Novel signaling axis for ROS generation during K-Ras-induced cellular transformation

Park, M-T,Kim, M-J,Suh, Y,Kim, R-K,Kim, H,Lim, E-J,Yoo, K-C,Lee, G-H,Kim, Y-H,Hwang, S-G,Yi, J-M,Lee, S-J Macmillan Publishers Limited 2014 CELL DEATH AND DIFFERENTIATION Vol.21 No.8

Reactive oxygen species (ROS) are well known to be involved in oncogene-mediated cellular transformation. However, the regulatory mechanisms underlying ROS generation in oncogene-transformed cells are unclear. In the present study, we found that oncogenic K-Ras induces ROS generation through activation of NADPH oxidase 1 (NOX1), which is a critical regulator for the K-Ras-induced cellular transformation. NOX1 was activated by K-Ras-dependent translocation of p47<SUP>phox</SUP>, a subunit of NOX1 to plasma membrane. Of note, PKCδ, when it was activated by PDPK1, directly bound to the SH3-N domain of p47<SUP>phox</SUP> and catalyzed the phosphorylation on Ser348 and Ser473 residues of p47<SUP>phox</SUP> C-terminal in a K-Ras-dependent manner, finally leading to its membrane translocation. Notably, oncogenic K-Ras activated all MAPKs (JNK, ERK and p38); however, only p38 was involved in p47<SUP>phox</SUP>-NOX1-dependent ROS generation and consequent transformation. Importantly, K-Ras-induced activation of p38 led to an activation of PDPK1, which then signals through PKCδ, p47<SUP>phox</SUP> and NOX1. In agreement with the mechanism, inhibition of p38, PDPK1, PKCδ, p47<SUP>phox</SUP> or NOX1 effectively blocked K-Ras-induced ROS generation, anchorage-independent colony formation and tumor formation. Taken together, our findings demonstrated that oncogenic K-Ras activates the signaling cascade p38/PDPK1/PKCδ/p47<SUP>phox</SUP>/NOX1 for ROS generation and consequent malignant cellular transformation.

DMNQ S-64 Induces Apoptosis via Caspase Activation and Cyclooxygenase-2 Inhibition in Human Nonsmall Lung Cancer Cells

LIM, E.-S.,RHEE, Y.-H.,PARK, M.-K.,SHIM, B.-S.,AHN, K.-S.,KANG, H.,YOO, H.-S.,KIM, S.-H. Wiley (Blackwell Publishing) 2007 Annals of the New York Academy of Sciences Vol.1095 No.1

<P>Shikonin has been reported to induce apoptosis and inhibit angiogenesis in vivo and in vitro. 6-(1-propoxyiminoalkyl)-5,8-dimethoxyoxy 1,4-naphtoquinone S-64 (DMNQ S-64) was synthesized as a shikonin derivative. In this article, the underlying apoptotic mechanism of DMNQ S-64 was examined. DMNQ S-64 exerted cytotoxicity against A549 lung carcinoma cells with IC(50) of 27.3 microM. Apoptotic bodies were observed in DMNQ S-64-treated A549 cells by 4'-6-diamidino-2-phenylindole (DAPI) staining assay. DMNQ S-64 also increased sub-G1 DNA portion in a concentration-dependent manner by flow cytometric analysis. Western blotting has revealed that DMNQ S-64 effectively activates the expression of caspase 8, 9, and 3, cleaves poly (ADP-ribose) polymerase, and increases the ratio of Bax/Bcl-2. Furthermore, cytochrome c was released in a concentration-dependent manner by DMNQ S-64. Similarly, DMNQ S-64 significantly increased caspase 3 activity by enzyme-linked immunosorbent assay (ELISA). It also significantly inhibited the level of prostaglandin E2 (PGE(2)) by ELISA and downregulated the expression of cyclooxygenase-2 (COX-2) in a concentration-dependent manner. Taken together, DMNQ S-64 may exhibit cytotoxicity against A549 cells via caspase activation and COX-2 inhibition.</P>

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation

Chung, C.,Yoo, G.,Kim, T.,Lee, D.,Lee, C.S.,Cha, H.R.,Park, Y.H.,Moon, J.Y.,Jung, S.S.,Kim, J.O.,Lee, J.C.,Kim, S.Y.,Park, H.S.,Park, M.,Park, D.I.,Lim, D.S.,Jang, K.W.,Lee, J.E. Academic Press 2016 Biochemical and biophysical research communication Vol.479 No.2

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance.

