• Biomolecules & Therapeutics
• /
• v.28 no.6
• /
• pp.549-560
• /
• 2020

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5'-serially deleted eNOS promoters revealed that Tax-responsive element site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser¹³³ level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser¹⁹⁸¹ and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser¹⁹⁸¹, p-Akt-Ser⁴⁷³, p-CREB-Ser¹³³, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

• Journal of Yeungnam Medical Science
• /
• v.3 no.1
• /
• pp.41-48
• /
• 1986

Band 3, the predominent 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneouly upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane(ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol(1 : 1 molar ratio) were dissolved in chloroform and then chloroform was removed by rotatory evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

• Microbiology and Biotechnology Letters
• /
• v.41 no.4
• /
• pp.391-397
• /
• 2013

A putative cyclomaltodextrinase (LLCD) gene was cloned from the genome of Lactococcus lactis subsp. lactis KCTC 3769 (ATCC 19435), which encodes 584 amino acids with the predicted molecular mass of 68.7 kDa. KCTC 3769 shares approximately 40% of amino acid sequence identity with the CDase-family of enzymes. The dimeric enzyme with C-terminal six-histidines was heterologously expressed and purified from recombinant E. coli. LLCD showed the highest activity against ${\beta}$-cyclodextrin (CD) at pH 7.0 and $37^{\circ}C$. In particular, LLCD exhibited extremely low activity against starch and pullulan, while its CD-hydrolyzing activity was about 80 times higher than starch. Due to its much higher activity on CD over starch, LLCD has been identified as a member of CDases. However, LLCD can be distinguished from the other common CDases on the basis of its extremely low hydrolyzing activity against starch, pullulan, and acarbose.

• Journal of Korean Society of Environmental Engineers
• /
• v.29 no.9
• /
• pp.989-996
• /
• 2007

In this study, abiotic transformation of 1-naphthol(1-NP) via oxidative-coupling reaction and its reaction products were investigated in the presence of Mn oxides. The reaction products were characterized for their relative polarity using solvent extraction experiment and reverse-phase HPLC, and for structure using CCMS and LC/MS, and for absorption characteristics using UV-Vis spectrometry. The reaction products present in aqueous phase were more polar than parent naphthol and comprised of 1,4-naphthoquinon(1,4-NPQ) and oligomers such as dimers and trimers. Hydrophilic component present in water phase after solvent$(CH_2Cl_2)$ extractions was identified as naphthol polymerized products having molecular weight(m/z) ranging from 400 to 2,000, and showed similar UV-Vis. absorption characteristics to that of foil fulvic acid. Transformation of 1,4-NPQ, which is non-reactive to Mn oxide, to the polymerized products via cross-coupling reaction in the presence of 1-NP was also verified. In this experimental conditions(20.5 mg/L, 1-NP, 2.5 g/L $MnO_2$, pH 5), the transformation of 1-NP into the oligomers and polymerized products were about 83% of initial 1-NP concentrations, and more than 30% of the reaction products was estimated to be water insoluble fractions, not extracted by $H_2O$ methanol. Results from this study suggest that Mn oxide-mediated treatment of naphthol contaminated soils can achieve risk reduction through the formation of oligomers md polymer precipitation.

• Korean Journal of Microbiology
• /
• v.53 no.3
• /
• pp.208-215
• /
• 2017

A cyclomaltodextrinase (SPCD) gene was cloned from Streptococcus pyogenes ATCC 700294. Its open reading frame consists of 567 amino acids (66.8 kDa), which shows less than 37% of amino acid sequence identity with the other CDase-family enzymes. The homo-dimeric SPCD with C-terminal six-histidines was expressed and purified from Escherichia coli. It showed the highest activity at pH 7.5 and $45^{\circ}C$, respectively. SPCD has the broad substrate specificities against ${\beta}$-cyclodextrin, starch, and maltotriose to produce mainly maltose, whereas it hydrolyzes pullulan to panose. It can also catalyze the hydrolysis of acarbose to glucose and acarviosine-glucose. Interestingly, it showed much higher activity on ${\beta}$-cyclodextrin and acarbose than that on starch, pullulan, or maltotriose, which makes SPCD distinguished from common CDase-family enzymes. Although SPCD has significantly high acarbose-hydrolyzing activity, it showed negligible transglycosylation activity.

