• Journal of Microbiology and Biotechnology
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• v.31 no.8
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• pp.1115-1122
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• 2021

Rotavirus (RV), as the main cause of diarrhea in children under 5 years, contributes to various childhood diseases. Valeriana jatamansi Jones is a traditional Chinese herb and possesses antiviral effects. In this study we investigated the potential mechanisms of V. jatamansi Jones in RV-induced diarrhea. MTT assay was performed to evaluate cell proliferation and the diarrhea mice model was constructed using SA11 infection. Mice were administered V. jatamansi Jones and ribavirin. Diarrhea score was used to evaluate the treatment effect. The enzyme-linked immunosorbent assay was performed to detect the level of cytokines. Western blot and quantitative reverse transcription-PCR were used to determine protein and mRNA levels, respectively. Hematoxylin-eosin staining was applied to detect the pathological change of the small intestine. TdT-mediated dUTP nick-end labeling was conducted to determine the apoptosis rate. The results showed V. jatamansi Jones promoted MA104 proliferation. V. jatamansi Jones downregulated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in protein level, which was consistent with the immunohistochemistry results. Moreover, V. jatamansi Jones combined with ribavirin regulated interleukin-1β (IL-1β), interferon γ, IL-6, tumor necrosis factor α, and IL-10, and suppressed secretory immunoglobulin A secretion to remove viruses and inhibit dehydration. V. jatamansi Jones + ribavirin facilitated the apoptosis of small intestine cells. In conclusion, V. jatamansi Jones may inhibit RV-induced diarrhea through PI3K/AKT signaling pathway, and could therefore be a potential therapy for diarrhea.

• Journal of Life Science
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• v.17 no.6 s.86
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• pp.783-790
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• 2007

G protein coupled receptors (GPCRs) transmit various extracellular signals into the cells. Upon binding of the ligands, conformational changes in the extracellular and/or transmembrane (TM) domains of CPCRs were propagated into the cytoplasmic (CP) domain of the molecule leading to the activation of their cognate heterotrimeric C proteins and kinases. Constitutively active GPCR mutants causing the activation of C Protein signaling even in the absence of ligand binding are of interest for the study of activation mechanism of GPCRs. Two classes of constitutively active mutations, categorized by their effects on the salt bridge between Ell3 and K296, were found in the TM domain of rhodopsin. Opsin mutants containing combinations of the mutations were constructed to study the conformational changes required for the activation of rhodopsin. Rhodopsin chromophore regenerated with 11-cis-retinal showed a thermal stability inversely correlated with its constitutive activity. In contrast, rhodopsin mutants exhibited a binding affinity to an agonist, all-trans-retinal, in a constitutive activity-dependent manner. In order to test whether the conformational changes responsible for the activation of trans-ducin (Gt) are the same as the conformation required for the recognition of rhodopsin kinase, analysis of the mutants were carried out with phosphorylation by rhodopsin kinase. Rhodopsin mutants containing combinations of different classes of the mutations showed a strong synergistic effect on the phosphorylation of the mutants in the dark as similar to that of Gt activation. The results suggest that at least two or three kinds of segmental and independent conformational changes are required for the activation of rhodopsin and the conformational changes responsible for activating rhodopsin kinase and Gt are similar to each other.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.4
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• pp.187-191
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• 2002

Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular $Ca^{2+}$ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular $Ca^{2+}$ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a $Ca^{2+}$ activated protein, and $Ca^{2+}$/calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of $Ca^{2+}$/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular $Ca^{2+}$ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and $Ca^{2+}$/calmodulin-dependent protein kinase II in HepG2 cells.

• BMB Reports
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• v.31 no.6
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• pp.611-614
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• 1998

Protein kinase CKII (CKII) is a protein Ser/Thr kinase that is ubiquitously distributed in eukaryotic cells. Although it has been suggested that CKII plays an critical role in cell growth and proliferation, its functional significance and regulation in the cells remain poorly understood. To investigate the exact biological function of CKII, we have identified proteins that interact with the subunits of CKII using the twohybrid system. In this report, we have identified cathepsin L, a lysosomal protease, as a cellular protein capable of interacting with the ${\beta}$ subunit of CKII. Cathepsin L does not interact with the ${\alpha}$ subunit of CKII, supporting the idea that the ${\beta}$ subunit can mediate the interaction of CKII with target proteins. We have found that cathepsin L has several putative CKII phosphorylation sites including Thr-84, Ser-160, Ser-270, Thr-288, and Ser-301. These data suggest that CKII is a possible protein kinase for cathepsin L phosphorylation.

• Journal of the Korea Institute of Military Science and Technology
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• v.7 no.4 s.19
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• pp.87-90
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• 2004

In order to elucidate the mechanism of infection on human macrophages, we peformed the 2-dimensional electrophoresis and the western blot analysis using the infected human macrophages with the spores of live and inactivated Sterne. We confirmed P21-activated kinase 2 protein which related to cell death(apoptosis) human macrophages at the early stage events. The inhibition of the P21-activated protein kinase 2 protein will be reduced apoptosis on infected human macrophages with Sterne spores.

