• 생명과학회지
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• 제20권10호
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• pp.1451-1457
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• 2010

Serotonin (5-hydroxytryptamine (5-HT))는 신경계의 세포-세포 간의 신호전달의 주요한 신경전달물질이다. 세포막에 존재하는 serotonin transporter (SERT)는 연접간격에 존재하는 5-HT를 세포 내로 재흡수 하여 세포외부의 5-HT 농도를 조절하지만 그 기전은 아직 밝혀지지 않았다. 본 연구에서는 yeast two-hybrid system을 사용하여 SERT의 C-말단이 protein kinase C-$\varepsilon$ (PKC-$\varepsilon$)과 특이적으로 결합함을 알았다. PKC-$\varepsilon$는 PKC의 isotype으로 calcium 비의존적이며 phorbol ester/diacylglycerol 민감성 serine/threonine kinase이다. $Na^+/Cl^-$ 의존성 SLC6 gene family의 다른 수송체는 PKC-$\varepsilon$과 결합하지 않았다. Deletion mutant들을 사용하여 SERT는 PKC-$\varepsilon$의 C-말단부위와 결합함을 알았으며, 또한 이 단백질간의 결합을 GST pull-down assay로 확인하였다. PKC-$\varepsilon$는 in vitro에서 SERT의 N-말단의 펩티드를 인산화시켰다. 이러한 결과들은 PKC-$\varepsilon$에 의한 SERT의 인산화가 세포막에 존재하는 SERT의 활성을 조절하는 역할을 할 가능성을 시사한다.

• Environmental Analysis Health and Toxicology
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• 제14권1_2호
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• pp.1-12
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• 1999

Recently, several new methods for the detection of genetic damages in vitro and in vivo based on molecular biological techniques were introduced according to the rapid progress in toxicology combined with cellular and molecular biology. Among these methods, mouse lymphoma thymidine kanase (tk) gene forward mutation assay, single cell gel electrophoresis (comet assay) and transgenic animal and cell line model as a target gene of lac I (Big Blue) and lac Z (Muta Mouse) gene mutation are newly introduced based on molecular toxicological approaches. The mouse lymphoma tk$\^$+/-/ gene assay (MOLY) using L5178Y tk$\^$+/-/ mouse lymphoma cell line is one of the mammalian forward mutation assays, and has many advantages and more sensitive than hprt assay. The target gene of MOLY is a heterozygous tk$\^$+/-/ gene located in 11 chromosome, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. The comet assay is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakages in mammalian cells, Also, transgenic animal and cell line models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease process, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Also in vivo acridine orange supravital staining micronucleus assay by using mouse peripheral reticulocytes was introduced as an alternative of bone marrow micronucleus assay. In this respect, there was an International workshop on genotoxicity procedure (IWGTP) supported by OECD and EMS (Environmental Mutagen Society) at Washington D. C. in March 25-26, 1999. The objective of IWGTP is to harmonize the testing procedures internationally, and to extend to finalization of OECD guideline, and to the agreement of new guidelines under the International Conference of Harmonization (ICH) for these methods mentioned above. Therefore, we introduce and review the principle, detailed procedure, and application of MOLY, comet assay, transgenic mutagenesis assay and supravital staining micronucleus assay.

• Toxicological Research
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• 제31권1호
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• pp.11-16
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• 2015

Our body's immune system has defense mechanisms against pathogens such as viruses and bacteria. Immune responses are primarily initiated by the activation of toll-like receptors (TLRs). In particular, TLR4 is well-characterized and is known to be activated by gram-negative bacteria and tissue damage signals. TLR4 requires myeloid differentiation factor 2 (MD2) as a co-receptor to recognize its ligand, lipopolysaccharides (LPS), which is an extracellular membrane component of gram-negative bacteria. Gambogic acid is a xanthonoid isolated from brownish or orange resin extracted from Garcinia hanburyi. Its primary effect is tumor suppression. Since inflammatory responses are related to the development of cancer, we hypothesized that gambogic acid may regulate TLR4 activation. Our results demonstrated that gambogic acid decreased the expression of pro-inflammatory cytokines ($TNF-{\alpha}$, IL-6, IL-12, and $IL-1{\beta}$) in both mRNA and protein levels in bone marrow-derived primary macrophages after stimulation with LPS. Gambogic acid did not inhibit the activation of Interferon regulatory factor 3 (IRF3) induced by TBK1 overexpression in a luciferase reporter gene assay using IFN-${\beta}$-PRD III-I-luc. An in vitro kinase assay using recombinant TBK1 revealed that gambogic acid did not directly inhibit TBK1 kinase activity, and instead suppressed the binding of LPS to MD2, as determined by an in vitro binding assay and confocal microscopy analysis. Together, our results demonstrate that gambogic acid disrupts LPS interaction with the TLR4/MD2 complex, the novel mechanism by which it suppresses TLR4 activation.

