• Toxicological Research
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• v.8 no.2
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• pp.343-357
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• 1992

Tumor necrosis factor-${\alpha}$(TNF), a polypeptide hormone secreted primarily by activated macrophages, was originally identified on the basis of its ability to cause hemorrhagic necrosis and tumor regression in vivo. Subsequently, TNF has been shown to be an important component of the host responses to infection and cancer and may mediate the wasting syndrome known as cachexia. These systemic actions of TNF are reflected in its diverse effects on target cells in vitro. TNF initiates its diverse cellular actions by binding to specific cell surface receptors. Although TNF receptors have been identified on most of animal cells, regulation of these receptors and the mechanisms which transduce TNF receptor binding into cellular responses are not well understood. Therefore, in the present study, the mechanisms how TNF receptors are being regulated and how TNF receptor binding is being transduced into cellular responses were investigated in rat liver plasma membranes (PM) and ME-180 human cervical carcinoma cell lines. $^{125}I$-TNF bound to high ($K_d=1.51{\pm}0.35nM$)affinity receptors in rat liver PM. Solubilization of PM with 1% