• Journal of Life Science
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• v.28 no.7
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• pp.835-840
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• 2018

Quantitative determination of the protein expression levels is one of the most important parts in assessment of the safety of foods derived from genetically modified (GM) crops. Overexpression of AtCYP78A7, a gene encoding cytochrome P450 protein, has been reported to improve tolerance to abiotic stress, such as drought and salt stress, in transgenic rice (Oryza sativa L.). In the present study, an enzyme-linked immunosorbent assay (ELISA) kit for diagnosing AtCYP78A7 protein including AtCYP78A7-specific monoclonal antibody was developed. GST-AtCYP78A7 recombinant protein was induced and purified by affinity column. Four monoclonal antibodies (mAb 6A7, mAb 4C2, mAb 11H6, and mAb 7E8) against recombinant protein were also produced and biotinylated with avidin-HRP. After pairing test using GST-AtCYP78A7 protein and lysate of rice samples, mAb 4C2 and mAb 7E8 were selected as a capture antibody and a detecting antibody, respectively, for ELISA kit. Product test using rice samples indicated that percentages of detected protein in total protein were greater than 0.1% in AtCYP78A7-overexpressing transgenic rice (Line 10B-5 and 18A-4), whereas those in negative control non-transgenic rice (Ilpum and Hwayoung) were less than 0.1%. The ELISA kit developed in this study can be useful for the rapid detection and safety assessment of transgenic rice overexpressing AtCYP78A7.

• Journal of Veterinary Clinics
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• v.33 no.4
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• pp.187-193
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• 2016

A tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to a protein in a cell. It functions as an "on" or "off" switch in many cellular functions. This study aims to show that the actions of growth factors associated with PDGFR-${\alpha}$, PDGFR-${\beta}$, VEGFR-2, c-KIT, and c-ABL, which are used in veterinary medicine, are expressed in canine intractable tumors. This study used archival cases of canine paraganglioma, gastrointestinal adenocarcinoma, hepatocellular carcinoma, and renal cell carcinoma. Tissues had been immunohistochemical analysis. The antibodies used were PDGFR-${\alpha}$, PDGFR-${\beta}$, c-kit, VEGFR-2, and c-Abl. PDGFR-${\alpha}$ was expressed only in HCC, and PDGFR-${\beta}$ was expressed in all tumors. VEGFR was also only expressed in HCC, and c-KIT has been expressed in HCC, paraganglioma, and small intestinal adenocarcinoma. c-Abl was expressed in all cancers, but was weakly expressed in paraganglioma, while more than moderately expressed in other tissues. In conclusion, this study investigated how TKIs used in human medicine can be applied to canine intractable tumors, through immunohistochemistry. The results indicate that there may be an application for TKIs in treating canine intractable tumors.

• Korean Journal of Acupuncture
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• v.32 no.2
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• pp.51-58
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• 2015

Objectives : Pinus densiflora gnarl, called Song-Jeol in Korean medicine, has been used to cure inflammatory diseases such as arthritis. In the present study, we evaluated inhibitory property of Song-Jeol pharmacopuncture(SJ) on C6 glioma cell migration. Methods : To evaluate cell viability on C6 glioma cells of SJ, the viability was assessed by using Ez-cytox assay kit. The cell migration was assessed by wound-healing assay and Boyden chamber assay, respectively. LPS-induced NO productions were determined by using the Griess reagent. The expression of iNOS and protein kinase $C(PKC)-{\alpha}$ were estimated by western blotting assay. Results : In the wound-healing assay and Boyden chamber assay, SJ showed a significant inhibition on serum-induced C6 glioma cell migration. In addition, NO production was decreased by SJ through suppression of iNOS expression in LPS-stimulated C6 glioma cell. Futhermore, LPS-induced protein kinase $C(PKC)-{\alpha}$ expression was effectively inhibited by SJ. Conclusions : These results demonstrated that SJ was useful for the suppression of the C6 glioma cell migration.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.4
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• pp.778-790
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• 2008