• Mycobiology
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• 제51권5호
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• pp.372-378
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• 2023

Lkh1, a LAMMER kinase homolog in the fission yeast Schizosaccharomyces pombe, acts as a negative regulator of filamentous growth and flocculation. It is also involved in the response to oxidative stress. The lkh1-deletion mutant displays slower cell growth, shorter cell size, and abnormal DNA content compared to the wild type. These phenotypes suggest that Lkh1 controls cell size and cell cycle progression. When we performed microarray analysis using the lkh1-deletion mutant, we found that only four of the up-regulated genes in the lkh1-deletion were associated with the cell cycle. Interestingly, all of these genes are regulated by the Mlu1 cell cycle box binding factor (MBF), which is a transcription complex responsible for regulating the expression of cell cycle genes during the G1/S phase. Transcription analyses of the MBF-dependent cell-cycle genes, including negative feedback regulators, confirmed the up-regulation of these genes by the deletion of lkh1. Pull-down assay confirmed the interaction between Lkh1 and Yox1, which is a negative feedback regulator of MBF. This result supports the involvement of LAMMER kinase in cell cycle regulation by modulating MBF activity. In vitro kinase assay and NetPhosK 2.0 analysis with the Yox1T40,41A mutant allele revealed that T40 and T41 residues are the phosphorylation sites mediated by Lkh1. These sites affect the G1/S cell cycle progression of fission yeast by modulating the activity of the MBF complex.

• IMMUNE NETWORK
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• 제15권4호
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• pp.206-211
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• 2015

Pulmonary edema is a major cause of mortality due to acute lung injury (ALI). The involvement of protein kinase C-${\delta}$ (PKC-${\delta}$) in ALI has been a controversial topic. Here we investigated PKC-${\delta}$ function in ALI using PKC-${\delta}$ knockout (KO) mice and PKC inhibitors. Our results indicated that although the ability to produce proinflammatory mediators in response to LPS injury in PKC-${\delta}$ KO mice was similar to that of control mice, they showed enhanced recruitment of neutrophils to the lung and more severe pulmonary edema. PKC-${\delta}$ inhibition promoted barrier dysfunction in an endothelial cell layer in vitro, and administration of a PKC-${\delta}$-specific inhibitor significantly increased steady state vascular permeability. A neutrophil transmigration assay indicated that the PKC-${\delta}$ inhibition increased neutrophil transmigration through an endothelial monolayer. This suggests that PKC-${\delta}$ inhibition induces structural changes in endothelial cells, allowing extravasation of proteins and neutrophils.

• Molecular & Cellular Toxicology
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• 제1권3호
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• pp.179-188
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• 2005

To develop the novel anti-allergic drug, many sophoricoside derivatives were synthesized. Among these derivatives, JSH-II-3, VI-3, VII-3, VIII-3, VII-20 and VII-20 (sodium salt) were selected and subjected to high throughput toxicity screening (HTTS) because they revealed strong IL-5 inhibitory activity and limitation of quantity. Single cell gel electrophoresis (Comet) assay, mouse lymphoma thymidine kinase ($tk^{+/-}$) gene assay (MOLY), chromosomal aberration assay in mammalian cells and Ames reverse mutation assay in bacterial system were used as simplified, inexpensive, short-term in vitro screening tests in our laboratory. Through the primary screening using the comet assay, we could choose the first candidates of sophoricoside derivatives with no genotoxic potentials as JSH-VI-3, VII-3, VII-20 and VII-20 (sodium salt). Also JSH-VII-3, VII-20 and VII-20 (sodium salt) are non-mutagenic in MOLY assay, while JSH-II-3 is mutagenic at high concentration with the presence of metabolic activation system in both comet assay and MOLY assay. The selected derivatives (JSH-VI-3, VII-3, VII-20 and VII-20 (sodium salt) are not mutagenic in S. typhimurium TA98 and TA100 strains both in the presence and absence of metabolic activation. From results of chromosomal aberration assay, 6 h treatment of JSH-VI-3, VII-3 and VII-20 (sodium salt) were not revealed clastogenicity both in the presence and absence of S-9 mixture. Therefore, we suggests that JSH-VI-3, VII-3, VII-20 and VII-20 (sodium salt), as the optimal candidates with both no genotoxic potential and IL-5 inhibitory effects must be chosen. To process the development into new anti-inflammatory drug of these derivatives, further investigation will need.

