• Journal of Life Science
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• v.27 no.12
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• pp.1393-1402
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• 2017

In plants, calcium ($Ca^{2+}$)-dependent protein kinases (CDPKs) are important sensors of $Ca^{2+}$ signals. Previous research demonstrated the expression of the OsCPK11 gene in various tissues at the transcription level, but its developmental and biochemical functions at the protein level were not determined. This study was aimed to identify biochemical characteristics of OsCPK11. GST- OsCPK11 was expressed in E. coli and used for an in vitro kinase assay. Biochemical analyses identified OsCPK11 as a CDPK. OsCPK11 autophosphorylated itself and transphosphorylated histone III-s and MBP as substrates in the presence of $Ca^{2+}$. The activity of the recombinant OsCPK11 was influenced by $Mg^{2+}$, with optimum activity detected at pH 7.0-7.5. OsCPK11 activity was not affected by $Mg^{2+}$, $Mn^{2+}$, or $Na^+$ in the presence of a high level of $Ca^{2+}$. Autophosphorylation of OsCPK11 decreased $Ca^{2+}$ sensitivity of OsCPK11. An anti-OsCPK11 rabbit antibody recognized 95.5 kD of GST-OsCPK11, as shown by an immunoblot analysis. These results shed light on the function of OsCPK11 in $Ca^{2+}$-mediated signaling in rice.

• Journal of Pharmacopuncture
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• v.9 no.2
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• pp.5-16
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• 2006

Objective : It has long been known about the osteogenic effect of CPC-HAS on bone tissues. However, it has not been determined the effect of CPC-HAS on cancer cells. The purpose of this study is to screen the CPC-HAS mediated differentially expressed genes in cancer cells such as HepG2 hepatoma cells. Oligonucleotide microarray and proteomics approaches were employed to screen the differential expression genes. Methods : CPC-HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of CPC-HAS (0.1, 0.5, 1.5, 10, 20mg/ml) for 24 h. Cell toxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with 1.5mg/ml of CPC-HAS. For oligonucleotide microarray assay, total RNA was used for gene expression analysis using oligonucleotide Genechip(Human genome Ul33 Plus 2.0., Affimatrix Co.). For proteomic analysis, total protein was analyzed by 2D gel electrophoresis and Q-TOF mass spectrometer. Results : It has no cytotoxic effects on both HepG2 cell in all concentrations(0.l, 0.5, 1.5, 10, 20mg/ml). In oligonucleotide microarray assay, the number of more than twofold differentially regulated known genes was 23 with 5 up-regulated and 18 down-regulated genes in HepG2 cells. In proteomic analysis, three spots were identified by 2D-gel electrophoresis and Q-TOF analysis. Two down-regulated proteins were aldehyde dehydrogenase 1 and enolase 1, and up-regulated protein was fatty acid binding protein 1 by 1.5mg/ml of CPC-HAS. Discussion : This study showed the screening of CPC-HAS mediated differentially regulated genes using combined approaches of oligonucleotide microarray and proteomic analysis. The screened genes will be used for the better understanding of the therapeutic effects of CPC-HAS on cancer fields.

• Journal of Plant Biotechnology
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• v.48 no.3
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• pp.165-172
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• 2021

The solubilization of isolated proteins into the adequate buffer containing of surfactants is primary step for proteomic analysis. Particularly, sodium dodecyl sulfate (SDS) is the most widely used surfactant, however, it is not compatible with mass spectrometry (MS). Therefore, it must be removed prior to MS analysis through rigorous washing, which eventually results in inevitable protein loss. Recently, photocleavable surfactant, 4-hexylphenylazosulfonate (Azo), was reported which can be easily degraded by UV irradiation and is compatible with MS during proteomic approach using animal tissues. In this study, we employed comparative label-free proteomic analysis for evaluating the solubilization efficacies of the Azo and SDS surfactants using rice leave proteins. This approach led to identification of 3,365 proteins of which 682 proteins were determined as significantly modulated. Further, according to the subcellular localization prediction in SDS and Azo, proteins localized in the chloroplast were the major organelle accounting for 64% of the total organelle in the SDS sample, while only 37.5% of organelle proteins solubilized in the Azo were predicted to be localized in chloroplast. Taken together, this study validates the efficient solubilization of total protein isolated from plant material for bottom-up proteomics. Azo surfactant is suitable as substitute of SDS and promising for bottom-up proteomics as it facilitates robust protein extraction, rapid washing step during enzymatic digestion, and MS analysis.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.195-203
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• 2018

