• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1124-1131
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• 2020

Techniques used for the regulation of gene expression facilitate studies of gene function and treatment of diseases via gene therapy. Many tools have been developed for the regulation of gene expression in mammalian cells. The Lac operon system induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) is one of the employed inducible systems. IPTG mimics the molecular structure of allolactose and has a strong affinity for the corresponding repressor. IPTG is known to rapidly penetrate into mammalian cells and exhibits low toxicity. In the present study, we developed a new inducible expression system that could regulate the expression of genes in mammalian cells using IPTG. Here we confirm that unlike other vector systems based on the Lac operon, this expression system allows regulation of gene expression with lactose in the mammalian cells upon transfection. The co-treatment with IPTG and lactose could improve the regulatory efficiency of the specific target gene expression. The regulation of gene expression with lactose has several benefits. Lactose is safe in humans as compared to other chemical substances and is easily available, making this technique very cost-effective.

• Molecular & Cellular Toxicology
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• 제5권3호
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• pp.187-192
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• 2009

Toll-like receptors (TLRs) play an important role in host defense by sensing invading microbial pathogens. The stimulation of TLRs by microbial components triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-$\beta$ (TRIF)-dependent downstream signaling pathways. TLR/MyD88 signaling pathway induces the activation of nuclear factor-kappa B (NF-${\kappa}B$) and the expression of inflammatory cytokine genes, including tumor necrosis factor-alpha, interleukin (IL)-6, IL-12, and IL-$1{\beta}$. On the other hand, TLR/TRIF signaling pathway induces the delayed-activation of NF-${\kappa}B$ and interferon regulatory factor 3 (IRF3), and the expression of type I interferons (IFNs) and IFN-inducible genes. The divalent heavy metal cadmium (Cd) is clearly toxic to most mammalian organ systems, especially the immune system. Yet, the underlying toxic mechanism(s) remain unclear. Cd inhibits the MyD88-dependent pathway by ceasing the activity of inhibitor-${\kappa}B$ kinase. However, it is not known whether Cd inhibits the TRIF-dependent pathway. Presently, Cd inhibited NF-${\kappa}B$ and IRF3 activation induced by lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid. Cd inhibited LPS-induced IRF3 phosphorylation and IFN-inducible genes such as interferon inducible protein-10 and regulated on activation normal T-cell expressed and secreted (RANTES). These results suggest that Cd can modulate TRIF-dependent signaling pathways of TLRs.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2005년도 국제학술심포지움 The 44th Annual Meeting of Korean Society for Life Science
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• pp.23-36
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• 2005

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed In yeast. To investigate whether .Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various orgarusms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs In detail. PBIl is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower lovels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. H$_{2O2}$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of H2O2 treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased In the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 i'n vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation In situ. Thus, AtNDPK2 appears to play a novel regulatory role in H2O2-mediated MAPK signaling in plants.

• BMB Reports
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• 제47권10호
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• pp.593-598
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• 2014

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of ${\beta}$-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain insight into the PTM of hSCP1, we used the Western blot, immunoprecipitation, succinylayed wheat germ agglutinin-precipitation, liquid chromatography-mass spectrometry analyses, and site-directed mutagenesis and identified the $Ser^{41}$ residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s).

• Environmental Analysis Health and Toxicology
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• 제15권3호
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• pp.69-80
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• 2000

ADP-rubosylation may be involved in the process of macrophage activation. Nitric oxide (NO) has emerged as an important intracellular and interacellular regulatory molecule with function as diverse as vasodilation, neural communication or host defense. NO is derived from the oxidation of the terminal guanidino nitrogen atom of L-arginine by the NADPH -dependent enzyme, nitric oxide synthase (NOS) which is one of the three different isomers in mammalian tissues. Since NO can exert protective or regulatory functions in the cell at a low concentration while toxic effects at higher concentrations, its role may be tightly regulated in the cell. Therefore, this paper was focused on signal transduction pathway of NO synthesis, role of endogenous TGF-$\beta$ in NO production. effect of NO on superoxide formation. Costimulation of murine peritoneal macrophages with interferon-gamma (IFN-γ) and phorbol 12-myristate 13-acetate (PMA) increased both NO secretion and mRNA expression of inducible nitric oxide synthase (iNOS) when PMA abolished costimulation. Pretreatmnet of the cells with PMA abolished costimuation effects due to the depletion of protein kinase C (PKC) activities . The involvement of PKC in NO secretion could be further confirmed by PKC inhibitor, stauroprine, and phorbol ester derivative, phorbol 12,13-didecanoate. Addition of actinomycine D in IFN-γ plus PMA stimulated cells inhibited both NO secretion and mRNA expression of iNOS indication that PMA stabilizes mRNA of iNOS . Exogenous TGF-$\beta$ reduced NO secretion in IFN -γ stimulated murine macrophages. However addition of antisense oligodeoxynucleotide (ODN) to TGF-$\beta$ to this system recovered the ability of NO production and inhibited mRNA expression of TGF-$\beta$. ACAS interactive laser cytometry analysis showed that transportation of FITC -labeled antisense ODN complementary to TGF-$\beta$ mRNA could be observed within 5 min and reached maximal intensity in 30 min in the murine macrophage cells. NO released by activated macrophages inhibits superoxide formation in the same cells . This inhibition nay be related on NO-induced auto -adenosine diphosphate (ADP) -ribosylation . In addition, ADP-ribosylation may be involved in the process of macrophage activation .