• BMB Reports
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• 제56권5호
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• pp.302-307
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• 2023

Lyn, a tyrosine kinase that is activated by double-stranded DNA-damaging agents, is involved in various signaling pathways, such as proliferation, apoptosis, and DNA repair. Ribosomal protein S3 (RpS3) is involved in protein biosynthesis as a component of the ribosome complex and possesses endonuclease activity to repair damaged DNA. Herein, we demonstrated that rpS3 and Lyn interact with each other, and the phosphorylation of rpS3 by Lyn, causing ribosome heterogeneity, upregulates the translation of p-glycoprotein, which is a gene product of multidrug resistance gene 1. In addition, we found that two different regions of the rpS3 protein are associated with the SH1 and SH3 domains of Lyn. An in vitro immunocomplex kinase assay indicated that the rpS3 protein acts as a substrate for Lyn, which phosphorylates the Y167 residue of rpS3. Furthermore, by adding various kinase inhibitors, we confirmed that the phosphorylation status of rpS3 was regulated by both Lyn and doxorubicin, and the phosphorylation of rpS3 by Lyn increased drug resistance in cells by upregulating p-glycoprotein translation.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.319-327
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• 2011

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM $CaCl_2{\cdot}2H_2O$), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM $CaCl_2{\cdot}2H_2O$) supplemented with 100, 200 or 300 ${\mu}$g/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}$sec. The activated embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 ${\mu}$g/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM $CaCl_2{\cdot}2H_2O$ (T1), 1.0 mM $CaCl_2{\cdot}2H_2O$ (T2) and 0.1 mM $CaCl_2{\cdot}2H_2O$ with 100 ${\mu}$g/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-${\alpha}$/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

• 대한한방부인과학회지
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• 제19권2호
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• pp.34-48
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• 2006

Purpose : This study was conducted to investigate the inhibitory effects of Dangguijakyaksan on cell proliferation in HeLa cells. Methods : Human uterine cervical carcinoma HeLa cells were cultured in the 1%, 5% and 10% concentration of Dangguijakyaksan extract solution for 24 hours, 48 hours and 72 hours for the direct inhibitory effects of Dangguijakyaksan. Afterwards, we executed the analysis of the effect of Dangguijakyaksan extract solution on cell proliferation inhibition using XTT assay, molecular biological method through MAP kinase activity and FACS analysis of caspase activity in the HeLa cells. Results : After 24, 48 and 72 hours cultivation, Dangguijakyaksan extract solution group showed significant decrease of HeLa cells except 1% solution after 24 hours compared with the control group. In the FACS analysis, Dangguijakyaksan extract solution groups showed increase of caspase activity except 1% solution after 48 hours compared with the control group. In the XTT assay, the caspase-3 activities were increased in Dangguijakyaksan extract solution groups except 1% solution after 24 hours in a dose-dependent manner. In the XTT study, cell activities were significantly decreased in 10% Dangguijakyaksan extract solution groups after 48 and 72 hours cultivation compared with the control group. In all Dangguijakyaksan extract solution groups, The activities of MAP kinase were decreased after 24, 48 and 72 hours cultivation compared with the control group. Conclusion : It could be concluded that Dangguijakyaksan is available to the inhibition of proliferation of human cervical carcinoma cell line in vitro.

• Biomolecules & Therapeutics
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• 제30권4호
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• pp.360-367
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• 2022

Tropomyosin receptor kinase A (TrkA) protein is a receptor tyrosine kinase encoded by the NTRK1 gene. TrkA signaling mediates the proliferation, differentiation, and survival of neurons and other cells following stimulation by its ligand, the nerve growth factor. Chromosomal rearrangements of the NTRK1 gene result in the generation of TrkA fusion protein, which is known to cause deregulation of TrkA signaling. Targeting TrkA activity represents a promising strategy for the treatment of cancers that harbor the TrkA fusion protein. In this study, we evaluated the TrkA-inhibitory activity of the benzoxazole compound KRC-108. KRC-108 inhibited TrkA activity in an in vitro kinase assay, and suppressed the growth of KM12C colon cancer cells harboring an NTRK1 gene fusion. KRC-108 treatment induced cell cycle arrest, apoptotic cell death, and autophagy. KRC-108 suppressed the phosphorylation of downstream signaling molecules of TrkA, including Akt, phospholipase Cγ, and ERK1/2. Furthermore, KRC-108 exhibited antitumor activity in vivo in a KM12C cell xenograft model. These results indicate that KRC-108 may be a promising therapeutic agent for Trk fusion-positive cancers.