Solubility of oxcarbazepine in eight solvents within the temperature range T=(288.15-308.15)K

Nam, K.,Ha, E.S.,Kim, J.S.,Kuk, D.H.,Ha, D.H.,Kim, M.S.,Cho, C.W.,Hwang, S.J. Academic Press 2017 The Journal of chemical thermodynamics Vol.104 No.-

In this study, the solubility of oxcarbazepine in pure methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, acetone, acetonitrile, and tetrahydrofuran was analysed across the temperature range of 288.15-308.15K under atmospheric pressure by using a solid-liquid equilibrium method. The experimental values obtained data were correlated using the modified Apelblat model at each temperature. The mole fraction solubility of oxcarbazepine in all eight pure solvents increased gradually in a temperature-dependent manner. The highest mole fraction solubility of 3.08x10<SUP>-3</SUP> at 308.15K was observed for tetrahydrofuran, followed by acetone (1.82x10<SUP>-3</SUP> at 308.15K), acetonitrile (1.22x10<SUP>-3</SUP> at 308.15K), methanol (1.11x10<SUP>-3</SUP> at 308.15K), ethanol (6.17x10<SUP>-4</SUP> at 308.15K), 1-butanol (6.17x10<SUP>-4</SUP> at 308.15K), 1-propanol (6.16x10<SUP>-4</SUP> at 308.15K), and 2-propanol (4.13x10<SUP>-4</SUP> at 308.15K). The experimental solubility in all solvents correlated well with that calculated using the modified Apelblat equation across the temperature range of (288.15-308.15)K. Therefore, the experimental solubility and correlation equations established in this study could be useful during the crystallization/purification, pre-formulation, and formulation stages of oxcarbazepine production in laboratories and related industries.

Relationship Between K_trans and K₁ with Simultaneous Versus Separate MR/PET in Rabbits with VX2 Tumors

Lee, K. H.,Kang, S. K.,Goo, J. M.,Lee, J. S.,Cheon, G. J.,Seo, S.,Hwang, E. J. INTERNATIONAL INSTITUTE OF ANTICANCER RESEARCH 2017 Anticancer research Vol.37 No.3

<P>Background/Aim: To compare the relationship between Ktrans from DCE-MRI and K1 from dynamic (NNH3)-N-13- PET, with simultaneous and separate MR/PET in the VX-2 rabbit carcinoma model. Materials and Methods: MR/PET was performed simultaneously and separately, 14 and 15 days after VX-2 tumor implantation at the paravertebral muscle. The Ktrans and K-1 values were estimated using an in-house software program. The relationships between Ktrans and K-1 were analyzed using Pearson's correlation coefficients and linear/non-linear regression function. Results: Assuming a linear relationship, Ktrans and K-1 exhibited a moderate positive correlations with both simultaneous ( r=0.54-0.57) and separate ( r=0.53-0.69) imaging. However, while the Ktrans and K-1 from separate imaging were linearly correlated, those from simultaneous imaging exhibited a non-linear relationship. The amount of change in K-1 associated with a unit increase in Ktrans varied depending on Ktrans values. Conclusion: The relationship between K-trans and K-1 may be mis-interpreted with separate MR and PET acquisition.</P>

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.

A facile method to prepare large quantities of active caspase-3 overexpressed by auto-induction in the C41(DE3) strain

Hwang, D.,Kim, S.A.,Yang, E.G.,Song, H.K.,Chung, H.S. Academic Press 2016 Protein expression and purification Vol.126 No.-

Since human Caspase-3, a member of the cysteine protease family, plays important roles not only in the apoptosis pathway as an executioner protein, but also in neurological disorders as a critical factor, biomedical researchers have been interested in the development of modulators of caspase-3 activity. Such studies require large quantities of purified active caspase-3. So far, purification of soluble caspase-3 from full-length human caspase-3 in Escherichia coli (E. coli) yields only several mg from a liter of culture media. Therefore, a number of alternative strategies to purify active caspase-3 have been described in the literature, including refolding and protein engineering. In this study, we systematically study the effects of host E. coli strains and growth conditions on purifications of active caspase-3 from full-length human caspase-3. Using a combination of conditions that include use of the C41(DE3) strain, low-temperature expression, and auto-induction that induces caspase-3 expression depending on metabolic state of the individual host cell, we are able to obtain 14-17 mg caspase-3 per liter of culture, an amount that is about 7 times larger than published results. This optimized expression and purification method for caspase-3 can be easily scaled up to facilitate the demand for active enzyme.