• 생명과학회지
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• 제15권4호
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• pp.607-612
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• 2005

열충격단백질 70 (HSP70)은 복수유전자족으로서 통상적으로 표현되는 Hsc70와 스트레스에 의하여 유도되는 Hsp70가 있다. 포유동물의 신경계통에서는 상당한 량의 HSP70가 정상조건에서도 표현되는 것으로 알려져 있다. 본 연구에서는 흰쥐의 소뇌 세포의 연접에서 Hsp70의 표현에 대한 연구를 하였다. 면역조직화학적으로 소뇌절편을 염색하여 관찰한 결과 Hsp70와 Hsc70 모두 표현되었는데, 소뇌 조롱박세포에서 가장 강하게 표현되었으며, 다음으로 소뇌 과립세포에서 강하게 표현되었다. 또한 깊은소뇌핵의 신경세포들도 강하게 염색되었다. 배양한 P1 소뇌신경 세포를 Hsp70 항체로 염색한 결과 Hsp70는 조롱박세포와 과립 세포에서 모두 표현되었으며, 세포체와 가지돌기를 따라 점박이를 형성하였다. 이들 점 박이들은 PSD95 점박이와 같이 위치하였다. 그리고 PSD 분획을 이용한 면역염색에서도 PSD70이 검출되었다. 본 연구결과는 Hsp70이 정상조건에서도 소뇌신경세포의 연접에 존재함을 의미한다.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1197-1202
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• 2008

Recent studies provide some evidence that the HtrA2 protein is intimately associated with the pathogenesis of neurodegenerative disorders and that endoplasmic reticulum (ER) quality control and ER stress-associated cell death play critical roles in neuronal cell death. However, little is known about the intimate relationship between HtrA2 and ER stress-associated cellular responses. In the present study, we have demonstrated that the HtrA2 protein level was gradually and significantly increased by up to to-fold in the mitochondria under tunicamycin (Tm)-induced ER stress, which eventually promoted cell death through the release of HtrA2 into the cytoplasm. Using an ecdysone-inducible mammalian expression system, we demonstrate that the extent of cell death in 293-HtrA2 cells was approximately 20 times higher under Tm-induced ER stress, indicating that the increase in the HtrA2 protein level in the mitochondria itself is necessary but not sufficient for the promotion of cell death. Taken together, these results suggest that HtrA2 may serve as a mediator of ER stress-induced apoptosis and ER-mitochondrial cross-talk in some cellular processes.

• Journal of Plant Biotechnology
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• 제5권3호
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• pp.143-148
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MSP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to playa novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2003년도 식물바이오벤처 페스티발
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• pp.65-71
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorho-damine 123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

• Radiation Oncology Journal
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• 제14권4호
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• pp.265-279
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• 1996

방사선은 DNA손상을 초래하고 세포 성장에 관련된 유전자의 발현 조절 및 apoptosis등을 유발한다고 알려져 있으며 본 연구는 신경계에 있어 방사선 조사 후 종양 발생율과 시간 경과의 관계 및 조사 양과 암 발생의 관계를 구명하기 위해 코발트 60의 전신조사에 따른 흰쥐 뇌 조직의 생체 반응을 연구하고자 하였다. 이를 위하여 상기조직의 jun 및 p53 유전자의 발현도를 1 Gy로 부터 100 Gy 범위의 감마선 용량별 및 1시간에서 6시간까지의 조사 훈 경과 시간 별로 Northern 분석하였다. Jun유전자 발현도는 ley이하에서 1시간 이내에 한계수준에 도달하였으며 30 Gy의 조사 1시간 째에 최대였다. 또한 조사 1시간 이후 1 Gy로부터 10 Gy 범위에서는 조사 5시간 및 6시간까지 점진적으로 증가되었으나 20 Gy로부터 100 Gy 범위에서는 조사 2시간까지 증가 후 감소되는 양상을 나타냈다. p53유전자의 발현도는 1 Gy이하에서 1시간 이내에 한계 수준에 근접했고 1 Gy의 조사 후 6시간 째에 최대였다. 1 Gy로부터 40 Gy까지의 범위에서는 조사 5시간 및 6시간까지 점진적으로 증가되는 반면 50 Gy에서 100 Gy범위에서는 조사 2시간 째까지 증가 후 감소되는 양상을 보였다. 따라서 감마선 조사양이 높을수록 jun 및 p53유전자는 신속하게 최대로 발현되었고 감마선 조사양이 낮을수록 서서히 증가되었다 그러나 jun유전자와 p53유전자의 감마선 조사에 따른 발현 양상에는 상호간의 연관성을 찾을 수 없었다.