mg/g was achieved at 2.5 klx in 48 h incubation. Subsequent analysis of the effect of photoperiod on the production of dry cell weight and total carotenoids at optimum light intensities (3 and 2.5 klx, respectively) revealed that 18 and 24 h were the optimum photoperiods for the production of dry cell weight and total carotenoids, respectively. The unique growth characteristics of the Af. marina strain ME can be exploited for biotechnology applications.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.10
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• pp.1579-1589
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• 2020

Objective: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction. Methods: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3). Results: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos. Conclusion: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.

• Animal cells and systems
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• v.1 no.1
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• pp.157-164
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• 1997

Ly-6E.1 antigen was proposed as a regulatory molecule of T lymphocyte activation, a hematopoietic stem cell marker, a memory cell marker, and an adhesion molecule. Though there were several reports suggesting the presence of Ly-6 ligand, the characterization of the ligand was not yet performed, As an attempt to screen the expression of Ly-6E.1 ligand, we prepared a probe for detecting Ly-6E.1 ligand by producing a fusion protein between Ly-6E.1 and $hlgC_{r1}$, A mammalian cell expression vector with Ly-6E.$1/hlgC_{r1}$ chimeric cDNA was transfected in SP2/0-Ag14 myeloma cells, and stable transfectants were selected. The fusion protein was produced as a dimer and maintained the epitopes for monoclonal antibodies specific for Ly-6E.1 and for anti-human lgG antibody. The purified fusion protein through Gammabind G column was used for FACS analyses for the expression of Ly-6E.1 ligand. The fusion protein interacted with several cell lines originating from B cells, T cells, or monocytes. The fusion Protein also strongly stained bone marrow, lymph node, and spleen cells, but thymic cells weakly, if any. The staining was more obvious in C57BL/6 $(Ly-6^b)$ than Balb/c $(Ly-6^a)$ mice. These results suggest that the interaction of Ly-6E.1 with Ly-6E.1 ligand may function both in the stem cell environment and in the activation of mature lymphocytes. The fusion protein may be a valuable tool in characterization of biochemical properties of the Ly-6E.1 ligand and, further, in isolating its cDNA.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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• pp.619-628
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• 2014

To identify lipase LipA (PFL_0617) from Pseudomonas protegens Pf-5, a lipA deletion mutant (Pf0617) and a complementary strain (Pf0617lipA) were constructed, and their effects on the lipase production were examined. Pf0617 remarkably decreased its whole-cell lipase activity, whereas Pf0617lipA made its whole-cell lipase activity not only restore to wild-type level but also get a further increment. However, the deletion and overexpression of lipA did not affect the extracellular lipase activity. In addition, the unbroken whole cells of these strains were able to catalyze the hydrolysis of membrane-permeable p-nitrophenyl esters, but could not hydrolyze the membrane-impermeable olive oil. These results confirmed that LipA was an intracellular lipase and Pf-5 could also be used as a natural whole-cell biocatalyst. To evaluate the potential of Pf-5 as a whole-cell biocatalyst and separately characterize the whole-cell LipA, the properties of the whole-cell lipases from Pf-5 and Top10lipA were characterized. The results demonstrated that both Pf-5 and Top10lipA exhibited high tolerance to alkaline condition, high temperature, heavy metal ions, surfactants, and organic solvents. Taken together, lipA can realize functional expression in E. coli Top10, and Pf-5 and Top10lipA as whole-cell biocatalysts may have enormous potential in applications.

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.398-405
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• 2002

Bifidobacteria have been previously shown to stimulate the immune functions and cytokine production in macrophages and T-lymphocytes. Accordingly, the RAW 264.7 murine macrophage cell line was used to assess the effects of Bifidobacterium on the proliferation and cytoskeleton reorganization of the cells. Cytokine production after exposure to Bifidobacterium was also monitored in both whole cells and cell-free extracts. When RAW 264.7 cells were cultured for 24 h in the presence of heat-killed Bifidobacterium bifidum BGN4, the proliferation of macrophages was slowed down in a dose-dependent manner and cell differentiation was observed by staining with the actin-specific fluorescent dye, rhodamin-conjugated phalloidin. Although EL-4 cells, a T-cell line, stimulated RAW 264.7 cells to produce TNF-${\alpha}$ and IL-6, the stimulatory activity of B. bifidum BGN4 decreased as the EL-4 cell number increased. When disrupted and fractionated BGN4 was used, the whole cell fraction was more effective than the other fractions for the TNF-${\alpha}$ production. In contrast, the cell-free extract exhibited the highest IL-6 production level among the fractions, which was evident even at a $1{\mu}g/ml$ concentration. The current results demonstrate that Bifidobacterium induced differentiation of the macrophages from the fast proliferative stage and that the cytokine production was differentially induced by the whole cells and cell-free extracts. The in vitro approaches employed herein are expected to be useful in further characterization of the effects of bifidobacteria with regards to gastrointestinal and systemic immunity.

• Molecules and Cells
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• v.46 no.4
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• pp.209-218
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• 2023

In induced pluripotent stem cells (iPSCs), pluripotency is induced artificially by introducing the transcription factors Oct4, Sox2, Klf4, and c-Myc. When a transgene is introduced using a viral vector, the transgene may be integrated into the host genome and cause a mutation and cancer. No integration occurs when an episomal vector is used, but this method has a limitation in that remnants of the virus or vector remain in the cell, which limits the use of such iPSCs in therapeutic applications. Chemical reprogramming, which relies on treatment with small-molecule compounds to induce pluripotency, can overcome this problem. In this method, reprogramming is induced according to the gene expression pattern of extra-embryonic endoderm (XEN) cells, which are used as an intermediate stage in pluripotency induction. Therefore, iPSCs can be induced only from established XEN cells. We induced XEN cells using small molecules that modulate a signaling pathway and affect epigenetic modifications, and devised a culture method which can produce homogeneous XEN cells. At least 4 passages were required to establish morphologically homogeneous chemically induced XEN (CiXEN) cells, whose properties were similar to those of XEN cells, as revealed through cellular and molecular characterization. Chemically iPSCs derived from CiXEN cells showed characteristics similar to those of mouse embryonic stem cells. Our results show that the homogeneity of CiXEN cells is critical for the efficient induction of pluripotency by chemicals.

• BMB Reports
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• v.37 no.1
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• pp.83-92
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• 2004

As a result of the enormous amount of information that has been collected with E. coli over the past half century (e.g. genome sequence, mutant phenotypes, metabolic and regulatory networks, etc.), we now have detailed knowledge about gene regulation, protein activity, several hundred enzyme reactions, metabolic pathways, macromolecular machines, and regulatory interactions for this model organism. However, understanding how all these processes interact to form a living cell will require further characterization, quantification, data integration, and mathematical modeling, systems biology. No organism can rival E. coli with respect to the amount of available basic information and experimental tractability for the technologies needed for this undertaking. A focused, systematic effort to understand the E. coli cell will accelerate the development of new post-genomic technologies, including both experimental and computational tools. It will also lead to new technologies that will be applicable to other organisms, from microbes to plants, animals, and humans. E. coli is not only the best studied free-living model organism, but is also an extensively used microbe for industrial applications, especially for the production of small molecules of interest. It is an excellent representative of Gram-negative commensal bacteria. E. coli may represent a perfect model organism for systems biology that is aimed at elucidating both its free-living and commensal life-styles, which should open the door to whole-cell modeling and simulation.