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.

Effects of estrogen and estrogenic compounds, 4-tert-octylphenol, and bisphenol A on the uterine contraction and contraction-associated proteins in rats

An, B.S.,Ahn, H.J.,Kang, H.S.,Jung, E.M.,Yang, H.,Hong, E.J.,Jeung, E.B. North-Holland 2013 Molecular and cellular endocrinology Vol.375 No.1

We examined the effects of estradiol (E2), 4-tert-octylphenol (OP), and bisphenol A (BPA) on uterine contractions in immature rats. The expression and localization of contraction-associated proteins (CAPs), and contractility of rat uterus with a collagen gel contraction assay were analyzed. E2, OP, and BPA all increased oxytocin (OT)-related pathway, while the prostaglandin-related signaling was reduced. Interestingly, E2 and estrogenic compounds showed distinct effects on the contractile activity of uterine cells. E2 enhanced the contractility, while OP and BPA significantly decreased it. Immunohistochemical analysis of CAPs showed distinct regulation of prostaglandin F receptor localization by E2 and estrogenic compounds, which may explain the different contractile activities of those reagents. In summary, we demonstrate that E2, OP, and BPA regulate CAP expression in a similar manner in the immature rat uterus, however, the effects on contractile activity were modulated differently. These findings suggest that OP and BPA interfere with uterine contractility.

Streptozotocin induces endoplasmic reticulum stress and apoptosis via disruption of calcium homeostasis in mouse pancreas

Ahn, C.,An, B.S.,Jeung, E.B. North-Holland 2015 Molecular and cellular endocrinology Vol.412 No.-

Calcium homeostasis refers to the regulation of calcium ion concentration in the body. This concentration is tightly controlled by a stabilizing system consisting of calcium channels and calcium buffering proteins. Calcium homeostasis is crucial for cell survival. Various forms of cell death (e.g., necrosis and apoptosis) also share calcium signaling pathways and molecular effectors. Calcium acts not only as a ubiquitous second messenger involved in apoptosis along with various cell death inducers but also a regulator for the synthesis of enzymes/hormones such as insulin. We hypothesized that streptozotocin disrupts calcium homeostasis and the altered intracellular calcium levels may induce cell death. After streptozotocin administration, blood glucose level was increased while insulin levels decreased. The expression of insulin response markers also decreased relative to the vehicle group. L-type voltage-gated calcium channel expression and sarcoplasmic reticulum Ca<SUP>2+</SUP> ATPase were increased by streptozotocin. Calcium buffering protein calbindin-<SUB>D</SUB>9k and calmodulin family members were also increased. The expression of genes involved in transporting calcium ions to the endoplasmic reticulum (ER) was decrease while the expression of those affecting the removal of calcium from the ER was increased. Depletion of calcium from the ER leads to ER-stress and can induce apoptosis. In the streptozotocin-treatment group, apoptosis markers were increased. Taken together, these results imply that the disruption of calcium homeostasis by streptozotocin induces ER-stress and leads to the apoptosis of pancreatic cells. Additionally, findings from this study suggest that imbalances in calcium homeostasis could promote pancreatic beta cell death and result in type I diabetes.

ERα/E2 signaling suppresses the expression of steroidogenic enzyme genes via cross-talk with orphan nuclear receptor Nur77 in the testes

Lee, S.Y.,Park, E.,Kim, S.C.,Ahn, R.S.,Ko, C.,Lee, K. North-Holland 2012 Molecular and cellular endocrinology Vol.362 No.1

Estrogen receptor alpha (ERα) has been reported to affect steroidogenesis in testicular Leydig cells, but its molecular mechanism remains unclear. Here, we investigate the effect of estrogen and ERα on Nur77, a major transcription factor that regulates the expression of steroidogenic enzyme genes. In MA-10 Leydig cells, estradiol (E2) treatment, and interestingly ERα overexpression, suppressed the cAMP-induced and Nur77-activated promoter activity of steroidogenic enzyme genes via the suppression of Nur77 transactivation. ERα physically interacted with Nur77 and inhibited its DNA binding activity. In addition, ERα/E2 signaling decreased Nur77 protein levels. Consistent with the above results, the testicular testosterone level was higher in Leydig cell-specific ERα knock-out mice (ERα<SUP>flox/flox</SUP>Cyp17iCre) than in wild-type mice (ERα<SUP>flox/flox</SUP>). Taken together, these results suggest that ERα/E2 signaling controls the Nur77-mediated expression of steroidogenic enzyme genes in Leydig cells. These findings may provide a mechanistic explanation for the local regulation of testicular steroidogenesis by estrogenic compounds and ERα.

