• Korean Journal of Environmental Agriculture
• /
• v.36 no.4
• /
• pp.241-248
• /
• 2017

BACKGROUND: RNA interference (RNAi) eliminates or decreases gene expression by disrupting the target mRNA or by interfering with translation. Recently, RNAi technique was applied to generate new crop traits which provide protection against pests. To establish the environmental risk assessment protocol of RNAi LMO in lab scale, we developed dsRNA expression system using E. coli and tested acute oral toxicity assay to honey. METHOD AND RESULTS: The dsRNA expression vector, L4440, was chosen and cloned 240 bp of Snf7 and GFP gene fragment. To develop the maximum dsRNA induction condition in E. coli, we tested induction time, temperature and IPTG concentration in media. To estimate the risk assessment of dsRNA to honey bee, it has been selected and cultured with dsRNA supplement for 48 hours according to OECD guideline. As a result, the optimum condition of dsRNA induction was $37^{\circ}C$, 4 hours and 0.4 mM IPTG concentration and the difference between Snf7 and GFP dsRNA molecules from E. coli was not significant in survival and behavior to honey bee. Furthermore, blast search results indicated that effective match of predicted dsRNA fragments were not existed in honey bee genome. CONCLUSION: In this study, we developed and tested the acute oral toxicity of dsRNA using E. coli expression system to honey bee.

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.2
• /
• pp.267-271
• /
• 1997

Apoptosis, programmed cell death, is posulated to occur in granulosa cells in ovarian follicular atresia. bcl-2 gene serves as protector from apoptosis and, thus, is associated with increased cell survival. TRPM-2 gene expression has been implicated as a trigger of apoptosis in rat prostate, uterus and mammary gland. Our objective was to determine if bcl-2 and TRPM-2 are expressed in luteinized human GC and, therefore, have regulatory functions for apoptosis in GC. Human GC were obtained via oocyte retrival from the infertile patients stimulated with exogeneous gonadotropins while undergoing IVF. GC were isolated from follicular fluid using Percoll gradient centrifugation. The GC were further purified with anti-CD45 magnetic beads to remove contaminating WBC's. RT-PCR were performed to analyze the mRNA expression of bcl-2 and TRPM-2 in the GC. The PCR primers were designed to amplify a 195 bp fragment of bcl-2 and a 174 bp fragment of TRPM-2. The PCR products were electrophoresed on 4% agarose gel. Three separate experiments indicated that both bcl-2 and TRPM-2 are concurrently expressed in human GC. We cultured granulosa cells with FSH (1 ng/ml) for 1 day to investigate the relative changes of TRPM-2 mRNA level with RNAse protection assay. When we cultured GC with serum free medium for 1 day TRPM-2 mRNA level increased with 1.3 fold, however it was decreased 0.64 fold with FSH. Therefore we conclude that bcl-2 and TRPM-2 are concurrently expressed and that the interaction of their products may be involved in GC apoptosis. And TRPM-2 may be regulated with FSH.

• Biomolecules & Therapeutics
• /
• v.29 no.5
• /
• pp.551-561
• /
• 2021

Thyroid cancer is the most common endocrine malignancy. Patients with well-differentiated thyroid cancers, such as papillary and follicular cancers, have a favorable prognosis. However, poorly differentiated thyroid cancers, such as medullary, squamous and anaplastic advanced thyroid cancers, are very aggressive and insensitive to radioiodine treatment. Thus, novel therapies that attenuate metastasis are urgently needed. We found that both PDGFC and PDGFRA are predominantly expressed in thyroid cancers and that the survival rate is significantly lower in patients with high PDGFRA expression. This finding indicates the important role of PDGF/PDGFR signaling in thyroid cancer development. Next, we established a SW579 squamous thyroid cancer cell line with 95.6% PDGFRA gene insertion and deletions (indels) through CRISPR/Cas9. Protein and invasion analysis showed a dramatic loss in EMT marker expression and metastatic ability. Furthermore, xenograft tumors derived from PDGFRA geneedited SW579 cells exhibited a minor decrease in tumor growth. However, distant lung metastasis was completely abolished upon PDGFRA gene editing, implying that PDGFRA could be an effective target to inhibit distant metastasis in advanced thyroid cancers. To translate this finding to the clinic, we used the most relevant multikinase inhibitor, imatinib, to inhibit PDGFRA signaling. The results showed that imatinib significantly suppressed cell growth, induced cell cycle arrest and cell death in SW579 cells. Our developed noninvasive apoptosis detection sensor (NIADS) indicated that imatinib induced cell apoptosis through caspase-3 activation. In conclusion, we believe that developing a specific and selective targeted therapy for PDGFRA would effectively suppress PDGFRA-mediated cancer aggressiveness in advanced thyroid cancers.

