• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.

• Applied Microscopy
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• v.37 no.4
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• pp.219-230
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• 2007

The morphological effects of intranasal zinc sulfate(5% solution) irrigation on the mouse olfactory epithelium and the regeneration process of olfactory receptor cells following nasal irrigation were studied with scanning and transmission electron microscope. The results were as follows: 1. The septal epithelium except some basal cells was wholly detached from the basement membrane, during the first 6 to 24 hours after 5% zinc sulfate irrigation. 2. 3 days after $ZnSO_4$ treatment, two layered septal epithelium was formed from basal cells. And microvilli were observed in the apical epithelium of newly formed olfactory epithelial cells. 3. 5 days after treatment, a lot of centrosomes and basal bodies were observed in the olfactory receptor cells, and cilia were lined up between microvilli on the apical membrane of olfactory receptor cells. And immature olfactory knob was first observed in the newly formed olfactory receptor cells. Mature olfactory knob was observed 1 week after treatment. 4. There are very many mature olfactory knobs in the olfactory receptor cells 2 weeks after intranasal zinc sulfate irrigation. These results support that treatment with 5% zinc sulfate is a good experimental model for the regeneration of mammalian nervous tissues because this method could thoroughly detach the septal epithelium. During the regeneration of olfactory receptor cells, the surface membrane of the olfactory receptor cells widen the surface with the microvilli. Then cilia, which arranged in a line, substituted for the microvilli. The part of the surface membrane with cilia protruded and finally formed the olfactory vesicle.

• Applied Microscopy
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• v.35 no.2
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• pp.65-72
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• 2005

The ultrastructure of submandibular gland was examined in the lesser white-toothed shrew, Crocidura suaveolens. The submandibular gland of C. suaveolens was a mixed gland composed of serous and mucous acinar cells. Secretory granules from the acini were discharged through the salivary ducts into the oral cavity. Serous and mucous acinar cells had well developed rough endoplasmic reticulum and mitochondria and large amount of granules. In case of serous acinar granules, an immature granule was formless and had only dense specks, and a matured granule was a complete round type delimiting by a single membrane and had a homogeneous dense center with dense specks on the border. In case of mucous acinar granules, while an immature granule was a round type and had an only homogeneous matrix and an indistinct limiting membrane, a mature granule was an even round type having a variety of pattern with several dense bands into the homogeneous matrix and had a distinct membrane. Therefore, a mature mucous acinar granule of C. suaveolens was not only distinct from those of the other mammalian species to have a variety of pattern but also from those of C. lasiura to have an even round type. A great serous-like secretory granules and Myelin-like body were observed in the cytoplasm and lumen of granular duct cells. Myelin-like body, a characteristic structure only reported in salivary gland of three shrews, was discharged from secretory cell into lumen by the manner of exocytosis which has little differences from discharging manner of secretory granules.

• Archives of Pharmacal Research
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• v.29 no.8
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• pp.666-676
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• 2006

Gangliosides are widely distributed in mammalian cells and play important roles in various functions such as cell differentiation and growth control. In addition, diabetes and obesity cause abnormal development of reproductive processes in a variety of species. However, the mechanisms underlying these effects, and how they are related, are not fully understood. This study examined whether the differential expression of gangliosides is implicated in the abnormal follicular development and uterine architecture of streptozotocin (STZ)-induced and db/db diabetic mice. Based upon the mobility on high-performance thin-layer chromatography, mouse ovary consisted of at least five different ganglioside components, mainly gangliosides GM3, GM1, GD1a and GT1b, and diabetic ovary exhibited a significant reduction in ganglioside expression with apparent changes in the major gangliosides. A prominent immunofluorescence microscopy showed a dramatic loss of ganglioside GD1a expression in the primary, secondary and Graafian follicles of STZ-induced and db/db diabetic mice. A significant decrease in ganglioside GD3 expression was also observed in the ovary of db/db mice. In the uterus of STZ-induced diabetic mice, expression of gangliosides GD1a and GT1b was obviously reduced, but gangliosides GM1, GM2 and GD3 expression was increased. In contrast, the uterus of db/db mice showed a significant increase in gangliosides GM1, GD1a and GD3 expression. Taken together, a complex pattern of ganglioside expression was seen in the ovary and uterus of normoglycemic ICR and $db/^+$ mice, and the correspoding tissues in diabetic mice are characterized by appreciable changes of the major ganglioside expression. These results suggest that alterations in ganglioside expression caused by diabetes mellitus may be implicated in abnormal ovarian development and uterine structure.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• Journal of Life Science
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• v.17 no.11
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• pp.1497-1504
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• 2007

To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.17-24
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• 2017

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 ${\beta}$-galactoside ${\alpha}$-2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a ($10{\mu}M$) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• Korean Journal of Clinical Laboratory Science
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• v.53 no.4
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• pp.353-362
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• 2021

Cancer is a global health problem. There are diverse types of cancers, but there are several common pathways which lead to the development of cancer. Changes in gene expression might be the most common similarity found in almost all cancers. An understanding of the underlying changes in gene expression during cancer progression could lay a valuable foundation for the development of cancer therapeutics and even cancer vaccines. In this study, a well-known carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was employed to induce changes in gene expression in normal stomach cells. MNNG is known to cause cancer by inducing damage to DNA in MNNG-treated mammalian cells and animals fed with this carcinogen. An analysis was performed by comparing the differentially expressed genes (DEGs) caused by MNNG treatment with DEGs in stomach cancer cell lines. To this end, methods of analysis for functional categorization and protein-protein interaction networks, such as gene ontology (GO), the database for annotation, visualization, and integrated discovery (DAVID), Kyoto encyclopedia of genes and genomics (KEGG) and search tool for the retrieval of interacting genes/proteins (STRING), were used. As a result of these analyses, MNNG-regulated specific genes and interaction networks of their protein products that contributed to stomach cancer were identified.

• Korean Journal of Food Science and Technology
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• v.53 no.6
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• pp.715-721
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• 2021

Cognitive impairment and Alzheimer's disease are serious social problems associated with the rising elderly population in Korea. 1,2,3,4,6-Penta-O-galloyl-β-ᴅ-glucopyranose (PGG) is a gallotannin isolated from medicinal plants such as Rhus chinensis. This study was performed to evaluate the effect of PGG on biomarkers related to cognitive function in human neuroblastoma SK-N-SH cells. Inhibition of acetylcholinesterase (AChE) activity is considered to be one of the main therapeutic strategies. PGG inhibited AChE activity in the test tube as well as in SK-N-SH cells. In addition, PGG induced protein and mRNA expression of brain-derived neurotrophic factor (BDNF), which is a mammalian neurotrophin that plays major roles in the development, maintenance, repair, and survival of neuronal populations. As one of the underlying molecular mechanisms that induce BDNF expression, PGG induced the activation of Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII)-cAMP response element binding protein (CREB) pathway. In conclusion, PGG may be an useful material for improving cognitive function.