• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.347-352
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• 1999

This cDNAs were cloned with the aid of the polymerase chain reaction (PCR) by sequences based on cloned rat LH/CG and FSH receptor cDNAs. A cDNAs of LHR and FSHR were transfected into the 293 cells. Several clonal cell lines were obtained expressing different numbers of cell surface receptors. One cell lines for each LHR and FSHR were chosen, and a corresponding cell lines expressing the wild type LHR and FSHR were selected based on the number of cell surface receptor for the particular LHR and FSHR. The abilities of the LHR and FSHR to transduce the hCG and FSH signals were measured by quantitating cAMP accumulation in cells incubated with increasing concentrations of hCG and FSH. The cAMP accumulation effects for these receptors were increased by the increasing concentrations of hCG and FSH. Thus, most of the receptors expressed in cells transfected with LHR and FSHR could be detected by measuring hormone binding and cAMP response, and can utilize to study the structure function and signal transduction of the choriogonadotropins and glycoprotein hormones.

• The Journal of Pediatrics of Korean Medicine
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• v.26 no.1
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• pp.36-45
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• 2012

Objectives Perillae Folium (PF) has been widely used in Korean herbal medicines used for treatment of acute and chronic inflammatory diseases, such as rhinitis, asthma, and enteritis. In this study, to investigate the protective effect of PF on type 1 allergic response, we determined whether PF inhibits early or late allergic responses. Methods The effect of PF was analyzed by ELISA,. RT-PCR and Western blot in RBL-2H3 cells. Levels of ${\beta}$-hexosaminidase, interleukin (IL)-4 and TNF-${\alpha}$ were measured using enzyme-linked immunosorbent assays (ELISAs). mRNA levels of cytokines and enzymes were analyzed with RT-PCR. Signal transduction was analyzed with Western blot. Results We found that PF suppressed ${\beta}$-hexosaminidase release in RBL-2H3 by the IgE-DNP-HSA stimulation. PF also significantly inhibited enzymes level, such as COX-1, COX-2, iNOS, and HDC2, along with reduced cytokine levels, such as IL-2, IL-3, IL-4, IL-6, IL-13, and TNF-${\alpha}$ in RBL-2H3. In addition, PF suppressed the phospholyation of ERK1/2, JNK1/2, and $I{\kappa}B{\alpha}$. Conclusions Our results indicate that PF protects against type 1 allergic response and exert an anti-inflammatory effect through the inhibition of degranulation and expression of cytokines and enzymes via the suppression of signal transduction.

• BMB Reports
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• v.31 no.4
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• pp.370-376
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• 1998

Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

• The Plant Pathology Journal
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• v.28 no.3
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• pp.227-238
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• 2012

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules in the eukaryotic cells. They are involved in many major cell processes in fungi such as stress responses, vegetative growth, pathogenicity, secondary metabolism and cell wall integrity. In this review, we summarized the advances of research on the MAPK signaling pathways in Alternaria species. As major phytopathogenic fungi, Alternaria species reduce crop production. In contrast to the five MAPK pathways known in yeast, only three MAPK pathways as Fus3/Kss1-type, Hog1-type, and Slt2-type have been characterized in Alternaria. The Fus3/Kss1-type MAPK pathway participates in regulation of vegetative growth, conidiation, production of some cell-wall-degrading enzymes and pathogenicity. The Hog1-type pathway is involved in osmotic and oxidative stress, fungicides susceptibility and pathogenicity. The Slt2-type MAP kinases play an important role on maintaining cell wall integrity, pathogenicity and conidiation. Although recent advances on the MAPK pathways in Alternaria spp. reveal many important features on the pathogenicity, there are many unsolved problems regarding to the unknown MAP kinase cascade components and network among other major signal transduction. Considering the economic loss induced by Alternaria spp., more researches on the MAPK pathways will need to control the Alternaria diseases.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.155-162
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• 2020