• Journal of the Korean Society of International Agriculture
• /
• v.23 no.5
• /
• pp.570-577
• /
• 2011

We developed insect-resistant GM rice(Bt transgenic rice) by inserting the mCry1Ac1 a modified gene from the soil bacterium, Bacillus thuringiensis. The Bt transgenic rice expressing the Bttoxin mCry1Ac1 was tested for the effects on survival of Misgurnus anguillicaudatus and Cyprinus carpio, commonly used as a model organism in ecotoxicological studies. M. anguillicaudatus and C. carpio fed 100% ground rice in suspension, using either Bt rice or non-GM counterpart rice(Nakdong). The Bt rice used for the test were confirmed to have the mCry1Ac1 gene expression by the immuno-strip and ELISA analysis. Feeding test showed that no significant differences in cumulative immobility and abnormal response of M. anguillicaudatus and C. carpio fed on between Bt rice and non-GM counterpart rice. The 96hr-LC₅₀ values showed no difference between Bt rice(>1,000mg/L) and non-GM rice(>1,000mg/L). We concluded that there was no significant difference in toxicity for non-target organisms(M. anguillicaudatus and C. carpio) between Bt rice and non-GM counterparts.

• Journal of Microbiology and Biotechnology
• /
• v.33 no.9
• /
• pp.1206-1212
• /
• 2023

The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation [1-3]. The intracellular β-catenin level, which is regulated by a proteasomal degradation pathway, is critical to Wnt/β-catenin pathway control [4]. Normally, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with the scaffolding protein Axin and the tumor suppressor protein adenomatous polyposis coli (APC), phosphorylate β-catenin at Ser45, Thr41, Ser37, and Ser33 [5, 6]. Phosphorylated β-catenin is ubiquitinated by the β-transducin repeat-containing protein (β-TrCP), an F-box E3 ubiquitin ligase complex, and ubiquitinated β-catenin is degraded via a proteasome pathway [7, 8]. Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis [9]. Genetic mutations in the APC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [10]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene were found in patients with colorectal cancer [11]. These mutations cause β-catenin to accumulate in the nucleus, where it forms complexes with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family to stimulate the expression of β-catenin responsive genes, such as c-Myc and cyclin D1, which leads to colorectal tumorigenesis [12-14]. Therefore, downregulating β-catenin response transcription (CRT) is a potential strategy for preventing and treating colorectal cancer. Plant cytokinins are N⁶-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid (isopentenyladenine, zeatin, and dihydrozeatin), aromatic (benzyladenine, topolin, and methoxytopolin), or furfural (kinetin and kinetin riboside), depending on their structure [15, 16]. Kinetin riboside was identified in coconut water and is a naturally produced cytokinin that induces apoptosis and exhibits antiproliferative activity in several human cancer cell lines [17]. However, little attention has been paid to kinetin riboside's mode of action. In this study, we show that kinetin riboside exerts its cytotoxic activity against colon cancer cells by suppressing the Wnt/β-catenin pathway and promoting intracellular β-catenin degradation.

• Research in Plant Disease
• /
• v.30 no.2
• /
• pp.148-156
• /
• 2024

Ralstonia pseudosolanacearum, a plant pathogenic bacterium that can survive for a long time in soil and water, causes lethal wilt in the Solanaceae family. Sigma S is a part of the RNA polymerase complex, which regulates gene expression during bacterial stress response or stationary phase. In this study, we investigated the role of sigma S in R. pseudosolanacearum under stress conditions using a rpoS-defective mutant strain of R. pseudosolanacearum and its wild-type strain. The phenotypes of rpoS-defective mutant were complemented by introducing the original rpoS gene. There were no differences observed in bacterial growth rate and exopolysaccharide production between the wild-type strain and the rpoS mutant. However, the wild-type strain responded more sensitively to nutrient deficiency compared to the mutant strain. Under the nutrient deficiency, the rpoS mutant maintained a high bacterial viability for a longer period, while the viability of the wild-type strain declined rapidly. Furthermore, a significant difference in pH was observed between the culture supernatant of the wild-type strain and the mutant strain. The pH of the culture supernatant for the wild-type strain decreased rapidly during bacterial growth, leading to medium acidification. The rapid decline in the wild-type strain's viability may be associated with medium acidification and bacterial sensitivity to acidity during transition to the stationary phase. Interestingly, the rpoS mutant strain cannot utilize acetic acid, D-alanine, D-trehalose, and L-histidine. These results suggest that sigma S of R. pseudosolanacearum regulates the production or utilization of organic acids and controls cell death during stationary phase under nutrient deficiency.