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

• Journal of Nutrition and Health
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• v.54 no.3
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• pp.321-333
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• 2021

Purpose: Reflux esophagitis is a disease caused by the reflux of stomach contents and stomach acid etc. into the esophagus due to defect in the lower esophageal sphincter and is currently increasing worldwide. This study was conducted to evaluate the effect of a mixture of Citrus Reticulata and Scutellariae Radix (CS) extract on acute reflux esophagitis in rats. Methods: Rats were divided into five groups for examination: normal group (Normal, n = 8), water-treated acute reflux esophagitis rats (Control, n = 8), tocopherol 30 mg/kg body weight-treated acute reflux esophagitis rats (Toco, n = 8), CS 100 mg/kg body weight-treated acute reflux esophagitis rats (CS100, n = 8), CS 200 mg/kg body weight-treated acute reflux esophagitis rats (CS200, n = 8). The experimental groups were administrated of each treatment compounds and after 90 min, acute reflux esophagitis was induced through surgery. Rats were sacrificed 5 h after surgery. We measured the level of reactive oxygen species (ROS) in serum and analyzed the expression of nicotinamide adenine dinucleotide phosphate, inflammatory, and tight junction-related proteins by western blot in the esophageal tissues. Results: CS administration significantly protected the esophageal mucosal damage due to reflux esophagitis, and the level of ROS in the serum was significantly reduced with CS administration as compared to Control. In addition, CS administration significantly suppressed mitogen-activated protein kinase (MAPK or MAP kinase) and nuclear factor-kappa B (NF-κB) pathways and increased protein expressions of tight junction protein. Conclusion: These results suggest that the CS not only regulates the expression of inflammatory proteins by inhibiting oxidative stress, but also reduces damage to the esophageal mucosa by inhibiting the expression of tight junction proteins.

• Animal Bioscience
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• v.35 no.7
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• pp.964-974
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• 2022

Objective: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines. Methods: Resistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing. Results: A gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1β, IL-6, IL-8), type I interferons (IFN-α, IFN-β), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens. Conclusion: Resistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.36 no.1
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• pp.21-39
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• 2023

Objectives : The purpose of this study was to investigate the anti-inflammatory effect of Goihwa-san water extract(GHS) in vitro & in vivo. Methods : In vitro, we evaluated the anti-inflammatory effect of GHS by comparing the Raw 264.7 cells with 10, 30, 100, 300㎍/㎖ of GHS for 1 hour before Lipopolysaccharide(LPS) to the single LPS treated group. We examined the relative cell viability by MTT assay and the relative level of LPS, Loxoribine(LOX), Peptidoglycan(PGN), Flagellin(FLA)-induced NO production by using Griess reagent and measured relative iNOS protein level and COX-2 protein level by using western blot and Image analyzing system. We measured the production of TNF-α, IL-1β, and IL-6 by each ELISA kits and then measured the relative levels of IκBα, p-IκBα in whole-cell lysate fraction and NF-κB in nuclear fraction by using western blot and Image analyzing system. In vivo, we induced the paw edema by subcutaneous injection of 100㎕/rat CA and measured the swelling volume of paw by using a plethysmometer and then measured the relative iNOS protein level by using western blot. Results : As a result, in vitro, LPS, PGN-induced NO production was significantly inhibited by pretreatment with GHS. GHS reduced LPS, PGN-induced iNOS expression, PGN-induced COX-2 expression and LPS-induced production of cytokine(TNF-α, IL-1β, IL-6). Expression of IκBα was increased by pretreatment with GHS 100㎍/㎖. And the expression of p-IκBα and NF-κB were decreased by pretreatment with GHS 100㎍/㎖. In vivo, CA-induced inflammation rat model was used for the evaluation of the anti-inflammatory effect of GHS. 0.3 or 1.0g/kg of GHS significantly reduced the increases of paw swelling and iNOS expression in paw tissues. Conclusions : These results show that GHS can decrease inflammatory response via inhibition of the NF-κB pathway in vitro. And in vivo, the anti-inflammatory effect suggest the clinical basis of GHS for the treatment of inflammatory diseases.

