• Molecules and Cells
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• v.39 no.10
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• pp.768-775
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• 2016

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

• Journal of Digestive Cancer Research
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• v.12 no.2
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• pp.106-114
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• 2024

Circulating tumor cells (CTCs) are a valuable biomarker for the diagnosis, prognosis, and therapeutic management of gastrointestinal (GI) cancers. A major challenge in GI cancer treatment is the high rate of metastasis, which significantly contributes to cancer-related mortality. CTCs are crucial in the metastatic cascade, serving as indicators of tumor progression. Therefore, the detection and molecular characterization of CTCs have prognostic potential for identifying early-stage GI cancers and assessing metastatic probability, enabling timely treatment. Moreover, CTC analysis offers a minimally invasive method for real-time monitoring of tumors. Clinicians can adjust therapeutic strategies accordingly by tracking changes in CTC count and molecular profile. Despite this promising application, no standardized protocol for CTC isolation in GI tract cancers has been established, which poses a barrier to routine clinical use. This review explores the current CTC detection methodologies, their clinical relevance in GI cancer management, and the potential integration of CTC analysis into personalized medicine. We also discuss the challenges and future directions in CTC research, focusing on clinical validation and the development of standardized procedures to fully realize the utility of CTC count for improving patient care.

• Microbiology and Biotechnology Letters
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• v.39 no.2
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• pp.111-118
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• 2011

For the strain improvement of the industrial polyploid yeast strain through hybridization and protoplast fusion, a dominant selection marker other than a recessive marker such as the auxotrophic marker was required for the selection of the resulting hybrids. In the present investigation, the aureobasidin A resistant gene was tested in relation to whether it can be used as the dominant selectable marker for the isolation of hybrids of the yeast Saccharomyces. The plasmid pAUR112, carrying the gene responsible for resistance to aureobasidin A, was introduced into the haploid yeast strain K114/YIp. From the rare-mating between polyploid C6 and haploid K114/YIp carrying pAUR112, many hybrids were obtained from the agar medium containing 0.5 ${\mu}g$/ml of aureobasidin A. The hybrids exhibited characteristics derived from both of the parental strains; and the cell sizes of the hybrids were larger than those of the parental strains. These results showed that the aureobasidin A resistant gene could be successfully used as the dominant selectable marker for the isolation of yeast hybrids resulting from rare-mating.

• Molecules and Cells
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• v.37 no.7
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• pp.562-567
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• 2014

Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-${\beta}1$. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth.

• Korean Journal of Microbiology
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• v.35 no.1
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• pp.61-64
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• 1999

Organophosphorus inseclicide phosphamidon-degrading bacteria were isolated from agricultural soils and identified using Biolog microtiter assay. All Gram-positive degrading bacterial strains belong to genus Bacillus and many Gram-negative bacteria were rare soil species. Among them fast growing strains on phosphamidon-containing minimal medium were sclected and their biodegrading capability wcre measured. YD-17 which was identified as Capnocytophaga gingivalis showed the highest biodegradation rate. It could incrcase the removal of phosphamidon up to 52%. During the biodegradation continuous increase of amount of cell protein was observed, which indicated that phosphamidon was utilized as a carbon source for phosphamidon-degrading bacteria.