• BMB Reports
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• v.42 no.3
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• pp.160-165
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• 2009

The temperate Staphylococcus aureus phage ${\Phi}11$ harbors cI and cro repressor genes similar to those of lambdoid phages. Using extremely pure ${\Phi}11$ Cro (the product of the ${\Phi}11$ cro gene) we demonstrated that this protein possesses a single domain structure, forms dimers in solution at micromolar concentrations and maintains a largely $\alpha$-helical structure even at $45^{\circ}C$. ${\Phi}11$ Cro was sensitive to thermolysin at temperatures ranging from $55-75^{\circ}C$ and began to aggregate at ${\sim}63^{\circ}C$, suggesting that the protein is moderately thermostable. Of the three homologous 15-bp operators (O1, O2, and O3) in the ${\Phi}11$ cI-cro intergenic region, ${\Phi}11$ Cro only binds efficiently to O3, which is located upstream of the cI gene. Our comparative analyses indicate that the DNA binding capacity, secondary structure and dimerization efficiency of thermostable ${\Phi}11$ Cro are distinct from those of P22 Cro and $\lambda$ Cro, the best characterized representatives of the two structurally different Cro families.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.10 no.3
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• pp.648-653
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• 2009

p62 is a key component of protein aggregates found in brains of neurodegenerative diseases in which oxidative stress is involved in the pathogenesis. p62 was induced in SH-SY5Y, a neuroblastoma cell line, by hydroxydoparnine or $C_2-ceramide$ known to be related to neurodegenerative diseases. The over-expression of p62 showed the neuroprotective effect against the ceramide induced cell death. In addition, p62 became insoluble and cleaved forms as time proceeded after the ceramide treatment, suggesting the mechanism by which p62 is associated with aggregates in neurodegenerative diseases.

• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.433-444
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• 1996

We describe a novel approach to evaluate quantitatively the amounts of denatured proteins in cells upon heat exposure. A thiol compound, diamide [azodicarboxylic acid bis (dimethylamide)] causes protein cross-linking with exposed sulfyhydryl residues of denatured proteins. Since denatured proteins expose normally well-hidden sulfhydryl groups, these will be preferentially cross-linked by diamide. Thus diamide acts to 'trap' denatured proteins. We observed that protein aggregates (high molecular weight protein aggregates, HMA) appeared on SDS-polyacrylamide gels run under non-reducing conditions and that the amount of HMA can be quantified by scanning the gels using a gas flow counter. Heating cells followed by a fixed dose of diamide exposure resulted in HMA increases in a heat-dose dependent manner, demonstrating that the quantitation of HMA could serve as a measure of heat-denatured proteins. We compared thermotolerant and nontolerant cells and found decreased HMA in tolerant cells upon heat treatment. As an attempt to examine the kinetics of protein renaturation (or 'repair'), we measured the amounts of aggregates formed by the addition of diamide at various times after heat shock. Such experiments demonstrate an equally rapid disappearance of HMA in previously unheated and in thermotolerant cells. Levels of HMA in tolerant cells increased significantly after electroporation of HSP70 specific mAbs, suggesting an involvement of HSP70 in reducing HMA levels in thermotolerant cells upon heat exposure. Immunoprecipitation studies using anti-HSP70 antibody indicated an association of HSP70 with heat-denatured proteins. Our results suggest that heat induces protein denaturation, and that elevated level of HSP70 present in thermotolerant cells protects them by reducing the level of protein denaturation rather than by facilitating the 'repair' (or degradation) process.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.313-318
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• 2010

Deinococcus radiodurans RecA protein, when bound to DNA, exhibits a DNA-dependent ATPase. In the absence of DNA, the rate of RecA protein-promoted ATP hydrolysis drops 1,000-fold under the physiological concentrations of salt. This DNA-independent activity can be stimulated to levels approximating those observed with DNA by adding high concentrations (approximately 1.6 M) of a wide variety of salts. This effect was characterized by varying salt concentration and comparing the effects of different ion types. The higher concentrations of salt stimulated the ATP hydrolysis by RecA protein in the absence of DNA. At 1.6 M chloride, the observed stimulation showed the following cation trend $K^+{\geq}Na^+$ > $NH_4^+$ and the following anion sequence was observed: $glutamate^- \; > \; C1^- \;> \; acetate^-\; > \;PO_4^-$ at 1.6 M $K^+$. The catalytic properties of the salt-stimulated ATP hydrolysis reaction was optimal between pH 7.0 and 8.0, which was similar to the double stran nded DNA-dependent ATPase activities of Deinococcus radiodurans RecA protein. In the absence of DNA the active species for ATP hydrolysis by RecA protein was shown to be an aggregate of three RecA protein molecules.

