• BMB Reports
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• v.50 no.12
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• pp.628-633
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• 2017

Gastrin-releasing peptide (GRP) has been reported to be implicated in the pathogenesis of inflammatory disorders. The migration and proliferation of vascular smooth muscle cells (VSMCs) are key components of vascular inflammation that leads to the development of atherosclerosis. The present study aimed to investigate the molecular effect of GRP on VSMC proliferation and migration. We report that GRP significantly enhanced the proliferation and migration of rat VSMCs. GRP increased mRNA and protein expression of matrix metalloproteinase-2 and -9 (MMP-2/9) in VSMCs. The induction of MMP-2/9 by GRP was regulated by the activation of the signal transducer and activator of transcription-3 (STAT3). In addition, STAT3-knockdown of VSMCs by siRNA or blockade of the GRP receptor inhibited GRP-induced migration of VSMCs. Taken together, our findings indicate that GRP promotes the migration of VSMCs through upregulation of MMP-2/9 via STAT3 activation.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.6
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• pp.671-676
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• 1998

In the present study, we first examined whether the changes in the DNA binding activities of the transcription factors, cAMP response element binding protein (CREB) and activator protein-1 (AP-1) mediate the long-term effects of morphine in differentiated SH-SY5Y human neuroblastoma cells. The increases in CREB and AP-1 DNA binding activities were time-dependent up to 6 days of morphine treatment (1, 4, and 6 days). However, the significant reduction in the DNA binding activities of CREB and AP-1 was observed after 10 days of chronic morphine $(10\;{\mu}M)$ administration. Secondly, we examined whether the changes of CREB and AP-1 DNA binding activities could be modulated by dopamine and haloperidol. Dopamine cotreatment moderately increased the levels of the CREB and AP-1 DNA binding activities induced by 10 days of chronic morphine treatment, and haloperidol cotreatment also resulted in a moderate increase of the CREB and AP-1 DNA binding activities. However, dopamine or haloperidol only treatment showed a significant increase or decrease of the CREB and AP-1 DNA binding activities, respectively. In the case of acute morphine treatment, the CREB and AP-1 DNA binding activities were shown to decrease in a time-dependent manner (30, 60, 90, and 120 min). Taken these together, in differentiated SH-SY5Y cells, morphine tolerance seems to involve simultaneous changes of the CREB and AP-1 DNA binding activities. Our data also suggest the possible involvement of haloperidol in prevention or reversal of morphine tolerance at the transcriptional level.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.148-156
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• 2019

The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by coinjecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.240-246
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• 2019

The aim of this study was to investigate effect of heat stress on expression levels of plasminogen activators (PAs) related mRNAs and proteins, and changes of PAs activity in porcine endometrial explants. The endometrial explants (200 ± 50 mg) were isolated from middle part of uterine horn at follicular phase (Day 19-21) and were pre-incubated in serum-free culture medium at 38.5℃ in 5% CO₂ for 18 h. Then, the tissues were transferred into fresh medium and were cultured at different temperature (38.5, 39.5, 40.5 or 41.5℃) for 24 h. The expression level of urokinase-type PA (uPA), type-1 PA inhibitor (PAI-1), type-2 PAI (PAI-2), and heat shock protein-90 (HSP-90) mRNA were analysis by reverse-transcription PCR and proteins were measured by western blotting. The supernatant were used for measurement of PAs activity. In results, mRNA and protein levels of HSP-90 was higher in 41.5℃ treatment groups than other treatment groups (p < 0.05). The expression of uPA, PAI-1, and PAI-2 mRNA were slightly increased by heat stress, however, there were no significant difference. Heat stress condition suppressed expression of active uPA and PAI-2 proteins (p < 0.05), whereas PAI-1 protein was increased (p < 0.01). Although PAI-1 protein was increased and active uPA was decreased, PAs activity was greatly enhanced by exposure of heat stress (p < 0.05). These results suggest that heat stress condition could change intrauterine microenvironment through regulation of PAs activity and other factors regarding with activation of PAs might be regulate by heat stress. Therefore, more studies regarding with regulatory mechanism of PAs activation are needed.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.299-303
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• 2015

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.

• Korean Journal of Environmental Biology
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• v.21 no.3
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• pp.308-312
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• 2003

Various pesticides known or suspected to interfere with steroid hormone function were screened toy effects in leydig cells on catalytic activity and mRNA expression of aromatase. Dichlorodiphenyltrichloroethane (DDT) is a widespread environmental pollutant. In this study, we investigated the effect of DDT on testosterone production through aromatase activity and its molecular mechanism in testicular leydig cell, R2C by using radioimmunoassay (RIA). As the results, the potent leydig: cell activator LH increased testosterone production compared to the control. DDT exposure significantly decreased testosterone production in R2C cell. In addition, DDT was found to increase aromatase gene expression and activity in R2C cell in a dose dependent manner. In order to assess whether the suppressive effects of DDT on LH-inducible testosterone (T) production might be influenced by the ER, ICI 182.780 was used, and it was found that these inhibitory effects of DDT were antagonized by ICI 182.780, implying that the estrogen receptor (ER) mediates the suppressive effects of DDT. Furthermore, the inducible effects of DDT on aromatase gene expression might be influenced by the ER, ICI 182.780 was used, and it was found that these enhancing effects of DDT were antagonized by ICI 182.780, implying that the ER mediates the inducible effects of DDT. Our results indicated that DDT inhibition of luteinizing hormone (LH) -inducible T production in R2C cell is mediated through aromatase. However, the precise mechanisms by which DDT enhance in R2C cell remains unknown. The current study suggests the possibility that DDT might act as a modulator aromatase gene transcription.