• The Korea Journal of Herbology
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• v.33 no.5
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• pp.89-103
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• 2018

Objectives : This study is aimed to compare the changes in Antioxidative capacity of Liriopis Tuber by steaming process and to compare the effects in hyperlipidemia induced rats fed high cholesterol diet between Simvastatin and Liriopis Tuber by steaming process. Methods : The SD rats were divided into six groups: normal diet (Nor), high cholesterol diet (Veh), high cholesterol diet plus Simvastatin 5 mg/kg (Sim), high cholesterol diet plus LT0 extract 200 mg/kg (LT0), high cholesterol diet plus LT6 extract 200 mg/kg (LT6) and high cholesterol diet plus LT9 extract 200 mg/kg (LT9). We compared the total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL) contents and reactive oxygen species (ROS) from each serums. Protein expression in liver tissues related to antioxidant and cholesterol was analyzed. Results : The Antioxidant activity of Liriopis Tuber increased by steaming process. In vivo, TC, TG, LDL-c, atherogenic index (AI) and cardiac risk factor (CRF) decreased and HDL-c increased with increasing steaming frequency. aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and blood urea nitrogen (BUN) decreased with increasing steaming frequency. ROS decreased only in LT9, and SOD, catalase and glutathione peroxidase (GPx) increased with increasing steaming frequency. phospho-AMP-activated protein kinase (p-AMPK) increased and sterol regulatory element-binding protein 2 (SREBP-2), Phospho-Acetyl-CoA Carboxylase (p-ACC) and HMG-CoA reductase (HMGCR) decreased with increasing steaming frequency. Liver staining showed a decrease in hepatic fat accumulation of LT9. LT9 showed significant results in all experiments. Conclusions : LT9 showed significance of anti-lipid effect and improved fatty liver of hyperlipemia induced rats fed on high cholesterol diet, In conclusion, LP9 can be effectively used for the treatment of hyperlipidemia.

• Journal of Life Science
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• v.33 no.5
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• pp.436-444
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• 2023

Scientific training, diet, and ergogenic aids are widely used to overcome the limits of humans' physical abilities and to achieve excellent sports records. The adoption of nutritional strategies is important for athletes to perform at their highest level, and one of the main factors determining endurance ability is increased fat metabolism. A ketogenic diet (high fat, low carbohydrates) has thus been proposed as an alternative strategy to maximize fatty acid oxidation during prolonged periods of exercise. However, studies have shown mixed results regarding the ergogenic value of a ketogenic diet. For this reason, exogenous ketone supplements (EKS, ingestible forms of ketone bodies, ketone esters, and/or salts) have been suggested to obtain nutritional ketosis, an acute transient increase in circulating acetoacetate (AcAc) and b-hydroxybutyrate (bHB) concentrations, without limiting carbohydrate intake. Some studies have suggested the beneficial effects of EKS on the performance of endurance exercises by providing an additional fuel substrate for peripheral tissues, such as cardiac and skeletal muscles, sparing carbohydrates/increasing fat oxidation and post-exercise recovery by increasing glycogen resynthesis in the liver/muscle, attenuating protein degradation, stimulating protein synthesis in the skeletal muscle, etc. However, many studies have failed to observe the beneficial effects of EKS as an ergogenic aid. As such, this review summarizes the theoretical basis of, as well as the proposed and proven effects of EKS on exercise performance and recovery to date.

• Korean Journal of Poultry Science
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• v.50 no.4
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• pp.261-266
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• 2023

The skeletal muscles of livestock play a crucial role as protein sources for humans, and the consumption of poultry meat is steadily increasing worldwide. Numerous genes, including myogenic regulatory factors, are involved in myogenesis, and precise regulation of them is essential. In this study, genes specifically expressed in muscles were selected, and their promoters were cloned and analyzed. The analysis of gene expression in various tissues of animals revealed that many genes exhibited specific expression patterns in skeletal muscles, with TNNT3, TNNC2, and MYF6 genes showing similar patterns in poultry. The promoter regions of three genes were amplified by polymerase chain reaction to sizes of 1.2 kb, 1.03 kb, and 1.43 kb, respectively. These fragments were then inserted at the front of the enhanced green fluorescent protein gene in vectors. It was confirmed that the sequences of three promoters closely matched the chicken genome sequences. Upon introducing vectors with each promoter into QM7 quail muscle cells, all three promoters successfully induced the expression of the green fluorescent protein. The brightness of the green fluorescence in each promoter was approximately seven times dimmer compared to the control, CMV-IE promoter. It is predicted that more than 230 transcription factors can bind to each promoter, especially various transcription factors expressed in muscles, including myogenic regulatory factors such as MYF5, MYOD, and MYOG. These promoters can be valuable for studying gene expression in poultry muscle cells, and further research is needed to precisely investigate the regulatory region of gene expression in promoters.