Double-layered Ag-Al back reflector on stainless steel substrate for a-Si:H thin film solar cells

Jung, K.H.,Yun, S.J.,Lee, S.H.,Lee, Y.J.,Lee, K.S.,Lim, J.W.,Kim, K.B.,Kim, M.,Schropp, R.E.I. North-Holland ; Elsevier Science Ltd 2016 Solar energy materials and solar cells Vol.145 No.3

An effective light trapping method for substrate-type hydrogenated amorphous silicon (a-Si:H) thin film solar cells is the use of a back reflector (BR) of high roughness, e.g., 'hot silver', which is deposited at temperatures higher than 450<SUP>o</SUP>C. In this work, textured silver-aluminum (Ag-Al) BR films were fabricated by depositing Ag on Al film at Ag-deposition temperatures (T<SUB>Ag</SUB>) ranging from 25 to 350<SUP>o</SUP>C. The surface morphology and roughness of Ag-Al films were strongly affected by T<SUB>Ag</SUB>. The Al and Ag films were formed entirely of Ag<SUB>2</SUB>Al alloy at T<SUB>Ag</SUB> of 330<SUP>o</SUP>C or higher, while the Ag-Al films maintained a double-layered structure at 290<SUP>o</SUP>C or below. Although the films did not undergo alloying at T<SUB>Ag</SUB> of 290<SUP>o</SUP>C, the Ag-Al films have a well-developed surface structure with high diffuse-reflectance, compared to Ag films deposited at the same temperature. The conversion efficiency of an a-Si:H thin film solar cell on a flexible stainless steel substrate increased from 7.63% to 8.44% as T<SUB>Ag</SUB> was increased from 25 to 290<SUP>o</SUP>C, as a result of more effective light scattering by Ag-Al BRs, producing increased short-circuit current. However, at higher T<SUB>Ag</SUB>, Ag<SUB>2</SUB>Al alloy films with sharp crystallite edges were formed, and were not appropriate as BRs. The present work clearly shows that double-layered Ag-Al films fabricated at temperatures as low as 290<SUP>o</SUP>C could be useful back reflectors for substrate-type thin film solar cells.

β-Galactosidase Gene of Thermus thermophilus KNOUC112 Isolated from Hot Springs of a Volcanic Area in New Zealand: Identification of the Bacteria, Cloning and Expression of the Gene in Escherichia coli

Nam, E.S.,Choi, J.W.,Lim, J.H.,Hwang, S.K.,Jung, H.J.,Kang, S.K.,Cho, K.K.,Choi, Y.J.,Ahn, J.K. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.11

To isolate the $\beta$-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of $\beta$-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72$^{\circ}C$, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding $\beta$-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of $\beta$-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase(KNOUC112$\beta$-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112$\beta$-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b.

