• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.2
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• pp.86-93
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• 2007

Chronic inflammation has been known to have a close relationship with several diseases including periodontitis, colitis, hepatitis and arthritis. Recently anti-inflammatory agents have been developed from marine natural resources. In this study, Ecklonia cava (EC) was found to have anti-inflammatory effect. Ethanolic extract of EC belonging to brown algae exhibited an excellent inhibitory effect on the production of inflammatory mediators such as tumor necrosis factor-${\alpha}$, interleukin-$1{\beta}$, interleukin-6 and prostaglandin $E_2$ by RA W264.7 cells. Furthermore, in reporter gene assay and western blot analysis, EC extract exerted anti-inflammatory effect via inactivation of NF-${\kappa}B$ transcription factor that regulates the expression of these inflammatory mediators in macrophages. In addition, EC extract inhibited the activity of matrix metalloproteinase that play an important role in chronic inflammation. These results suggest that EC extract may provide a pharmaceutical potential in inhibiting chronic inflammation.

• Molecules and Cells
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• v.22 no.1
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• pp.44-50
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• 2006

We investigated the possible role of p38 MAPK and $ET_B$ receptors in ET-1 induction of cyclooxygenase-2 (COX-2) and prostaglandin $E_2$ ($PGE_2$) in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated with 10 nM ET-1 and expression of COX-1 and COX-2, involvement of receptors, and activation of p38 MAPK, were examined by Western blot analysis. Levels of $PGE_2$ induced by ET-1 were measured by Elisa. Using $ET_A$and $ET_B$ antagonists (BQ-123 and BQ-788, respectively), the contribution of the ET receptors to COX-1 and COX-2 expression induced by ET-1 was determined. Western blot analysis revealed that treatment of ESMC with ET-1 resulted in transient expression of COX-2 and activation of p38 MAPK. Activation of p38 MAPK was maximal after 1 h. SB202190, a p38 MAPK inhibitor, reduced expression of COX-2, but not COX-1. ET-1-induced release of $PGE_2$ was also blocked by SB202190. COX-2 expression was upregulated only via the $ET_B$ receptor, and COX-1 expression was not affected by either antagonist. Taken together, our data suggest that ET-1 causes p38 MAPK-dependent expression of COX-2 by interacting with $ET_B$ receptors on ESMC.

• KSBB Journal
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• v.9 no.5
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• pp.532-540
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• 1994

Secretion efficiency is generally affected by promoter, signal sequence, characteristics of foreign protein and host. Secretion efficiencies of glucoamylase in recombinant plasmid-harboring yeast and chromosome-integrated yeast which had STA signal sequences were 74% and 65% at the 4th day of incubation, respectively. The high secretion efficiencies of the yeasts were obtained due to the fact that the expression levels were not reached at the secretory apparatus capacities of the host yeasts. In both yeasts, most of the intracellular glucoamylase were detected in cytoplasm and small portion (below 10%) of glucoamylase were located in periplasm. The characteristics of secreted heterologous glucoamylase from recombinant Saccharomyces cerevisiaes were investigated by using Western blot analysis. The secreted mature glucoamylase was heterogeneous and its molecular weight was about 200 to 300 kilodalton. The carbohydrate content of mature glucoamylase was higher than 80%, and several bands of about 55 to 65 kilodalton indicate the endoplasmic reticulum forms of intracellular glucoamylase.

• Korean Journal of Plant Resources
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• v.28 no.3
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• pp.297-304
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• 2015

The flower buds of Sophora japonica L (SF), as a well-known traditional Chinese medicinal herb, have been used to treat bleeding-related disorders such as hematochezia, hemorrhoidal bleeding, dysfunctional uterine bleeding, and diarrhea. However, no specific anti-cancer effect and its molecular mechanism of SF have been described. Thus, we performed in vitro study to investigate if treatment of SF affects activating transcription factor 3 (ATF3) expression and ATF3-mediated apoptosis in human colorectal cancer cells. The effects of SF on cell viability and apoptosis were measured by MTT assay and Western blot analysis against cleaved poly (ADP-ribose) polymerase (PARP). ATF3 activation induced by SF was evaluated using Western blot analysis, RT-PCR and ATF3 promoter assay. SF treatment caused decrease of cell viability and increase of apoptosis in a dose-dependent manner in HCT116 and SW480 cells. Exposure of SF activated the levels of ATF3 protein and mRNA via transcriptional regulation in HCT116 and SW480 cells. Inhibition of extracellular signal-regulated kinases (ERK) 1/2 by PD98059 and p38 by SB203580 attenuated SF-induced ATF3 expression and transcriptional activation. Ectopic ATF3 overexpression accelerated SF-induced cleavage of PARP. These findings suggest that SF-mediated apoptosis may be the result of ATF3 expression through ERK1/2 and p38-mediated transcriptional activation.