• BMB Reports
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• v.36 no.5
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• pp.488-492
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• 2003

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for neuronal survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of copper and peroxide in the modification of NF-L. When disassembled NF-L was incubated with copper ion and hydrogen peroxide, then the aggregation of protein was proportional to copper and hydrogen peroxide concentrations. Dityrosine crosslink formation was obtained in copper-mediated NF-L aggregates. The copper-mediated modification of NF-L was significantly inhibited by thiol antioxidants, N-acetylcysteine, glutathione, and thiourea. A thioflavin-T binding assay was performed to determine whether the copper/$H_2O_2$ system-induced in vitro aggregation of NF-L displays amyloid-like characteristics. The aggregate of NF-L displayed thioflavin T reactivity, which was reminiscent of amyloid. This study suggests that copper-mediated NF-L modification might be closely related to oxidative reactions which may play a critical role in neurodegenerative diseases.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.311-320
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• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.246-254
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• 2022

Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and anti-inflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient's synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RA-hMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.245-252
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• 2001

Erm proteins, MLS (macrolide-lincosamide-streptogramin B) resistance factor proteins, show high degree of amino acid sequence homology and comprise of a group of structurally homologous N-methyltransferases. On the basis of the recently determined structures of ErmC` and ErmAM, ErmSF was divided into two domains, N-terminal end catalytic domain and C-terminal end substrate binding domain and attempted to overexpress catalytic domain in E. coli using various pET expression systems. Three DNA fragments were used to express the catalytic domain: DNA fragment 1 encoding Met 1 through Glu 186, DNA fragment 2 encoding Arg 60 to Glu 186 and DNA fragment 3 encoding Arg 60 through Arg 240. Among the pET expression vectors used, pET 19b successfully expressed the DNA fragment 3 and pET23b succeeded in expression of DNA fragment 1 and 2. But the overexpressed catalytic domains existed as inclusion body, a insoluble aggregate. To assist the soluble expression of ErmSF catalytic domains, Coexpression of chaperone GroESL or Thioredoxin and lowering the incubation temperature to $22^{\circ}C$ were attempted, as did in the soluble expression of the whole ErmSF protein. Both strategies did not seem to be helpful. Solubilization with guanidine-HCl and renaturation with gradual removal of denaturant and partial digestion of overexpressed whole ErmSF protein (expressed to the level of 126 mg/ι culture as a soluble protein) with proteinase K, nonspecific proteinase are under way.

• Journal of Nutrition and Health
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• v.29 no.1
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• pp.9-21
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• 1996

This study was carried out to evaluate the fatty acid intakes of employees in employee feeding operations in Seoul and to provide prudent dietary guidelines with special concern on dietary fat. Four establishments were selected in large scale group and other four were selected as small scale group according to feeding numbers and food cost. Food intake was measured by substracting the leftover from the averaged portion amount. The leftover was measured by the modified aggregate selection plate waste measurement technique. The results were as follows : Employees from the large scale institution consumed more energy, protein, carbohydrate and niacin compared to those from the small scale institution(p<0.05). The mean calorie compositions of carbohydrate, protein and fat of all subjects were 66.7, 16.4 and 16.9%. The mean fat intake was 12.1g/lunch. Linoleic acid(C18:2 $\omega$6, 3.67g) was the most abundant fatty acid contained in the diet, followed by oleic acid (C18:1 $\omega$9, 3.53g) and palmitic acid(C16:0, 1.83g). The subjects consumed 5.2g polyunsaturated fatty acids(PUFA), 4.6g monounsaturated fatty acid(MUFA), 3.2g saturated fatty acid(SFA) per lunch per person. The average ratios of P/M/S and $\omega$6/$\omega$3 fatty acids were 1.6/1.5/1.0 and 8.5/1/0., respectively. the dietary $\omega$3 fatty acid status can be improved, even though the ratios found belong to the desirable range, by including $\omega$3 fatty acid rich-foods such as bean products and seafoods more frequently in the diet. Caution is needed for higher unsaturated nature of $\omega$3 series fatty acids to be prevented from peroxidation.