새로 국내에서 밝혀진 균주(R19)와 전통적 균주를 이용하여 혈청학적으로 진단된 쯔쯔가무시병의 임상상 및 항체반응 양상

이수기,이은경,허충,임병욱,김용림,강재승,이진관,조준탁,박동철,박경현 대한감염학회 1991 감염 Vol.23 No.1

From September to December 1989, eighteen cases of tsutsugamushi disease were diagnosed by indirect immunofluorescent antibody test in Ulsan-Ulchu area. Their clinical features and patterns of antibody response to R. tsutsugamuschi were analyzed. It was most prevailing in female over 30 years old. Most of patients (16 patients) were rural inhabitants. Two patients in urban area had reaped the rice in the rice field at several days before onset of the disease. It occurred between September and November with a peak in October. Chief complaints of patients were febrile sensation with or without chills or headache. All patients had fever and chills. Headache and myalgia were common. In one patient, delirium with visual hallucination was seen. The major physical findings were skin rash (83%), eschr (78%), lymphadenopathy (11%). The skin rash appeared on whole body or on the trunk. The eschar appeared on the chest or abdomen or in the genial region. The antobody titer to R 19 that had been newly isolated strain in Korea was universally high. There often were anemia (31%), leukocytosis (23%). Microscopic hematuria and pyuria were also seen in routine urinalysis. Liver function tests showed eleveted aminotransferases and LDH. There were erythrophagocytic histiocytes in the bone marrows of six patients among the ten patients whose bone marrow had been aspirated. Chloramphenicol had been used in all patients. The mean time to alleviation of fever was 2.5 days. All patients were cured.

Effects of β-Glucan on the Release of Nitric Oxide by Macrophages Stimulated with Lipopolysaccharide

Choi, E.Y.,Lee, S.S.,Hyeon, J.Y.,Choe, S.H.,Keum, B.R.,Lim, J.M.,Park, D.C.,Choi, I.S.,Cho, K.K. Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.11

This research analyzed the effect of ${\beta}$-glucan that is expected to alleviate the production of the inflammatory mediator in macrophagocytes, which are processed by the lipopolysaccharide (LPS) of Escherichia. The incubated layer was used for a nitric oxide (NO) analysis. The DNA-binding activation of the small unit of nuclear factor-${\kappa}B$ was measured using the enzyme-linked immunosorbent assay-based kit. In the RAW264.7 cells that were vitalized by Escherichia coli (E. coli) LPS, the ${\beta}$-glucan inhibited both the combatant and rendering phases of the inducible NO synthase (iNOS)-derived NO. ${\beta}$-Glucan increased the expression of the heme oxygenase-1 (HO-1) in the cells that were stimulated by E. coli LPS, and the HO-1 activation was inhibited by the tin protoporphyrin IX (SnPP). This shows that the NO production induced by LPS is related to the inhibition effect of ${\beta}$-glucan. The phosphorylation of c-Jun N-terminal kinases (JNK) and the p38 induced by the LPS were not influenced by the ${\beta}$-glucan, and the inhibitory ${\kappa}B-{\alpha}$ ($I{\kappa}B-{\alpha}$) decomposition was not influenced either. Instead, ${\beta}$-glucan remarkably inhibited the phosphorylation of the signal transducer and activator of transcription-1 (STAT1) that was induced by the E. coli LPS. Overall, the ${\beta}$-glucan inhibited the production of NO in macrophagocytes that was vitalized by the E. coli LPS through the HO-1 induction and the STAT1 pathways inhibition in this research. As the host immune response control by ${\beta}$-glucan weakens the progress of the inflammatory disease, ${\beta}$-glucan can be used as an effective immunomodulator.

말단비대증 환자의 뇌하수체 종양조직에서 H - ras 유전자 변이의 가능성

임승길(S . K . Lim),권이현(Y . H . Kwon),정윤석(Y . S . Chung),안광진(K . J . Ahn),이은직(E . J . Lee),김경래(K . R . Kim),이현철(H . C . Lee),허갑범(K . B . Huh),김태승(T . S . Kim) 대한내과학회 1993 대한내과학회지 Vol.45 No.3