Equine follicle stimulating hormone receptor (eFSHR) has a large extracellular domain and an intracellular domain containing approximately 10 phosphorylation sites within the G protein-coupled receptor. This study was conducted to analyze the function of phosphorylation sties at the eFSHR C-terminal region. We constructed a mutant of eFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 641 (eFSHR-t641). This removed 10 potential phosphorylation sites from the C-terminal region of the intracellular loop. The eFSHR-wild type (eFSHR-wt) and eFSHR-t641 cDNAs were subcloned into the pCMV-ARMS1-PK2 expression vector. These plasmids were transfected into PathHunter CHO-K1 Parental cells expressing β-arrestin 2 enzyme acceptor fusion protein and analyzed for agonist-induced cAMP response. The cAMP response in cells expressing eFSHR-t641 was lower than the response in cells expressing eFSHR-wt. EC₅₀ values of eFSHR-wt and eFSHR-t641 were 1079 ng/mL and 1834 ng/mL, respectively. eFSHR-t641 was approximately 0.58-fold compared with that of eFSHR-wt. The maximal response in eFSHR-wt and eFSHR-t641 was 24.7 nM and 16.7 nM, respectively. The Rmax value of phosphorylation sites in eFSHR-t641 was also decreased to approximately 68.4% of that in eFSHR-wt. The collective data implicate that the phosphorylation sites in the eFSHR C-terminal region have a pivotal role in signal transduction in PathHunter CHO-K1 cells, and indicate that β-arrestin is involved in coupling the activated receptors to the internalization system.

• Reproductive and Developmental Biology
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• v.42 no.1
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• pp.1-6
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• 2018

In this study, we analyzed signal transduction by equine follicle-stimulating hormone receptor (eFSHR) on sti- mulation with recombinant $eelFSH{\beta}/{\alpha}$ ($rec-eelFSH{\beta}/{\alpha}$), natural porcine FSH (pFSH), and natural human FSH (hFSH). cAMP stimulation in CHO-K1 cells expressing eFSHR was determined upon exposure to different doses (0-1450 ng/mL) of these hormones. The $EC_{50}$ value of $rec-eelFSH{\beta}/{\alpha}$ was 53.35 ng/mL. The Rmax values of $rec-eelFSH{\beta}/{\alpha}$ and pFSH were 28.12 and 2.88 ng/mL, respectively. The activity of $rec-eelFSH{\beta}/{\alpha}$ was much higher than that of natural pFSH. However, signal transduction in CHO PathHunter Parental cells expressing eFSHR was not enhanced by stimulation with natural hFSH. Thus, $rec-eelFSH{\beta}/{\alpha}$ was completely active in cells expressing eFSHR. However, natural hFSH did not invoke a signal response in cells expressing eFSHR. Particularly, natural pFSH was weakly active in the same cells. These results showed that $eelFSH{\beta}/{\alpha}$ has potent activity in cells expressing eFSHR. Thus, $rec-eelFSH{\beta}/{\alpha}$ may efficiently bind to eFSHR, where as natural hFSH does not bind to eFSHR.

• BMB Reports
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• v.51 no.10
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• pp.538-543
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• 2018

Pancreatic beta cell destruction and dysfunction induced by cytokines is a major cause of type 1 diabetes. Paraoxonase 1 (PON1), an arylesterase with antioxidant activity, has been shown to play an important role in preventing the development of diabetes in transgenic mice. However, no studies have examined the anti-diabetic effect of PON1 delivered to beta cells using protein transduction. In this study, we expressed the cell-permeable PON1 fused with PEP-1 protein transduction domain (PEP-1-PON1) to investigate whether transduced PEP-1-PON1 protects beta cells against cytokine-induced cytotoxicity. PEP-1-PON1 was effectively delivered to INS-1 cells and prevented cytokine-induced cell destruction in a dose-dependent manner. Transduced PEP-1-PON1 significantly reduced the levels of reactive oxygen species (ROS) and nitric oxide (NO), DNA fragmentation, and expression of inflammatory mediators, endoplasmic reticulum (ER) stress proteins, and apoptosis-related proteins in cytokine-treated cells. Moreover, transduced PEP-1-PON1 restored the decrease in basal and glucose-stimulated insulin secretion induced by cytokines. These data indicate that PEP-1-PON1 protects beta cells from cytokine-induced cytotoxicity by alleviating oxidative/nitrosative stress, ER stress, and inflammation. Thus, PEP-1-mediated PON1 transduction might be an effective method to reduce the extent of destruction and dysfunction of pancreatic beta cells in autoimmune diabetes.

