A novel chimeric promoter that is highly responsive to hypoxia and metals

Lee, J-Y,Lee, Y-S,Kim, J-M,Kim, K L,Lee, J-S,Jang, H-S,Shin, I-S,Suh, W,Jeon, E-S,Byun, J,Kim, D-K Nature Publishing Group 2006 Gene Therapy Vol.13 No.10

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 × HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS–MRE-3 × HRE (E–M–H) gave a hypoxia induction ratio of 69. The expression induced from E–M–H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E–M–H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1α, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E–M–H chimeric promoter. E–M–H was also induced by hypoxia mimetics such as Co<SUP>2+</SUP> and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E–M–H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E–M–H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E–M–H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.Gene Therapy (2006) 13, 857–868. doi:10.1038/sj.gt.3302728; published online 9 February 2006

p34 is a novel regulator of the oncogenic behavior of NEDD4-1 and PTEN

Hong, S-W,Moon, J-H,Kim, J-S,Shin, J-S,Jung, K-A,Lee, W-K,Jeong, S-Y,Hwang, J J,Lee, S-J,Suh, Y-A,Kim, I,Nam, K-Y,Han, S,Kim, J E,Kim, K-p,Hong, Y S,Lee, J-L,Lee, W-J,Choi, E K,Lee, J S,Jin, D-H,Kim, Macmillan Publishers Limited 2014 CELL DEATH AND DIFFERENTIATION Vol.21 No.1

PTEN is one of the most frequently mutated or deleted tumor suppressors in human cancers. NEDD4-1 was recently identified as the E3 ubiquitin ligase for PTEN; however, a number of important questions remain regarding the role of ubiquitination in regulating PTEN function and the mechanisms by which PTEN ubiquitination is regulated. In the present study, we demonstrated that p34, which was identified as a binding partner of NEDD4-1, controls PTEN ubiquitination by regulating NEDD4-1 protein stability. p34 interacts with the WW1 domain of NEDD4-1, an interaction that enhances NEDD4-1 stability. Expression of p34 promotes PTEN poly-ubiquitination, leading to PTEN protein degradation, whereas p34 knockdown results in PTEN mono-ubiquitination. Notably, an inverse correlation between PTEN and p34/NEDD4-1 levels was confirmed in tumor samples from colon cancer patients. Thus, p34 acts as a key regulator of the oncogenic behavior of NEDD4-1 and PTEN.

Efficient proteolytic cleavage by insertion of oligopeptide linkers and its application to production of recombinant human interleukin-6 in Escherichia coli

Lee, E.G.,Baek, J.E.,Lee, S.H.,Kim, T.W.,Choi, J.H.,Rho, M.C.,Ahn, J.O.,Lee, H.W.,Jung, J.K. IPC Science and Technology Press ; Elsevier Scienc 2009 Enzyme and microbial technology Vol.44 No.5

Efficient expression and purification of bioactive recombinant human interleukin-6 (hIL6) was successfully achieved in Escherichia coli (E. coli) by fusion of the maltose-binding protein (MBP) with hIL6 and the insertion of oligopeptide linkers. MBP/hIL6 was over-expressed in the soluble form at a concentration of approximately 2.5g/L. For hIL6 recovery, enterokinase, factor Xa, and thrombin were employed to cleavage MBP from the fusion constructs. However, undesired and non-specific cleavage fragments as well as rhIL6 were obtained following the cleavage. The introduction of oligopeptide linkers at the C-terminal end of the fusion construct could improve the efficiency and the rate of the enzymatic cleavage reaction, and the rhIL6 purification was achieved by using MBP affinity chromatography, factor Xa cleavage, and reverse-phase chromatography, resulting in an overall yield as high as 33% (equivalent to 0.27ghIL6/L) at purity over 98%. The biological activity of the purified recombinant hIL6 was demonstrated by confirming the presence of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This study suggests that the optimized peptide linker specifically designed for both fusion partner and target molecule has a great potential for efficient recombinant protein production.

Base tolerant polybenzimidazolium hydroxide membranes for solid alkaline-exchange membrane fuel cells

Lee, J.Y.,Lim, D.H.,Chae, J.E.,Choi, J.,Kim, B.H.,Lee, S.Y.,Yoon, C.W.,Nam, S.Y.,Jang, J.H.,Henkensmeier, D.,Yoo, S.J.,Kim, J.Y.,Kim, H.J.,Ham, H.C. Elsevier Scientific Pub. Co 2016 Journal of membrane science Vol.514 No.-