• Korean Chemical Engineering Research
• /
• v.55 no.3
• /
• pp.369-378
• /
• 2017

Vascular endotherial growth factor (VEGF) is a potent mitogen that stimulates vascular permeability and angiogenesis and has a potential in therapeutic applications. An industrial production method that provides high yield as well as purity is needed. Researches for various factors of mild solubilization with combination of ubiquitin fusion protein to increase solubility were carried out as well as by changing pH and denaturant concentration. Usage of pET28-a bacteral expression vector in BL21 (DE3) host cell was capable of producing approximately 14 g/L VEGF fusion protein in 20L fermentor. A purification process consisting of four chromatography steps including refolding and digestion with UBP1 resulted in mild solublization under the conditions of 2M urea and pH 10.0 due to ubiquitin fusion tag protein that increases in solubility of target protein VEGF. High yield of refolding and dimerization could be obtained between two step Ni-affinity chromatography. Multimeric and misfolded proteins and endotoxin were removed by DEAE anion exchange chromatography. Final monomers were removed from dimers by gel filtration chromatography. Characterization analysis of purified dimeric VEGF was performed using SDS-PAGE and RP-HPLC with a purity of 97%.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
• /
• 2008.11a
• /
• pp.430-430
• /
• 2008

Using multi plasma enhanced chemical vapor deposition system (Multi-PECVD), p-a-Si:H deposition layer as a $p^+$ region which was annealed by laser (Q-switched fiber laser, $\lambda$ = 1064 nm) on an n-type single crystalline Si (100) plane circle wafer was prepared as new doping method for single crystalline interdigitated back contact (IBC) solar cells. As lots of earlier studies implemented, most cases dealt with the excimer (excited dimer) laserannealing or crystallization of boron with the ultraviolet wavelength range and $10^{-9}$ sec pulse duration. In this study, the Q-switched fiber laser which has higher power, longer wavelength of infrared range ($\lambda$ = 1064 nm) and longer pulse duration of $10^{-8}$ sec than excimer laser was introduced for uniformly deposited p-a-Si:H layer to be annealed and to make sheet resistance expectable as an important process for IBC solar cell $p^+$ layer on a polished n-type Si circle wafer. A $525{\mu}m$ thick n-type Si semiconductor circle wafer of (100) plane which was dipped in a buffered hydrofluoric acid solution for 30 seconds was mounted on the Multi-PECVD system for p-a-Si:H deposition layer with the ratio of $SiH_4:H_2:B_2H_6$ = 30:120:30, at $200^{\circ}C$, 50 W power, 0.2 Torr pressure for 20 minutes. 15 mm $\times$ 15 mm size laser cut samples were annealed by fiber laser with different sets of power levels and frequencies. By comparing the results of lifetime measurement and sheet resistance relation, the laser condition set of 50 mm/s of mark speed, 160 kHz of period, 21 % of power level with continuous wave mode of scanner lens showed the features of small difference of lifetime and lowering sheet resistance than before the fiber laser treatment with not much surface damages. Diode level device was made to confirm these experimental results by measuring C-V, I-V characteristics. Uniform and expectable boron doped layer can play an important role to predict the efficiency during the fabricating process of IBC solar cells.