• Journal of Veterinary Clinics
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• v.25 no.5
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• pp.355-358
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• 2008

The aim of this study was to investigate whether trophic factor supplementation (TFS) enhance the survival of kidney cell during cold ischemic storage and rewarming. The effect of TFS on the phosphorylation of p44/42 and p38 mitogen activated protein kinases (MAPK) signaling pathway was determined by Western blot. Apoptotic changes after cold ischemic storage and rewarming was determined by 4',6'-diamino-2-phenylindole (DAPI) staining. The cell viability was evaluated by live assay. TFS significantly decreased p44/42 and p38 MAPK activity induced by cold ischemic injury and rewarming (p < 0.05). The number of apoptotic cells was decreased after 5 minute rewarming in the presence of TFS. TFS significantly increased the cell viability after 5 minute rewarming (p < 0.05). Therefore, it was concluded that trophic factor supplementation protects kidney tubule cells from cold ischemic and rewarming injury via the inhibition of p44/42 and p38 MAPK activation and reducing apoptotic change.

• Korean Journal of Veterinary Research
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• v.41 no.2
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• pp.197-202
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• 2001

Although several methods have been developed and applied to control swine respiratory diseases, the disease induces severe economic impact to swine industry worldwide. As one of the new trials, application of egg yolk antibody(IgY) was attempted for the purposes and immune response in sera and egg yolk was analysed with ELISA in previous study. In this study, immunological specificity of the IgY was analysed by Western blot analysis. In the analysis of causative agents of atrophic rhinits, B bronchiseptica and P multocida 4D, proteins of 33, 40, 43, 67 and 141 kDa were specifically reacted with IgY Also, 40 and 110 kDa proteins were identified as the major immunogens in P multocida 3A. In A pleuropneumoniae serotypes 2 and 5, 40 kDa and 47 kDa proteins were found to be the major reactive ones. These results suggested that egg yolk antibodies from immunized hens was specific with antigens injected into hens and partially purified antigens, outer membrane proteins and dermonecrotic toxin, were more effective than bacterin for the production of specific antibody.

• Journal of Life Science
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• v.21 no.4
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• pp.534-541
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• 2011

Retinoic acid (RA), a metabolite of vitamin A, is known to regulate dedifferentiation of rabbit articular chondrocytes. The regulatory mechanism of dedifferentiation by RA is not yet understood. Thus, the effect of RA on the regulation of nitric oxide (NO)-induced dedifferentiation was investigated in rabbit articular chondrocytes. RA caused loss of the differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen expression and proteoglycan synthesis. RA also accelerated NO-induced dedifferentiation in rabbit articular chondrocytes as detected by expression of type II collagen and Sox-9 using Western blot analysis and production of sulfated proteoglycan using Alcain blue staining. Further, RA potentiated NO-induced activation of ERK. Inhibition of ERK with PD98059 (PD) recovered the expression of type II collagen and Sox-9 and production of sulfate proteoglycan in NO-induced dedifferentiated chondrocytes by RA treatment. Our findings suggest that RA accelerates NO-induced dedifferentiation of rabbit articular chondrocytes via the ERK pathway.

• Korean Journal of Plant Resources
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• v.28 no.5
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• pp.582-590
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• 2015

In the present study, we investigated the effect of 70% EtOH extract from Hippophae Rhamnoides L. leaves (HRL) on the anti-obesity effect in 3T3-L1 cells. The effects of HRL on lipid accumulation in 3T3-L1 cells were examined using Oil Red O staining. In addition, we examined the gene expression levels by using RT-PCR and western blot. The results of this analysis showed that 100 ㎍/㎖ HRL significantly increased the inhibition of lipid accumulation by 82.25%; significantly decreased the mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and fatty acid synthase (FAS) in 3T3-L1 cells as well as the stimulated protein expression of AMP-activated protein kinase (AMPK); and suppressed the expression level of PPARγ. These results suggest that HRL can prevent adipogenesis through activation of AMPKα and inhibition of adipogenesis transcription factors.

• Herbal Formula Science
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• v.23 no.2
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• pp.171-187
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• 2015

Objective This study aimed to evaluate the effects of Sagan-tang (SGT) on COPD mouse model. Methods The study was carried out by two ways (in vitro, in vivo). In vitro RAW264.7 cells (mouse macrophage) were used and analysed by flow cytometry, ELISA, Western blot. In vivo LPS and CSS challenged mice were used and its BALF had been analysed by cytospin image, FACS, ELISA, lung tissue by real-time PCR. Results In vitro, SGT maintained 80-100% rate of viablilty on 10 ~ 500 ㎍/㎖ concentration. In ELISA analysis with RAW264.7 cells, SGT significantly decreased NO over 30 ㎍/㎖. In flow cytometry, SGT 100 ㎍/㎖ dosage group displayed a tendency for decrease ROS. In Western blot analysis, SGT 100 ㎍/㎖ dosage group decreased NF-κB. In ELISA analysis, SGT significantly decreased TNF-α, IL-6 over 200 ㎍/㎖. In vivo SGT 200 ㎎/㎏ dosage group, application of SGT significantly decreased increase of neutrophils, TNF-α, IL-6 in BALF, muc5AC, TGF-β, TNF-α, expression of mRNA in lung tissue and histological lung injury. Conclusion This Study suggests usability of SGT for COPD patients by controlling lung tissue injury.