N/A Backround: Little is known about the mechanism of tumorigenesis in pituitary adenomas. An important finding in somatotroph adenomas is that a somatic mutation may convert a G protein, Gs(α) into a putative oncogene termed gsp via point mutations at two critcal sites. The ras protooncogenes are structurally related to the G-protein family and are involved in cell proliferation and differentiation. Although ras oncogene mutations have been indentified in a wide variety of human neoplasm, only one case was reported as containting single point mutation in a patient with invasive prolactinoma, In this report we used oligonucleotide-specific hybridization to screen ras mutations in 13 acromegalic tumors. Methods: Pituitary tissue samples were derived from a central portion of the paraffin embedded pituitary tumor to minimize the possibility of contamination with normal tissue. Genomic DNA was isolated and purified from tumor tissue and amplified by the standard PCR method. Amplified DNAs from each of the region of H-ras genes (12/13 and 61) were analyzed for potential ras mutations using oligonucleotide-specific hybridization as described previously. Results: Wild type radiolabelled oligoncleotides were hybridized to the amplified DNAs from the patients' tumor and to the positive specimens. They were, however, easily striped out at 68℃ by nonstringent washing procedures except control (wild type) specimens. All radiolabelled mutant oligonucleotides could be easily striped out of 13 specimens except a control mutant specimen by the same procedure. Conclusion: We could not find any H-ras mutation that might not be frequently found in acromegalic patients, and that gsp (Gsa mutation) or mutations in the PKA system-related proteins might be the main oncogene in acromegalic patients. However further efforts to find the other somatic mutations including K-ras and N-ras should be given to these patients for more precise understanding of pathogenesis and for planning of the better treatment.

Magnetoresistance of Si1−xMnx Semiconductor Thin Films Grown by Using Molecular Beam Epitaxy

T. T. Lan Anh,H. K. Lim,B. C. Lee,D. H. Kim,K. J. Baek,D. J. Kim,H. J. Kim,J. H. Kim,Y. E. Ihm 한국물리학회 2009 THE JOURNAL OF THE KOREAN PHYSICAL SOCIETY Vol.55 No.1

The magnetoresistance of Si1−xMnx (x = 0.065, 0.076, and 0.113) thin films grown by using molecular beam epitaxy has been studied. The electrical resistivities of Si0.935Mn0.065 and Si0.924Mn0.076 thin films show semiconductor behaviors. The magnetoresistance of Si0.935Mn0.065 and Si0.924Mn0.076 thin films is negative at low temperatures and gradually transits to positive as the temperature increases. The conduction is dominated by variable range hopping, and the negative magnetoresistance is attributed, in part, to spin-dependent scattering. The magnetoresistance of the Si0.887Mn0.113 thin film increases with temperature, revealing metallic characteristics at temperatures below 240 K. The magnetoresistance of the Si0.887Mn0.113 thin film is also negative at low temperatures and positive at 300 K, but the magnetoresistance has a minimum at the Curie temperature of the ferromagnetic SiMn phase. An intriguing anomalous magnetoresistance was also observed at room temperature. The magnetoresistance of Si1−xMnx (x = 0.065, 0.076, and 0.113) thin films grown by using molecular beam epitaxy has been studied. The electrical resistivities of Si0.935Mn0.065 and Si0.924Mn0.076 thin films show semiconductor behaviors. The magnetoresistance of Si0.935Mn0.065 and Si0.924Mn0.076 thin films is negative at low temperatures and gradually transits to positive as the temperature increases. The conduction is dominated by variable range hopping, and the negative magnetoresistance is attributed, in part, to spin-dependent scattering. The magnetoresistance of the Si0.887Mn0.113 thin film increases with temperature, revealing metallic characteristics at temperatures below 240 K. The magnetoresistance of the Si0.887Mn0.113 thin film is also negative at low temperatures and positive at 300 K, but the magnetoresistance has a minimum at the Curie temperature of the ferromagnetic SiMn phase. An intriguing anomalous magnetoresistance was also observed at room temperature.

Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD

Eom, G.T.,Oh, J.Y.,Park, J.H.,Lim, H.J.,Lee, S.J.,Kim, E.Y.,Choi, J.E.,Jegal, J.,Song, B.K.,Yu, J.H.,Song, J.K. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.122 No.3

<P>An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33 degrees C. To isolate a mutant TliDEF capable of secreting TliA at 35 degrees C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type WE, tliF and tliA in Escherichia con. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35 degrees C. At the growth temperature of 35 degrees C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20 degrees C, 25 degrees C and 30 degrees C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.</P>