• Journal of the Korean Chemical Society
• /
• v.54 no.6
• /
• pp.687-695
• /
• 2010

Complexes of thiocyanato(L)cobaloximes where L is urea, acetamide, semicrabazide and formamide were synthesized and characterized. The reaction of thiocyanato (L) cobaloximes (SCNCo$(DH)_2$(L)) with benzyl (aquo) cobaloxime $PhCH_2Co(DH)_2(OH_2)$ was found to produce a series of thiocyanato bridged dicobaloximes of a general formula of $PhCH_2Co(DH)_2SCNCo(DH_2)(L)$. Evidence for formulation as dicobaloximes containing thiocyanato ligand bridges was obtained from infrared data which show $20-45cm^{-1}$ increase in vCN upon formation of the dicobaloxime from the corresponding terminal thiocyanocobaloxime (SCNCo$(DH)_2$(L)). Further characterization of these two series was done on the basis of ($^1H$,$^{13}C$)NMR, LCMS and elemental analysis. Anti-microbial activity of thiocyanato(L)cobaloximes and thiocyanato bridged dicobaloximes were screened against E. Coli. The DNA-binding behaviors of both monomers and dimers were investigated by spectroscopic methods and viscosity measurements. The results indicated that the dimer complexes bind with calf-thymus DNA in an intercalative mode via the terminal benzyl ring into the base pairs of DNA. It was observed that the monomer complexes did not interact with DNA. Fluorescence spectra for the interaction between thiocyanato bridged dicobaloximes and DNA were also studied.

• Korean journal of applied entomology
• /
• v.34 no.3
• /
• pp.181-190
• /
• 1995

Malate dehydrogenase in the mosquito ovary after a blood meal, Aedes aegypti, was purified and characterized. MDH purification steps involved DEAE-Sepharose, S-Sepharose and Cibacron blue affinity chromatography. The purified MDH was 70,000 daltons in molecular weight and was a homodimer consisting of tow identical subunits. Optimal activity of purified MDH was obtained pH 9.0-9.2 in malate-oxaloacetate reaction and pH 9.8-10.2, in oxaloactate-malate reaction. With obtained pH 9.0-92 in malate-oxaloacetate reaction and pH 9.8-10.2, in oxaloactate-malate reaction. With malate as substrate, purified mitochondrial MDH (1.28$\times$${10}^{-4}$ M) had lower Km value than cytoplasmic MDH (8.92x${10}^{-3}$ M). MDH activity was inhibited by citrate, $\alpha$-ketoglutarate, and ATP. Inhibition of MDH activity by ATP and citrate was less in malate-oxaloacetate reaction and in oxaloacetate-malate reaction. MDH activity was completely inhibited by ATP in oxaloacetate-malate reaction and not inhibited by citrate in malate-oxaloacetate reaction. Temporal activity change of MDH is similar to that of isocitrate dehydrogenase in the ovary after blood feeding; their activities in the ovary began to rise at 18 hours after a blood meal, and reached at the maximal level at 48 hours.

• Development and Reproduction
• /
• v.7 no.2
• /
• pp.105-112
• /
• 2003

Previously, we discovered a new MMP-2 isoform GA110, of which appearance in human follicular fluid(FF) and serum was increased by EDTA. The present study was conducted to investigate how GAI 10 can appear by EDTA. To examine possible involvement of protein disulfide isomerase(PDI), an enzyme responsible for the dimerization of protein via disulfide formation, effect of PDI inhibitor on the appearance of GA110 by EDTA was investigated. When PDI inhibitor added to FF before EDTA treatment, the gelatinolytic activity of GA110 was abolished in a concentration dependent manner. By contrast, the activity of 72 kDa gelatinase increased. However, the PDI inhibitor added to FF after EDTA treatment, the gelatinolytic activity of GA110 was unaffected. To find out the nature of the enzyme which converts 72 kDa gelatinase into GAI 10, chromatographic separation method of FF proteins was done. Using hydroxyapatite column, fractions rich in 72 kDa gelatinase were isolated and pooled. By using this pool as substrate for the 72 kDa converting enzyme, protein fractions containing the converting activity were obtained from chromatographic separation of FF onto glutathione sepharose fast flow column. When immunoblotting was performed on this enzymatically active protein fractions against polyclonal anti-PDI antibody, distinct immunoreactivity was observed, although appeared in smaller molecular weight region. Based on these observations, it is suggested that the appearance of GAI 10 in FF by EDTA treatment could be due to an activation of PDI-like enzyme, which dimerizes 72 kDa gelatinase into GAI 10 via the formation of disulfide bond between molecules.