• Journal of Ginseng Research
• /
• v.47 no.5
• /
• pp.627-637
• /
• 2023

Background: Damage to the healthy intestinal epithelial layer and regulation of the intestinal immune system, closely interrelated, are considered pivotal parts of the curative treatment for inflammatory bowel disease (IBD). Plant-based diets and phytochemicals can support the immune microenvironment in the intestinal epithelial barrier for a balanced immune system by improving the intestinal microecological balance and may have therapeutic potential in colitis. However, there have been only a few reports on the therapeutic potential of plant-derived exosome-like nanoparticles (PENs) and the underlying mechanism in colitis. This study aimed to assess the therapeutic effect of PENs from Panax ginseng, ginseng-derived exosome-like nanoparticles (GENs), in a mouse model of IBD, with a focus on the intestinal immune microenvironment. Method: To evaluate the anti-inflammatory effect of GENs on acute colitis, we treated GENs in Caco2 and lipopolysaccharide (LPS) -induced RAW 264.7 macrophages and analyzed the gene expression of proinflammatory cytokines and anti-inflammatory cytokines such as TNF-α, IL-6, and IL-10 by real-time PCR (RT-PCR). Furthermore, we further examined bacterial DNA from feces and determined the alteration of gut microbiota composition in DSS-induced colitis mice after administration of GENs through 16S rRNA gene sequencing analysis. Result: GENs with low toxicity showed a long-lasting intestinal retention effect for 48 h, which could lead to effective suppression of pro-inflammatory cytokines such as TNF-α and IL-6 production through inhibition of NF-κB in DSS-induced colitis. As a result, it showed longer colon length and suppressed thickening of the colon wall in the mice treated with GENs. Due to the amelioration of the progression of DSS-induced colitis with GENs treatment, the prolonged survival rate was observed for 17 days compared to 9 days in the PBS-treated group. In the gut microbiota analysis, the ratio of Firmicutes/Bacteroidota was decreased, which means GENs have therapeutic effectiveness against IBD. Ingesting GENs would be expected to slow colitis progression, strengthen the gut microbiota, and maintain gut homeostasis by preventing bacterial dysbiosis. Conclusion: GENs have a therapeutic effect on colitis through modulation of the intestinal microbiota and immune microenvironment. GENs not only ameliorate the inflammation in the damaged intestine by downregulating pro-inflammatory cytokines but also help balance the microbiota on the intestinal barrier and thereby improve the digestive system.

• Journal of Life Science
• /
• v.16 no.2 s.75
• /
• pp.289-296
• /
• 2006

In order to establish highly efficient gene transfer condition at early stage of soybean transformation, various experiments were performed and compared their efficiencies by transient GUS analysis; those conditions are genotype determination of Korean soybean cultivars for amenability to Agro-infection, appropriate agar and selective agent concentration, orientation of explant placement, hormone pre-culture, and liquid selection condition. In the genotype screen of Korean soybean varieties, 14 amenable genotypes were selected. For efficient Agrobacterium washing, cefotaxime was chosen and hygromycin at the concentration of 10 and 15 ppm was used as selection agent in the media. Agar concentration was slightly better in 0.6% and 0.8% for both shoot and callus formation, and explant placement with adaxial side down showed high frequency of GUS expression. For wounding treatment, oriental needle was efficient than scalpel for shoot formation and gene transfer. To increase the frequency of gene transfer, hormone pre-treatment was applied. BA at the concentration of 5 and 10 ppm resulted in better survival at the late stage of selection in shoot elongation media. Selection in liquid media after hormone pre-treatment seemed to be effective to remove the escaped non-transformants at early stage of procedure. Considering the results obtained, Eunhakong could be the first choice as a material for soybean transformation among Korean soybean genotypes.

• Reproductive and Developmental Biology
• /
• v.35 no.1
• /
• pp.33-45
• /
• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

• Journal of Life Science
• /
• v.24 no.4
• /
• pp.343-351
• /
• 2014

Phosphoinositide 3-kinase (PI3K) plays a central role in cell signaling and leads to cell proliferation, survival, motility, exocytosis, and cytoskeletal rearrangements, as well as specialized cell responses, superoxide production, and cardiac myocyte growth. PI3K is divided into three classes; type I PI3K is preferentially expressed in leukocytes and activated by ${\beta}{\gamma}$ subunits of heterotrimeric G-proteins. In this study, the cDNAs encoding the $PI3K{\gamma}$ gene were isolated from a brain cDNA library constructed using the flounder (Paralichthys olivaceus). The sequence of the isolated $PI3K{\gamma}$ was 1341 bp, encoding 447 amino acids. The nucleotide sequence of the $PI3K{\gamma}$ gene was analyzed with that of other species, including Oreochromis niloticus and Danio rerio, and it turned out to be well conserved during evolution. The $PI3K{\gamma}$ gene was subcloned into the expression vector pET-44a(+), and expressed in the E. coli BL21 (DE3) codon plus cell. The resulting protein was expressed as a fusion protein of approximately 49 kDa containing a C-terminal six-histidine extension that was derived from the expression vector. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to $PI3K{\gamma}$. The binding of wortmannin to $PI3K{\gamma}$, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. The results obtained from this study will provide a wider base of knowledge on the primary structure and characterization of the $PI3K{\gamma}$ at the molecular level.