Relation of N-Terminal Pro-B-Type Natriuretic Peptide and Left Ventricular Diastolic Function to Exercise Tolerance in Patients With Significant Valvular Heart Disease and Normal Left Ventricular Systolic Function

Hwang, J.w.,Park, S.J.,Cho, E.J.,Kim, E.K.,Lee, G.Y.,Chang, S.A.,Choi, J.O.,Lee, S.C.,Park, S.W. Cahners Pub. Co., etc.] ; Elsevier Science Ltd 2017 The American Journal of Cardiology Vol.119 No.11

<P>An association between N-terminal prohormone brain natriuretic peptide (NT-proBNP) and exercise tolerance in patients with valvular heart disease (VHD) has been suggested; however, there are few data available regarding this relation. The aim of this study is to evaluate the correlation between exercise tolerance and NT-proBNP in patients with asymptomatic or mildly symptomatic significant VHD and normal left ventricular ejection fraction (LV EF). A total of 96 patients with asymptomatic or mildly symptomatic VHD and normal LV EF (>= 50%) underwent cardiopulmonary exercise echocardiography. NT-proBNP levels were determined at baseline and after exercise in 3 hours. Patients were divided in 2 groups based on lower (<26 ml/kg/min, n = 47) or higher (>= 26 ml/kg/min, n = 49) peak oxygen consumption (VO2) as a representation of exercise tolerance. In the 2 groups, after adjusting for age and gender, the NT-proBNP level after exercise in 3 hours, left atrial volume index before exercise, right ventricular systolic pressure before exercise, E velocity after exercise, and E/e' ratio after exercise varied significantly. In addition, peak V02 was inversely related to NT-proBNP before (r = 0.352, p <0.001) and after exercise (r = 0.351, p <0.001). The NT-proBNP level before exercise was directly related to the left atrial volume index, E/e' ratio, and right ventricular systolic pressure before and after exercise. NT-proBNP after exercise was also directly related to the same parameters. NTproBNP levels both before and after exercise were higher in the group with lower exercise tolerance. In conclusion, through the correlation among exercise tolerance, NT-proBNP, and parameters of diastolic dysfunction, we demonstrated that diastolic dysfunction and NT-proBNP could predict exercise tolerance in patients with significant VHD and normal LV EF. (C) 2017 Elsevier Inc. All rights reserved.</P>

Approximating bone ECM: Crosslinking directs individual and coupled osteoblast/osteoclast behavior

Hwang, M.P.,Subbiah, R.,Kim, I.G.,Lee, K.E.,Park, J.,Kim, S.H.,Park, K. IPC Science and Technology Press 2016 Biomaterials Vol.103 No.-

<P>Osteoblast and osteoclast communication (i.e. osteocoupling) is an intricate process, in which the biophysical profile of bone ECM is an aggregate product of their activities. While the effect of microenvironmental cues on osteoblast and osteoclast maturation has been resolved into individual variables (e.g. stiffness or topography), a single cue can be limited with regards to reflecting the full biophysical scope of natural bone ECM. Additionally, the natural modulation of bone ECM, which involves collagenous fibril and elastin crosslinking via lysyl oxidase, has yet to be reflected in current synthetic platforms. Here, we move beyond traditional substrates and use cell-derived ECM to examine individual and coupled osteoblast and osteoclast behavior on a physiological platform. Specifically, preosteoblast-derived ECM is crosslinked with genipin, a biocompatible crosslinker, to emulate physiological lysyl oxidase-mediated ECM crosslinking. We demonstrate that different concentrations of genipin yield changes to ECM density, stiffness, and roughness while retaining biocompatibility. By approximating various bone ECM profiles, we examine how individual and coupled osteoblast and osteoclast behavior are affected. Ultimately, we demonstrate an increase in osteoblast and osteoclast differentiation on compact and loose ECM, respectively, and identify ECM crosslinking density as an underlying force in osteocoupling behavior. (C) 2016 Elsevier Ltd. All rights reserved.</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.

Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II

Lee, K‐,E,Kang, H‐,Y,Lee, S‐,K,Yoo, S‐,H,Lee, J‐,C,Hwang, Y‐,H,Nam, KH,Kim, J‐,S,Park, J‐,C,Kim, J‐,W Blackwell Publishing Ltd 2011 Clinical genetics Vol.79 No.4

<P>Lee K‐E, Kang H‐Y, Lee S‐K, Yoo S‐H, Lee J‐C, Hwang Y‐H, Nam KH, Kim J‐S, Park J‐C, Kim J‐W. Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II.</P><P>The dentin sialophosphoprotein (<I>DSPP</I>) gene encodes the most abundant non‐collagenous protein in tooth dentin and DSPP protein is cleaved into several segments including the highly phosphorylated dentin phosphoprotein (DPP). Mutations in the <I>DSPP</I> gene have been solely related to non‐syndromic form of hereditary dentin defects. We recruited three Korean families with dentinogenesis imperfecta (DGI) type II and sequenced the exons and exon–intron boundaries of the <I>DSPP</I> gene based on the candidate gene approach. Direct sequencing of PCR products and allele‐specific cloning of the highly repetitive exon 5 revealed novel single base pair (bp) deletional mutations (c.2688delT and c.3560delG) introducing hydrophobic amino acids in the hydrophilic repeat domain of the DPP coding region. All affected members of the three families showed exceptionally rapid pulp chambers obliteration, even before tooth eruption. Individuals with the c.3560delG mutation showed only mild, yellowish tooth discoloration, in contrast to the affected individuals from two families with c.2688delT mutation. We believe that these results will help us to understand the molecular pathogenesis of DGI type II as well as the normal process of dentin biomineralization.</P>

Characterization of the sensor domain of QseE histidine kinase from Escherichia coli

Yeo, K.J.,Park, J.W.,Kim, E.H.,Jeon, Y.H.,Hwang, K.Y.,Cheong, H.K. Academic Press 2016 Protein expression and purification Vol.126 No.-

In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6xHis tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4-5.0, the folded protein existed in a monomeric form with a predominantly helical structure. <SUP>1</SUP>H-<SUP>15</SUP>N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE.

Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H₂O₂ stability

Ryu, K.,Hwang, S.Y.,Kim, K.H.,Kang, J.H.,Lee, E.K. Elsevier Science Publishers 2008 Journal of biotechnology Vol.133 No.1

One of the major problems of wild-type lignin peroxidase (LiP) is its inactivity at the presence of excess H<SUB>2</SUB>O<SUB>2</SUB> and high concentration of aromatic compounds. Little is known about the substrate-binding site of LiP, and functionality improvement of LiP was not actively tried by genetic engineering and directed evolution. In order to improve LiPs functionality, we performed directed evolution with a colorimetric screening method. Finally, three types of LiP mutants were screened. The catalytic efficiency of the variants toward 2,4-dichlorophenol (DCP) degradation activity and the stability against H<SUB>2</SUB>O<SUB>2</SUB> was increased over the wild type. The K<SUB>m</SUB> value of the variants toward H<SUB>2</SUB>O<SUB>2</SUB> was increased, but K<SUB>m</SUB> value toward 2,4-DCP degradation was reduced. Overall, The K<SUB>cat</SUB>/K<SUB>m</SUB> values of the mutants toward 2,4-DCP was increased ca. 4-fold, and that toward H<SUB>2</SUB>O<SUB>2</SUB> was increased ca. 89-fold. Amino acid sequence analysis indicated that the most of the mutations were located on the enzyme surface. We expect that these results coupled with recombining mutation can be successfully applied to the molecular evolution cycles for screening of LiPs and other oxidative enzymes with improved functionality and stability.