• Journal of Life Science
• /
• v.30 no.3
• /
• pp.285-290
• /
• 2020

Foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. FMDV causes various clinical symptoms, including severe inflammation in infected tissue. Genome RNA of FMDV shows a positive single-strand chain approximately 8.3 kb long and encodes a single long open reading frame (ORF). The ORF is translated into structural and non-structural proteins by viral proteases. The FMDV 2C protein is one of the non-structural proteins encoded by FMDV and plays a critical role in FMD pathogenesis, including inflammation, apoptosis, and viral replication. In this study, we examined whether FMDV 2C induces intracellular expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). FMDV 2C expression in pig IBRS-2 cells increased mRNA and protein expression of TNFα at the transcriptional level via activation of TNFα promoter. Treatment with 4-phenylbutyric acid, an endoplasmic reticulum (ER) stress reducer, decreased TNFα expression induced by FMDV 2C. Activating transcription factor 4 (ATF4), a transcription factor mediating ER stress response, induced transactivation of TNFα promoter and expression of mRNA and protein of TNFα. However, the dominant negative mutant of ATF4 did not induce FMDV 2C-mediated TNFα expression. The results indicate that FMDV 2C protein increases clinical inflammation via ATF4-mediated TNFα expression and is associated with ER stress induction.

• Journal of Life Science
• /
• v.16 no.3 s.76
• /
• pp.400-408
• /
• 2006

Nutritional components of 4 kinds of red seaweeds, Meristotheca papulosa, Chondrus ocellatus, Gracilaria verrucosa, Gigartina tenella, were investigated to elucidate their functionality. Antioxidant activity and nitrite scavenging activity were analyzed from 70% ethanol extracted from these red seaweeds. Large difference in ash contents was found to exhibit among all samples analyzed in this study; $9.8{\pm}0.2g/100g$ for Gracilaria verrucosa and Gigartina tenella for $17.8{\pm}0.6g/100g$. While its crude fiber content was almost the same as those in other sample within a range between $2.0{\pm}0.4g/100g$ and $6.0{\pm}0.7g/100g$. Phenolic compounds content of Gracilaria verrucosa was also the highest as $78.4{\pm}1.0mg/g$, while the total flavonoids contents of Chondrus ocellatus and Gracilaria verrucosa were $14.9{\pm}0.5mg/g$ and $13.9{\pm}0.8mg/g$, respectively. These amounts were two folds higher than Meristotheca papulosa and Gigartina tenella. The total content of minerals was the highest in Meristotheca papulosa(12,107.7 mg/kg). The amount of glutamic acid was relatively high despite of small variation in measured levels of composition amino acid ($49.1{\sim}125.6mg/g$) for most samples investigated. SOD-like ability was significantly increased with increasing sample concentration, but its activity was lower. Gigartina tenella with highest electron donation ability exhibited increases in activity as $53.96{\pm}0.98%$ in concentration of 250 ${\mu}g/ml$ and $70.52{\pm}1.09%$ in 1000 ${\mu}g/ml$, respectively. In case of concentration of 100 ${\mu}g/ml$, particularly, the level of hydroxy radical scavenging activity were $57.87{\pm}1.70{\sim}62.07{\pm}0.87%$ which was significantly higher activity than ascorbic acid and BHT. Nitrite scavenging activity was the highest in Gracilaria verrucosa. Its activity was also increased from $24.04{\pm}1.9{\sim}27.52{\pm}0.82%$ in $100{\sim}500{\mu}g/ml$ concentration tp $34.81{\pm}1.36%$ in concentration of 1000 ${\mu}g/ml$.