This experiment investigated the effect of mixed extracts obtained from Mylabris phalerata Pall. and Drynariae Rhizoma on hair growth activity of the normal and spontaneous alopecia areata model of C57BL/6N mice for 16 days. First, we examined morphological regrowth of hair in normal and spontaneous alopecia model of C57BL/6N mice. Second, we examined immunoreactive density of vascular endothelial growth factor(VEGF), c-kit and protein kinase $C-{\alpha}(PKC-{\alpha})$ in skin of normal C57BL/6N mice by immunohistochemical methods. Third, we investigated expression of $TGF-{\beta}$, prolactin and placenta lactogen after topical application of mixed extracts of Mylabris phalerata Pall. and Drynariae Rhizoma to skin by RT-PCR. The results were as follows: Hair growth effect from middle and high concentration of mixed extracts of Mylabris phalerata Pall. and Drynariae Rhizoma was observed in 80% of normal mice in whose hair had been clipped in 15th days. Hair growth effect of all concentrations of mixed extracts of Mylabris phalerata Pall. and Drynariae Rhizoma was observed in 100% of spontaneous alopecia model of C57BL/6N mice in 15th days. Immunoreactive density of VEGF, c-kit and $PKC-{\alpha}$ in skin of all concentrations of mixed extracts of Mylabris phalerata Pall. and Drynariae Rhizoma were strongly stained in epidermis, bulge, secondary hair germ cells and cutaneous trunci m. compare to control group in 10th day. In experimental III group, Immunoreactive density of VEGF, c-kit and $PKC-{\alpha}$ in skin were strongly stained in inner and outer root sheath of skin. The treatment of mixed extracts of Mylabris phalerata Pall. and Drynariae Rhizoma increased the expression of $TGF-{\beta}$, placenta lactogen and prolactin in the skin of normal C57BL/6N mice compared to control group. These experiments suggest that mixed extracts of Mylabris phalerata Pall. and Drynariae Rhizoma may stimulate the topical hair growth activity and it can be useful for treatment of alopecia areata.

• Microbiology and Biotechnology Letters
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• v.41 no.1
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• pp.128-134
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• 2013

Salmonella enterica serovar typhi, a Gram-negative food-borne pathogen, causes typhoid fever in humans. OmpC is an outer membrane porin of S. typhi expressed throughout the infection period. OmpC is potentially an attractive antigen for multivalent vaccines and diagnostic kit designs. In this study we combined in silico, in vitro and in vivo approaches to analyze various aspects of OmpC's antigenic properties. The conserved region, in addition to secondary and tertiary structures, and linear B cell epitopes, were predicted. A number of results obtained from in silico analyses were validated by experimental studies. OmpC was amplified, cloned and then expressed, with the recombinant protein then being purified. BALB/c mice were immunized by purified denatured OmpC. The titer of antibody was raised. Results of challenges with the pathogen revealed that the immunity is non-protective. Most of the theoretical and experimental results were in consensus. Introduced linear B cell epitopes can be employed for the design of diagnostic kits based on antigen-antibody interactions.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.351-359
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• 2014

Phasianus colchicus is not only a beautiful bird but also a great value in science and under the threat of endanger. Hence, the aim of this study was to isolate the pheasant male germ cells (mGCs) and then induce them into elongated sperm-like cells in vitro. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the c-kit positive cells and c-kit negative cells examined by flow cytometry analysis (FCA) was 92.87% and 2.57%, respectively. Subsequently, the mGCs were induced for 48h in DMEM/F12 medium supplemented factors such as retinol acid, testosterone and bovine FSH, followed by 5 weeks in culture. We found that some elongated sperm-like cells appeared initially in vitro under inducement of stimulated factors. The elongated sperm-like cells showed in the expression of changed morphology and post-transcriptional marker such as spermatid associated (SPERT), spermatid perinuclear RNA binding protein (STRBP), round spermatid basic protein 1 (RSBN1) and SPER1L. Moreover, in DNA content identified assay, induced cells showed that the 1C DNA population markedly increased in differentiated group but it was not change in undifferentiated group. Successful in vitro differentiation of pheasant testicular germline cells into spermatids appears to offer extremely attractive potential for the conservation of endangered birds and treatment of male infertility.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.317-324
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• 2008

Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis. Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.9
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• pp.3901-3906
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• 2014