• Journal of Microbiology and Biotechnology
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• v.21 no.2
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• pp.176-182
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• 2011

The study objective was to prepare biodegradable branched dextran microspheres encapsulated with His-tagged interferon-alpha (BDM-hIFN-${\alpha}$) and evaluate its activity in vitro and in vivo. The glycidyl methacrylate derivatized dextrans (Dex-GMA) as precursor was primarily synthesized by substituting hydroxyl groups of either the branched or linear type of dextran with GMA. Dex-GMA microspheres loaded with hIFN-${\alpha}$ was then prepared by the water-in-water emulsion technique. In vitro release and Western blotting experiments demonstrated the retained activity of hIFN-${\alpha}$ released from branched dextran microspheres at 24 h by inducing phosphorylation of signal transducer and activator transcription-1 (STAT-1), a down-stream effector of IFN-${\alpha}$, in HepG2 cells. Animal data further revealed a peak of plasma levels of IFN-${\alpha}$ in rats injected intravenously with BDM-hIFN-${\alpha}$ at 10 min post-injection, but a sharp decline at 2 h. High plasma levels of neopterin, a plasma protein induced by IFN-${\alpha}$, were also detected in rats injected with BDM-hIFN-${\alpha}$ at 10 min post-injection. Notably, plasma levels of neopterin remained high at 4 h, but largely declined thereafter.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.189-196
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• 2017

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with formation of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Replication and transcription activator (RTA) genes are expressed upon reactivation of KSHV, which displays a biphasic life cycle consisting of latent and lytic replication phases. RTA protein expression results in KSHV genome amplification and successive viral lytic gene expression. Transcriptional activity of viral lytic genes is regulated through epigenetic modifications. In Raji cells latently infected with Epstein-Barr virus, various modifications, such as acetylation and methylation, have been identified at specific lysine residues in histone H4 during viral reactivation, supporting the theory that expression of specific lytic genes is controlled by histone modification processes. Data obtained from chromatin immunoprecipitation and quantitative real-time PCR analyses revealed alterations in the H4K8ac and H4K20me3 levels at lytic gene promoters during reactivation. Our results indicate that H4K20me3 is associated with the maintenance of latency, while H4K8ac contributes to KSHV reactivation in infected TREx BCBL-1 RTA cells.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1047-1055
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• 2015

For the search of a potent first-in-class compound to inactivate macrophages responsible for inflammatory responses, in the present study, we investigated the anti-nflammatory effects of YH-1118, a novel synthetic compound, in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, Raw 264.7. YH-1118 inhibited LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression at both the protein and mRNA levels. The suppression of LPS-induced iNOS expression by YH-1118 was mediated via nuclear factor kappa B (NF-κB), but not activator protein-1 (AP-1) transcription factor. This was supported by the finding that YH-1118 attenuated the phosphorylation of inhibitor of κBα (IκBα) and nuclear translocation and DNA binding activity of NF-κB. Through the mechanisms that YH-1118 inhibited the activation of IκB kinases (IKKs), upstream activators of NF-κB, or p38 MAPK, YH-1118 significantly suppressed LPS-induced production of pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 (p < 0.05). In conclusion, our results suggest that YH-1118 inhibits LPS-induced inflammatory responses by blocking IKK and NF-κB activation in macrophages, and may be a therapeutic candidate for the treatment of various inflammatory diseases.

• The Korea Journal of Herbology
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• v.27 no.4
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• pp.9-16
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• 2012

Objective : Canavalia gladiata DC semen (CGS) have been used to improve hematopoietic activity. In the current study, we investigated whether CGS regulate hemato-potentiating function using hematopoietic stem cells (HSCs) as a testing system. Methods : HSCs isolated from femur in mice with leukopenia and thrombocytopenia induced induced by CTX. Then, Real-time PCR was performed to measure the mRNA expression and hematopoietic related gene (EPO, IL-3, SCF, c-kit, GM-CSF), the phoaphorylation of GATA-1 and STAT-5a/b were observed by ELISA method, and the number of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E), semisolid clonogenic assay was performed. Result : When HSCs were treated with CGS, the expression of hematopoietic related genes (EPO, IL-3, SCF, c-kit, and GM-CSF) were significantly increased at the levels of mRNA as well as production in HSCs. Additionally, CGS enhanced phosphorylation of STAT-1 and signal transducer and activator of transcription-5a/b (STAT-5a/b) in HSCs. Furthermore, CGS significantly enhanced the growth rate of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E) in vitro. Conclusion : These result suggest that CGS has hematopoietic enhancement via hematopoietic cytokine-mediated GATA-1/STAT-5a/b pathway.