Characterization of quality assurance properties of biogenic volatile organic compounds with an emphasis on the breakthrough behavior, recovery, and temporal stability

Ahn, J.H.,Kim, K.H.,Szulejko, J.E.,Kwon, E.E.,Deep, A. Academic Press ; Elsevier Science Ltd 2016 Microchemical journal Vol.125 No.-

In this research, we investigated the breakthrough volume (BTV) and temporal performance of two types of sorbent tube (ST) sorbent beds toward 10 (target) biogenic volatile organic compounds (BVOCs) ((1) isoprene, (2) (+)-α-pinene, (3) camphene, (4) (+)-β-pinene, (5) (+)-3-carene, (6) α-phellandrene, (7) α-terpinene, (8) ®-(+)-limonene, (9) γ-terpinene, and (10) p-cymene) and two (reference) anthropogenic volatile organic compounds (AVOCs) ((11) benzene and (12) toluene). The analysis of their vaporized liquid-phase working standards was carried out using thermal desorption-gas chromatography-mass spectrometry (TD-GC/MS). To this end, the performance of two ST types (CC (Carbopack C) and CBX (Carbopack C, Carbopack B, and Carbopack X)) was tested as a function of a few key variables, e.g., sorbent type, N<SUB>2</SUB> gas purge volume, and sampling temperature. The CBX ST gave recoveries of 100+/-10% at 60<SUP>o</SUP>C for two BVOCs (camphene and α-terpinene). However, three compounds (isoprene, (+)-α-pinene, and (+)-β-pinene) showed poor recoveries (0.7, 59.3, and 11.3%, respectively), whilst p-cymene recorded an excess recovery (~190%). In contrast, for the CC ST, BT for (+)-α-pinene and camphene increased with purge volume, while isoprene was not detected. Accordingly, the range of BTV<SUB>5%</SUB> and BTV<SUB>50%</SUB> values (L/g) for each compound with CC ST were 1.7 (toluene)-17 (camphene) and 15 (toluene)-570 ((+)-α-pinene), respectively. In summary, a three-bed CBX with the higher BTV is the preferred choice for environmental sampling for a wide range of BVOCs compared to a one-bed CC ST. The recovery of CBX ST for 10 out of 12 analytes (after >150 reconditioning/loading/TD cycles) remained constant in terms of response factor, while the response factors of isoprene and β-pinene were highly variable. Both the present work and the reported literature recoveries showed similar and divergent results which are discussed in terms of high temperature on-sorbent reactions.

CCl₄를 投與한 家兎에 있어 人蔘抽出物이 脂質代謝에 미치는 影響

安在鏞,成樂應,南宮垠,權寧韶 中央醫學社 1968 中央醫學 Vol.14 No.5

The serum lipid contents, i.e. total cholesterol, phospholipids and triglycerides were determining at various time intervals after allowing inhalation of carbon tetrachloride to control rabbits and those administered Ginseng for 8 weeks. At the same time, comparison was made between the control and Ginseng-fed animals in regard to the liver lipids determined. 1) The elevation of serum total cholesterol and triglyceride contents were less marked in Ginseng-fed than in the control. 2) Although no marked change was observed in serum phospholipid contents in the control at Ginseng-fed, a slight increase was noted in the latter. 3) The lipids in the liver tissue were generally elevated in both control and Ginseng fed, the elevation being less marked in the Ginseng-fed animals.

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>

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.

Improving Soluble Expression of β-Galactosidase in Escherichia coli by Fusion with Thioredoxin

Nam, E.S.,Jung, H.J.,Ahn, J.K. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.12

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble $\beta$-galactosidase, the gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase (KNOUC112 $\beta$-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 $\beta$-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The $\beta$-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion $\beta$ -galactosidase expressed via pThioHis C at 37$^{\circ}C$ was about 5 times higher than that of unfused $\beta$-galactosidase expressed via pET-5b at 37$^{\circ}C$. Inclusion body of $\beta$-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 $\beta$ -gal was expressed via pET-5b at 37$^{\circ}C$. Fusion $\beta$ -galactosidase expressed at 37$^{\circ}C$ via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25$^{\circ}C$ prevented the formation of inclusion body, optimally at 25$^{\circ}C$. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25$^{\circ}C$. The soluble production of Thermus thermophilus KNOUC112 $\beta$-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

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.