• Molecules and Cells
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• v.24 no.1
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• pp.125-131
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• 2007

von Willebrand factor (vWF) is a multimeric glycoprotein which functions within the coagulation system. It colocalizes with factor VIII (FVIII) by non-covalent interaction and alters its intracellular trafficking. vWF is also instrumental in maintaining the stability of secreted FVIII. The principal objective of this study was to generate a lentivirus-based vWF expression vector for gene therapy of hemophilia A. We inserted a vWF of 8.8 Kb into a lentiviral vector thereby producing VSV-G-pseudotyped vEx52. However, its titer was quite low, presumably because the length of vWF gene exceeds the size limit of the lentiviral vector. In order to overcome the low-titer, we concentrated the vEx52 and thus increased the efficiency of transduction approximately 6-fold with $1/100^{th}$ of the volume. However, as concentration requires an additional laborious step, we attempted to enhance the transduction efficiency by deleting exons 24-46 and 29-46 in pRex52 to construct pRex23 and pRex28, and in pvEx52, yielding pvEx23 and pvEx28, respectively. The transfected pRex52 had a profound effect on the activity of secreted FVIII, and this activity declined as domains of vWF were deleted. However, when the domain-deleted vWF-lentiviruses were transduced into K562 cells, the vEx28 increased the activity of the secreted FVIII compared to what was observed with vEx52. This result is probably due to higher efficiencies of transduction and expression while retaining the essential domains required for proper interaction with FVIII.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5925-5928
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• 2013

Objective: This study aimed to examine the relationship between expression of mammal target of rapamycin (mTOR) and phosphorylation of mTOR (p-mTOR) protein in the PI3K/Akt/mTOR signaling pathways in gastrointestinal stromal tumors and relatiuonships with clinical factors. Methods: Immunohistochemistry was used to detect the expression of the associated proteins mTOR, p-mTOR, and phosphorylation of the tumor suppressor genes PTEN, P27, VEGF, and EGFR in 40 cases of gastrointestinal stromal tumors, with division into a very low and low risk group as well as a moderate and high risk group. Results: The positive rate of mTOR and p-mTOR was significantly increased in the moderate and high risk group compared with the very low and low risk group. The difference was statistically significant (P<0.05). When grouped according to size, the positive mTOR expression rate exhibited a statistical difference (P<0.05), which was significantly increased in the group of tumors larger than 5 cm. The difference in the positive mTOR and p-mTOR expression rate exhibit no statistical significance among the PTEN, P27, VEGF, and EGFR expression subgroups (P>0.05). Conclusion: The different expressions of mTOR and p-mTOR in the signal transduction pathway of gastrointestinal stromal tumor in the different degree-of-risk groups suggested that the mTOR and p-mTOR of the signal transduction pathway serve an important function in the occurrence and development of gastrointestinal stromal tumors.

• Korean Journal of Microbiology
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• v.45 no.1
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• pp.10-16
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• 2009

A diverse range of stresses such as heat, cold, salt, ethanol, oxygen starvation or nutrient starvation induces same stress-responsive proteins. This general stress response enhances bacterial survival significantly. In Bacillus subtilis and closely related Gram-positive bacteria Listeria monocytogenes, the general stress response is controlled by the alternative transcription factor ${\sigma}^B$. The activity of ${\sigma}^B$ is regulated post-translationally by a signal transduction network that has been extensively studied in B. subtilis, and serve as a model for L. monocytogenes. The proposed model of L. monocytogenes signal transduction network is similar to that of B. subtilis, but the energy stress pathway is missing. More than 150 general stress proteins belong to ${\sigma}^B$ regulon of B. subtilis and L. monocytogenes. In both bacteria, ${\sigma}^B$ function is primarily important for resistance to diverse stresses. In addition, ${\sigma}^B$ function contributes to the control of important virulence genes in food-borne pathogen L. monocytogenes. Therefore, understanding of the general stress response is important not only for bacterial physiology, but also for pathogenicity.