Poly(dibenzylated benzimidazolium) bromides (Bz-PBI-Br) were converted successfully to OH<SUP>-</SUP> ion conducting poly(dibenzylated benzimidazolium) hydroxides (Bz-PBI-hydroxides) by the treatment of KOH. The Bz-PBI-hydroxides obtained in this study showed an excellent alkali tolerance compared to previously synthesized poly(dimethylated benzimidazolium) hydroxides (Me-PBI-hydroxides). According to <SUP>1</SUP>H-NMR analysis, Me-PBI-hydroxides were decomposed during KOH treatment. In order to find out the reason, density functional theory (DFT) calculations of two benzimidazolium structures, e.g., dimethylated benzimidazolium (Me-BI<SUP>+</SUP>) and dibenzylated benzimidazolium (Bz-BI<SUP>+</SUP>), were performed. Bz-BI<SUP>+</SUP> showed lower electron affinity and OH<SUP>-</SUP>-binding energies at the C2 position of the benzimidazolium ring than Me-BI<SUP>+</SUP>. These DFT results strongly confirm that Bz-BI<SUP>+</SUP> is less vulnerable to an OH<SUP>-</SUP> attack than Me-BI<SUP>+</SUP>; this contributes to the enhanced stability and OH<SUP>-</SUP> ion conductivity of the Bz-PBI-hydroxides.

NMR study of hydrogen exchange during the B-Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

Lee, E.H.,Seo, Y.J.,Ahn, H.C.,Kang, Y.M.,Kim, H.E.,Lee, Y.M.,Choi, B.S.,Lee, J.H. North-Holland Pub ; Elsevier Science Ltd 2010 FEBS letters Vol.584 No.21

The Yaba-like disease viruses (YLDV) are members of the Yatapoxvirus family and have double-stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N-terminal Z-DNA binding domain and a C-terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZα<SUB>E3L</SUB>) bound to Z-DNA revealed that the overall structure of yabZα<SUB>E3L</SUB> and its interaction with Z-DNA are very similar to those of hZα<SUB>ADAR1</SUB>. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZα<SUB>E3L</SUB> and d(CGCGCG)<SUB>2</SUB> with a variety of protein-to-DNA molar ratios. This study revealed that yabZα<SUB>E3L</SUB> could efficiently change the B-form helix of the d(CGCGCG)<SUB>2</SUB> to left-handed Z-DNA via the active-mono B-Z transition pathway like hZα<SUB>ADAR1</SUB>1.

How do extreme wet events affect rice quality in a changing climate?

Lee, M.S.,Kang, B.M.,Lee, J.E.,Choi, W.J.,Ko, J.,Choi, J.E.,An, K.N.,Kwon, O.D.,Park, H.G.,Shin, H.R.,Lee, I.,Kim, J.K.,Kim, H.Y. Elsevier 2013 Agriculture, ecosystems & environment Vol.171 No.-

Despite the growing body of evidence that ongoing warming modifies the cereal quality of several crop species, little is known about how it interacts with other climate change drivers. Here, we examined how elevated [CO<SUB>2</SUB>], warming, and extreme wet events (EWEs) would affect the grain quality of a temperate rice (Oryza sativa L.). In 2009 and 2010, plants were grown at two levels of [CO<SUB>2</SUB>] (c. 390 and 650μll<SUP>-1</SUP>) and three levels of warming (ambient, +1.2<SUP>o</SUP>C and +2.2/2.4<SUP>o</SUP>C) over the seasons in six independent temperature gradient field chambers (three each for ambient and elevated [CO<SUB>2</SUB>]). From 20 days after heading, plants were exposed to EWE with a simulated rainfall of 30mm a day and cloud cover by shading (c. 18% of full sunlight) for 10 consecutive days. There were also control plots, which were non-treated (NT) in terms of EWE. We found that warming and EWE, both independently and in their interaction, lead to a significant reduction in the percentages of sound grains, whereas elevated [CO<SUB>2</SUB>] does not seem to do so. Not only warming and EWE, but also elevated [CO<SUB>2</SUB>] resulted in a significant increase of immature chalky grains (ICGs), which mostly consisted of milky-white chalky grains, degrading the grain quality. In addition, warming interacted positively with elevated [CO<SUB>2</SUB>] on ICGs, whereas the warming sensitivity of IGCs was moderated in EWEs: e.g., on average, the % of ICGs in ambient vs. elevated [CO<SUB>2</SUB>] were 8.5% vs. 9.4%, 10.7% vs. 18.1% and 17.8% vs. 29.8% at ambient (24.7<SUP>o</SUP>C), +1.2<SUP>o</SUP>C and +2.4<SUP>o</SUP>C, respectively, whereas they in NT vs. EWE were 1.2% vs. 16.6%, 7.7% vs. 21.1% and 19.4% vs. 28.2%. Our results tightly confirmed that elevated [CO<SUB>2</SUB>] leads to a significant reduction in rice grain protein content (PC). While warming had little effect on the PC, EWE had a significant positive effect, and the EWE-induced increase in PC was smaller in elevated (+3.3%) than in ambient [CO<SUB>2</SUB>] (+10.9%). Given the rice quality predictor of ICGs and PC, our findings imply that expected climate change and weather extremes have a great potential to degrade the quality of rice, in terms of its milling, cooking, eating, and nutritional quality, as well as market value.

