• Molecules and Cells
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• v.27 no.4
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• pp.391-396
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• 2009

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events - for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5$^{TM}$ and CyTRAK Orange$^{TM}$. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.

• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1269-1274
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• 2014

Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

• Journal of the Korean Chemical Society
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• v.55 no.1
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• pp.132-135
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• 2011

• International Journal of Oral Biology
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• v.35 no.3
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• pp.107-111
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• 2010

Treponema denticola is a gram-negative anaerobe that can cause periodontal disease. The adhesion of this bacterium to host tissues is considered to be the primary event in the colonization and infection of a host. Fibrinogen is generally found in damaged tissues resulting from periodontitis. The binding ability of T. denticola to fibrinogen may therefore be an important virulence factor in inducing periodontal diseases. It has been reported recently that oral spirochetes can be labeled with fluorescent fatty acids and we speculated that this labeling method could be used in an oral spirochete binding assay. The binding of several different strains of T. denticola to immobilized human fibrinogen was therefore tested using the fluorescent fatty acid labeling method. In the case of immobilized fibrinogen, the T. denticola ATCC 35405 strain showed saturable binding to immobilized fibrinogen. Indeed, all four different T. denticola strains tested in this experiment, T. denticola ATCC 35405, T. denticola ATCC 33520, T. denticola ATCC 35404 and T. denticola OTK showed binding to fibrinogen. The fluorescent fatty acid labeling method thus shows utility in binding assays for T. denticola, different strains of which can generally bind to immobilized fibrinogen.

• Molecules and Cells
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• v.40 no.11
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• pp.828-836
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• 2017

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the subcellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

• Bulletin of the Korean Chemical Society
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• v.29 no.5
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• pp.943-947
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• 2008

A phosphate-specific and fluorescent probe was prepared for label-free phosphatase assays based on fluorescence polarization. By using the probe, dephosphorylation reactions of DNA and protein substrates by calf intestinal alkaline phosphatase (CIP) could effectively be monitored in real-time. Since this assay method does not require additional materials such as labeled substrates and phosphospecific antibodies to obtain fluorescence polarization signals, it is simple, cost-effective, and expected to be useful not only for measuring activity of phosphatases but also for high-throughput screening of phosphatase inhibitors.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.208-211
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• 2000

The recombinant fluorescent chinese hamster ovary (CHO) cell line was developed and optimized through this study for biomonitoring system. This cell line, called KFC-A10, contains recombinant plasmid(pKCFG) constructed in this study for detecting toxic conditions (Mitomicyn C, EDCs, ${\gamma}-ray$, etc.). It is known that c-Fos is involved in proliferation and differentiation of the signal transduction and overexpression of this gene can lead cell to death under the toxic conditions including apoptosis status. Therefore, pKCFG which has the c-fosSRE::GFP is induced by toxic chemicals, especially DNA damage agents and apoptotic chemicals, and produces green fluorescence protein(GFP) under these toxic conditions. Through the characterization of KFC-A10 using fluorescent assays of GFP, it was shown that KFC-A10 cell line had a manifest GFP expression pattern due to various toxicants especially mitomycin C, ${\gamma}-ray$ and bisphenol A. Therefore this study proved the possibility of using GFP as a reporter for detecting various toxicants

• Fisheries and Aquatic Sciences
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• v.15 no.4
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• pp.325-335
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• 2012

The present study characterized the fast skeletal myosin light chain-2 gene (mlc2f) in the euryhaline Javanese ricefish (Oryzias javanicus: Beloniformes). Coding nucleotide and deduced amino acid sequences of Javanese ricefish mlc2f were well conserved in the vertebrate lineage. Javanese ricefish mlc2f showed a typical seven-exon structure, and its promoter exhibited transcription factor binding motifs common to most muscle-specific genes. However, Javanese ricefish mlc2f also displayed tandem duplications of intronic sequences in both intron 1 and intron 3. Based on quantitative reverse transcription-polymerase chain reaction, the mlc2f transcripts were highly predominant in skeletal muscles of adults and were differentially modulated during embryonic development. Microinjection of the mlc2f promoter-driven red fluorescent protein (RFP) reporter construct successfully exhibited heterologous expression of the fluorescent reporter, primarily in muscular areas of hatchlings, although the distribution pattern of RFP signals was not uniform due to the mosaic nature of the introduced transgene. Data from this study indicate that the Javanese ricefish mlc2f gene has undergone "intra-intronic" duplication events in a species-specific manner and that the mlc2f regulator may also be useful in heterologous expression assays of the skeletal muscles of this species.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.3
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• pp.179-188
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• 2007

Objectives: Many neurological diseases are known to be caused by expansion of trinucleotide repeats (TNRs). It is hard to diagnose the alteration of TNRs with single cell level for preimplantation genetic diagnosis (PGD). In this study, we describe methods optimized for PGD of TNRs related diseases such as Huntington's disease (HD), spinocerebellar ataxia 3 (SCA3) and fragile X syndrome (FXS). Methods: We performed the preclinical assays with heterozygous patient's lymphocytes by single cell PCR strategy. Fluorescent semi-nested PCR and fragment analysis using automatic genetic analyzer were applied for HD and SCA 3. Whole genome amplification with multiple displacement amplification (MDA) method and fluorescent PCR were carried out for FXS. Amplification and allele drop-out (ADO) rate were evaluated in each case. Results: The fluorescent semi-nested PCR of single lymphocyte showed 100.0% of amplification and 14.0% of ADO rate in HD, and 94.7% of amplification and 5.6% of ADO rate in SCA3, respectively. We could not detect the PCR product of CGG repeats in FXS using the fluorescent semi-nested PCR alone. After applying the MDA method in FXS, 84.2% of amplification and 31.3% of ADO rate were achieved. Conclusions: Fluorescent semi-nested PCR is a reliable method for PGD of HD and SCA3. The advanced MDA method overcomes the problem of amplification failure in CGG repeats of FXS case. Optimization of methods for single cell analysis could improve the sensitivity and reliability of PGD for complicated single gene disorders of TNRs.

• Journal of the Optical Society of Korea
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• v.16 no.1
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• pp.42-46
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• 2012

We have developed a fluorescence optical detection system using a digital micromirror device (DMD) for monitoring 3D cell culture matrices in situ. Full 3D imaging with fast scanning speed was implemented by the combined action of a DMD and a motorized stage. Imaging results with fluorescent microbeads measure the minimum axial resolution of the system as $6.3{\mu}m$, while full 1-mm scanning through 3D alginate-based matrix was demonstrated. For cell imaging, improved images were obtained by removing background fluorescence although the scanning distance was reduced because of low intracellular fluorescence efficiency. The system is expected to be useful to study various dynamics and behaviors of 3-dimensionally cultured cells in microfluidic systems.