• Journal of the Korea Society of Computer and Information
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• v.21 no.9
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• pp.101-106
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• 2016

The epidermal growth factor receptor(EGFR) protein kinase signaling is an important pathway in cancer development and recently reported that EGFR and its kinase domain molecules are mutated in various of cancers including head and neck cancer. Functional deregulation of EGFR due to mutations in coding exons and copy number amplification is the most common event in cancers, especially among receptor tyrosine kinases(TK). We have analyzed Korean oral squamous cell carcinomas (OSCC) cell lines for mutations in EGFRTK. Exons encoding the hot-spot regions in the TK domain of EGFR (exons 17 to 23) were amplified by using polymerase chain reaction(PCR) and sequenced directly. EGFR expression was also analyzed in 8 OSCC cell lines using western blotting. Data analysis of the EGFR exons 17 to 23 coding sequences did not show any mutations in the 8 OSCC cell lines that were analyzed. The absence of mutations indicate that protein overexpression might be responsible for activation rather than mutation.

• Biomedical Science Letters
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• v.9 no.2
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• pp.59-65
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• 2003

When cells are exposed to proteotoxic stresses such as heat shock, amino acid analogs, and heavy metals, they increase the synthesis of the heat shock proteins (HSPs) by activating the heat shock transcription factor 1 (HSF1), whose activity is controlled via multiple steps including homotrimerization, nuclear translocation, DNA binding, and hyperphosphorylation. Under unstressed conditions, the HSF1 activity is repressed through its constitutive phosphorylation by glycogen synthase kinase 3$\beta$ (GSK3$\beta$), extracellular regulated kinase 1/2 (ERK1/2), and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). However, the protein kinase (s) responsible for HSF1 hyperphosphorylation and activation is not yet identified. In the present study, we observed that profile of p38 mitogen-activated protein kinase (p38MAPK) activation in response to heat shock was very similar to those of HSF1 hyperphosphorylation and nuclear translocation. Therefore, we investigated whether p38MAPK is involved in the heat shock-induced HSF1 activation and HSP expression. Here we show that the p38MAPK inhibitors, SB202190 and SB203580, but not other inhibitors including the MEK1/2 inhibitor PD98059 and the PI3-K inhibitor LY294002 and wortmannin, suppress HSF1 hyperphosphorylation in response to heat shock and L-azetidine 2-carboxylic acid (Azc), but not to heavy metals. Furthermore, heat shock-induced HSF1-DNA binding and HSP72 expression was specifically prevented by the p38MAPK inhibitors, but not by the MEK1/2 inhibitor and the PI3-K inhibitors. These results suggest that SB202190- and SB203580-sensitive p38MAPK may positively regulate HSP gene regulation in response to heat shock and amino acid analogs.

• BMB Reports
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• v.31 no.4
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• pp.350-354
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• 1998

To understand the role of protein kinase C (PKC) in the regulation of chondrogenesis, we examined proteins which are phosphorylated by PKC. Stage 23/24 chick embryo wing mesenchymes were micromass-cultured to induce chondrogenesis and cell extracts were phosphorylated in a condition that activates PKC. Several proteins including 63 and 66 kDa proteins were phosphorylated. The 66 kDa protein was phosphorylated only in the presence of phorbol 12-myristate 13-acetate (PMA) and phosphatidylserine CPS), and the phosphorylation was almost completely diminished by bisindolylmaleimide, a PKC inhibitor. In addition, partially purified PKC increased the phosphorylation of the 66 kDa protein. Treatment of cultures with lysophosphatidylcholine (LPC) promoted chondrogenesis and phosphorylation of 66 kDa protein, while PMA and thymeleatoxin inhibited both of the two events. Our results suggest that the 66 kDa protein is a putative substrate of PKC, and phosphorylation of the 66 kDa protein, probably by $PKC\alpha$ is required for chondrogenesis.

• Molecules and Cells
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• v.24 no.2
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• pp.276-282
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• 2007

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.

• The Korea Journal of Herbology
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• v.22 no.2
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• pp.155-161
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• 2007

Objectives: Evidences suggests that Ginkgo biloba, a widely used traditional medicine, shows a hypoglycemic effect. Thus, we investigatd the effect of G. biloba extract (GB) on glucose uptake in L6 rat skeletal muscle cells. Method : Effect of GB on glucose uptake and phosphatidylinositol (PI) 3-kinase activity were assessed using Glucose uptake assay and PI 3-kinase assay, respectively. Also, AMP-activated protein kinase (AMPK), p38 mitogen activated protein kinase (p38 MAPK) expression were identified by Western blot. Results : Glucose uptake assay revealed that GB increased glucose uptake about 2.5-fold compared to thecontrol. GB stimulated the activity of PI 3-kinase which is a major switch element on the glucose uptake pathway. About a 6.5-fold increase in activity of PI 3-kinase was observed with GB. We then assessed the activity of AMPK, another regulatory molecule on the glucose uptake pathway. The result was that GB increased the phosphorylation level of both AMPK ${\alpha}$l and ${\alpha}$2. The activity of p38 MAPK, a downstream mediator of AMPK, was also increased by CB. Conclusion : These results suggest that GB may stimulate glucose uptake through both PI 3-kinase and AMPK mediated pathways in L6 skeletal muscle cells thereby contributing to glucose homeostasis.