Association of Extracellular Cleavage of E-Cadherin Mediated by MMP-7 with HGF-Induced in vitro Invasion in Human Stomach Cancer Cells

Lee, K.H.,Choi, E.Y.,Hyun, M.S.,Jang, B.I.,Kim, T.N.,Kim, S.W.,Song, S.K.,Kim, J.H.,Kim, J.-R. S. Karger AG 2007 European surgical research Vol.39 No.4

<P><I>Background:</I> Proteolytic shedding of the ectodomain of a variety of transmembrane proteins, including cell-to-cell adhesion molecules, has been observed in solid cancers. We have investigated whether extracellular cleavage of E-cadherin mediated by matrix metalloproteinase-7 (MMP-7) is involved in hepatocyte growth factor (HGF) induced in vitro invasion in stomach cancer cells. <I>Methods:</I> The effects of HGF on the expression of E-cadherin/β-catenin and MMP-7 at both the protein and mRNA levels were assessed in stomach cancer cells, NUGC-3 and MKN-28, and in cells in which the expression of MMP-7 was downregulated by transfection with a MMP-7 short hairpin RNA plasmid. <I>Results:</I> Treatment with HGF increased the extracellular cleavage of E-cadherin and the release of MMP-7 and reduced the level of E-cadherin in a dose- and time-dependent manner. HGF treatment repressed the phosphorylation of β-catenin in a Triton-soluble fraction, but enhanced this phosphorylation in a Triton-insoluble fraction. The association of E-cadherin with β-catenin was decreased by HGF treatment in the Triton-soluble fraction. In addition, treatment of MMP-7 short hairpin RNA transfected NUGC-3 cells with HGF resulted in no extracellular cleavage of E-cadherin and also decreased the in vitro cell invasion. <I>Conclusions:</I> These results suggest that incubation with HGF mediated the release of MMP-7, resulting in extracellular cleavage of E-cadherin from stomach cancer cells. This might be a key mechanism in HGF-induced in vitro invasion and metastasis.</P><P>Copyright © 2007 S. Karger AG, Basel</P>

Effects of Interferon-τ on the Secretion of Prostaglandins and Cyclooxygenase-2 In Vitro and Release of Progesterone In Vivo in Cows

J. E. Lee,Y. S. Lee,H. J. Yoo,K. J. Lee,J. J. Park,B. K. Yang,H. T. Cheong,C. K. Park 한국동물번식학회 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s

In ruminants, Interferon-τ (IFN-τ) has the role of recognizing pregnancy signals produced by the embryo and it may have an important role during the luteolysis. Therefore, the purpose of the present study was to investigate the effect of IFN-τ on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and secretion of progesterone (P4) in vivo. The epithelial and stromal cells isolated from bovine endometrium were cultured with different doses of IFN-τ (0, 0.02, 0.2 and 2 μg/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin E2 and F2α levels in the culture media were analyzed by enzyme immunoassays, and total RNA was extracted from the cells for RT-PCR. P4 concentrations in blood samples were assayed by chemiluminescent immunoassay system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN-τ (p<0.05), but it was not significantly different in all groups of stromal cells except 2 μg/ml IFN-τ group (p<0.05). Although IFN-τ did not affect PGE2 and PGF2α production in epithelial cells, it decreased PGE2 and PGF2α production significantly in stromal cells (p<0.05). In vivo experiment, the P4 concentrations in blood sample was significantly increased after injection of 1 μg/ml IFN-τ. These results indicate that PG production was mediated by COX-2 expression in the stromal cells but it did not affect in the epithelial cells, and suggest that treatment of IFN-τ was to improve the implantation environment of uterine by maintenance of high P4 concentration. * This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ907008)” Rural Development Administration, Republic of Korea.

Rotational viscosity calculation method for liquid crystal mixture using molecular dynamics

Kim, J.S.,Jamil, M.,Jung, J.E.,Jang, J.E.,Lee, J.W.,Ahmad, F.,Woo, M.K.,Kwak, J.Y.,Jeon, Y.J. The Korean Infomation Display Society 2011 Journal of information display Vol.12 No.3

This paper presents the directly obtained rotational viscosity values of E7, which includes pentylcyanobiphenol, heptylcyanobiphenol, 4-cyano-4'-n-octyloxy-1,1'-biphenyl, and 4-cyano-4"-n-pentyl-1,1',1"-terphenyl, at various tempe using molecular dynamics computer simulation. The director mean squared displacement was achieved from the squared displacement of the mean director using the concept of the mean director of various nematic liquid crystals. The calculated values were compared with the experiment results that predicted a good agreement. Additional points that must be considered for further study are also discussed.

EZH2 Generates a Methyl Degron that Is Recognized by the DCAF1/DDB1/CUL4 E3 Ubiquitin Ligase Complex

Lee, J.,Lee, Jason S.,Kim, H.,Kim, K.,Park, H.,Kim, J.Y.,Lee, S.,Kim, I.,Kim, J.,Lee, M.,Chung, C.,Seo, S.B.,Yoon, J.B.,Ko, E.,Noh, D.Y.,Kim, K.,Kim, K.,Baek, S. Cell Press 2012 Molecular cell Vol.48 No.4

Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing ''methyl degron''; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.