• Journal of Life Science
• /
• v.30 no.4
• /
• pp.359-369
• /
• 2020

Although many studies on immune modulatory materials have used RAW 264.7 cells, few have used T cell-derived TK-1 cell lines. Moreover, although some studies have investigated the efficacy of plant-derived β-sitosterol, few have examined the immunomodulatory activity of its analogue, daucosterol. In this study, β-sitosterol and daucosterol were isolated from D. batatas and identified by nuclear magnetic resonance spectroscopy. To evaluate the immune-enhancing or inhibitory effects of the isolated phytosterols, the expression levels of the inflammatory response genes COX-2, TNF-α, IL-6, and iNOS were analyzed by RT-PCR. The relative expression levels of TNF-α and iNOS in RAW 264.7 cells were increased more than threefold with β-sitosterol treatment comparing to those of untreated control. In the case of TK-1 cells, the expression level of TNF-α was decreased and the expression level of iNOS was increased in a β-sitosterol concentration-dependent manner. The expression levels of COX-2, TNF-α, and IL-6 increased by approximately 0.7-1.2 times in RAW 264.7 cells treated with daucosterol compared to those of untreated control, but iNOS expression decreased by 0.8-0.18 times. In the case of daucosterol-treated TK-1 cells, the expression levels of TNF-α, IL-6, and iNOS were markedly reduced from those of TK-1 cells treated only with lipopolysaccaride. As a conclusion, β-sitosterol treatment increased TNF-α and iNOS expression levels in RAW 264.7 cells, thus exerting an immune- boosting effect. However, in TK-1 cells, iNOS expression increased while TNF-α expression decreased, indicating an immunosuppressive activity of β-sitosterol. Daucosterol appears to exert an immunosuppressive effect in both macrophages and T cell lines by inhibiting iNOS expression in RAW 264.7 cells and greatly inhibiting the expression of TNF-α, IL-6, and iNOS in TK-1 cells.

• Journal of Korea Spatial Information System Society
• /
• v.3 no.2 s.6
• /
• pp.21-43
• /
• 2001

The environment to development information system including a GIS has been drastically changed in recent years in the perspectives of the complexity and diversity of the software, and the distributed processing and network computing, etc. This leads the paradigm of the software development to the CBD(Component Based Development) based object-oriented technology. As an effort to support these movements, OGC has released the abstract and implementation standards to enable approaching to the service for heterogeneous geographic information processing. It is also common trend in domestic field to develop the GIS application based on the component technology for municipal governments. Therefore, it is imperative to adopt the component technology considering current movements, yet related research works have not been made. This research is to propose a component-based GIS development methodology-ATOM(Advanced Technology Of Methodology)-and to verify its adoptability through the case study. ATOM can be used as a methodology to develop component itself and enterprise GIS supporting the whole procedure for the software development life cycle based on conventional reusable component. ATOM defines stepwise development process comprising activities and work units of each process. Also, it provides input and output, standardized items and specs for the documentation, detailed instructions for the easy understanding of the development methodology. The major characteristics of ATOM would be the component-based development methodology considering numerous features of the GIS domain to generate a component with a simple function, the smallest size, and the maximum reusability. The case study to validate the adoptability of the ATOM showed that it proves to be a efficient tool for generating a component providing relatively systematic and detailed guidelines for the component development. Therefore, ATOM would lead to the promotion of the quality and the productivity for developing application GIS software and eventually contribute to the automatic production of the GIS software, the our final goal.

• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.167-173
• /
• 2007

To study the transcriptional mechanisms by which expression of the murine glial cell-derived neurotrophic factor inducible transcription factor (mGIF) gene is regulated, a murine genomic clone was iso-lated using a mGIF cDNA as probe. A 13-kb genomic fragment, which comprises 4-kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Spl. The mGIF gene also has consensus sequences for AP2 binding sites. The transcriptional activity of five deletion mutants of a 2.1-kb fragment was analyzed by modulating transcription of the heterologous luciferase gene in the promoterless plasmid pGL2-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3. Transient expression assays suggested the presence of a positive regulator between -213 and -129 while a negative regulator was found in the region between -806 and -214. Relatively strong transcriptional activity was observed in neuronal NB41A3, glial C6 cells and hepatic HepG2, but very weak activity in skeletal muscle C2C12 cells. These findings confirm the tissue-specific activity of the mGIF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.

• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.198-203
• /
• 2007

DNA transfection is a powerful tool for studying gene functions. The $Ca^{2+}$-phosphate precipitation remains one of the most popular and cost-effective transfection techniques. Mature neurons are more resistant to transfection than young ones and most other cell types, and easy to die if microenvironment changes. Here, we report a transfection protocol for mature neurons. The critical modifications are inclusion of glial cells in culture and careful control of $Ca^{2+}$-phosphate precipitation under microscope. Cerebral glial cells were grown until ${\sim}70-80%$ confluence in DMEM/10% horse serum, which was thereafter replaced with serum-free Neurobasal/Ara-C, and 319 hippocampal neurons were plated onto the glial layer Formation of fine $DNA/Ca^{2+}$-phosphate precipitates was induced using Clontech $CalPhos^{TM}$ Mammalian Transfection Kit, and the size ($0.5-1\;{\mu}m$ in diameter) and density(about 10 particles/$100\;{\mu}m^2$) were carefully controlled by the time of incubation in the medium. This modified protocol can be reliably applied for transfection of mature neurons that are maintained longer than two weeks in vitro, resulting in 10-15 healthy transfected neurons per a well of 24-well plates. The efficacy of the protocol was verified by punctate expression of $pEGFP-CaMKII{\alpha}$, a synaptic protein, and diffuse expression of pDsRed2. Our protocol provides a reliable method for transfection of mature neurons in vitro.

• Journal of Life Science
• /
• v.20 no.1
• /
• pp.1-8
• /
• 2010

We recently reported the distributions of AMPA ($\alpha$-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptor subtypes glutamate receptors (GluR) 1 and GluR4 in the superior colliculi (SC) of hamsters with antibody immunocytochemistry and the effect of enucleation on these distributions. We also compared these labelings to those of calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. In the present study, we investigated whether the GluR1- and GluR4-immunoreactive (IR) neurons are interneurons or projection neurons by injection of the retrograde tracer horseradish peroxidase (HRP) into one of each major ascending and descending pathways of the SC. HRP injections were made into a tecto-reticulospinal pathway (TRS) and dorsal lateral geniculate nucleus (dLGN). Animals were then allowed to recover and to survive for 48 hr before perfusion. Sections containing retrograde-labeled neurons were then treated for GluR-immunoreactivity. HRP injections proved that only a small population of the GluR1-IR cells project into TRS (1.4%) and dLGN (2.6%). However, a large subpopulation of GluR4-IR cells project into TRS (32.7%). The differential compositions of inter/projection neurons, along with our previous studies on the separate distribution of the GluR subunits, its differential co-localization with calcium-binding proteins and GABA, and differential reactions to enucleations, strongly imply the functional variety of the receptor subunits in visual behavior responses.

• Journal of Life Science
• /
• v.19 no.8
• /
• pp.1039-1046
• /
• 2009

The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.

• Journal of Life Science
• /
• v.21 no.12
• /
• pp.1752-1760
• /
• 2011

In this study, the effect of the Opuntiahumifusa extracts on proliferation, alkaline phosphatase (ALP) activity, collagen synthesis and ROS level of a cell was investigated using an osteoblast. Opuntiahumifusawas separated intoOpuntiahumifusapeel (OH-P), seed (OH-Se) and stem (OH-St).These were subjected to extraction by using hot water and ethanol. The proliferation of the MC3T3-E1 osteoblastic cells that were treated with OH-Se water extract were increased by approximately 120%. Regarding the effects of OH-Se on ALP activity, the $50{\mu}g/ml$ ethanol extract group showed the highest activity. The synthesis of collagen increased significantly in response to treatment with OH-Se water extract. The ROS scavenging effects of Opuntiahumifusawere investigated for involvement of oxidativedamage, cell culture and staining. Also, when OH-Se water extract $100{\mu}g/ml$ was added, the ROS level decreased by 54%. These results indicate that Opuntiahumifusa extracts have an anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.

• Journal of Mushroom
• /
• v.19 no.3
• /
• pp.225-233
• /
• 2021

In this study, we evaluated the anti-inflammatory effect of extract from Cudrania tricuspidata twig sawdust fermented with Ganoderma lucidum mycelium. Fermented Cudrania tricuspidata twig sawdust extracted with 70% ethanol and elucidated the potential signaling pathway in lipopolysaccharide (LPS)-induced RAW264.7 cells. Fermented Cudrania tricuspidata twig sawdust inhibits LPS-stimulated nitric oxide (NO) production without affecting cell viability in a dose-dependent manner and production of LPS-induced pro-inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and prostaglandin2 (PGE₂). Fermented Cudrania tricuspidata twig sawdust also suppressed the expression of the pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 cells. Moreover, Fermented Cudrania tricuspidata twig sawdust significantly attenuated LPS-induced IkappaB (IκB) degradation and suppressed nuclear factor kappa B (NF-κB) nuclear translocation. These results suggest that fermented Cudrania tricuspidata twig sawdust may have great potential for the development of anti-inflammatory agent.