Aim: The purpose of this study was to investigate anti-tumor effects and safety of DH332, a new ${\beta}$-carboline alkaloids derivatives in vitro and in vivo. Materials and Methods: The effects of DH332 on human (RAMOS RA.1) and mouse (J558) B lymphoma cell lines were detected using a CCK-8 kit (Cell Counting Kit-8), and apoptosis was detected by flow cytometry with PI/annexinV staining. Western blotting was used to detected caspase-3 and caspase-8. Neurotoxic and anti-tumor effects were evaluated in animal experiments. Results: DH332 exerts a lower neurotoxicity compared with harmine. It also possesses strong antitumor effects against two B cell lymphoma cell lines with low $IC_{50s}$. Moreover, DH332 could inhibit the proliferation and induce the apoptosis of RAMOS RA.1 and J558 cell lines in a dose-dependent manner. Our results suggest that DH332 triggers apoptosis by mainly activating the caspase signaling pathway. In vivo studies of tumor-bearing BALB/c mice showed that DH332 significantly inhibited growth of J558 xenograft tumors. Conclusions: DH332 exerts effective antitumor activity in vitro and in vivo, and has the potential to be a promising drug candidate for lymphoma therapy.

• Journal of Nutrition and Health
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• v.55 no.1
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• pp.36-46
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• 2022

Purpose: Quercetin is a polyphenolic flavonoid abundant in many fruits and vegetables. It has potential health-beneficial properties, such as antioxidant, anti-obesity, anti-cancer, anti-diabetic and anti-inflammatory effects. The purpose of this study was to investigate whether the lipid metabolism improvement effect of quercetin affected the regulation of AMP-activated protein kinase (AMPK) activity and microRNA (miR)-21 expression in the liver of mice fed a high-cholesterol diet. Methods: Male C57BL/6J mice were fed with normal diet, quercetin-free diet and diets containing 0.05% or 0.1% quercetin for six weeks. Hypercholesterolemia was induced by adding 1% cholesterol and 0.5% cholic acid to all diets. Serum and liver triglyceride (TG), and total cholesterol (TC) concentrations were analyzed using a commercial enzymatic colorimetric kit. AMPK activity was quantified using an AMPK kinase assay kit. The levels of miR-21 and genes involved in lipid metabolism were measured by real-time quantitative polymerase chain reaction. Results: Supplementation of quercetin reduced serum and hepatic TG and TC levels without changing body weight and food intake. Dietary quercetin significantly inhibited the mRNA levels of hepatic sterol-regulatory element binding protein-1c, acetyl-CoA carboxylase 1 and fatty acid synthesis, which are involved in hepatic lipogenesis. Dietary quercetin enhanced AMPK activity and suppressed miR-21 expression, promoting hepatic lipid accumulation. Conclusion: These results suggest that the lipid-lowering effect of quercetin on the serum and liver of mice may be partially mediated by the regulation of lipogenic gene expression, AMPK activity and miR-21 expression in the liver of mice fed a high-cholesterol diet.

• Biomedical Science Letters
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• v.16 no.4
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• pp.349-357
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• 2010

We have examined the histological changes of skin during hair follicle growth after depilation in C57BL/6N mice. We first studied on histological changes of number of mast cells and thickness of skin during hair follicle growth periods (telogen, 1 day, 3 day, 5 day, 10 day, 14 day, 17 day and 21 day after depilation) by toluidine blue, Giemsa and H&E staining methods. We second studied immunoreactive density of cytokines and Brdu labeled cells in skin during hair follicle growth periods after depilation in C57BL/6N mice by immunohistochemical methods. The histological changes on skin thickness was increased from telogen to 14 day. The number of mast cells was decreased in 3,5 and 10 day and increased in 14, 17 and 20 day after depilation. Immunoreactive density of cytokines [protein kinase C-${\alpha}$ (PKC-${\alpha}$), c-kit, and vascular endothelial growth factor (VEGF)] in 1, 3, 5, 10, and 14 day after depilation was mildly stained in bulge and cutaneous trunci m., but immunoreactive density of cytokines in 17 and 21 day was heavily stained in epidermis, bulge, outer root sheath (ORS), inner root sheath (IRS) and cutaneous trunci m.. Immunoreactive density of Brdu labeled cells in skin in 1 and 3 day was heavily stained in bulge, epidermis and connective tissue under the cutaneous trunci m.. In all periods, immunoreactive density of Brdu labeled cells in skin was heavily stained in bulge, subcutaneous tissue, cutaneous trunci m, ORS and IRS. These experiments suggest that histological changes related to hair follicle growth elevated mast cell counts, skin thickness and epidermis thickness and heavily stained immunoreactive density of cytokines and Brdu labeled cutaneous trunci m. and connective tissue under the cutaneous trunci m. after depilation in C57BL/6N mice.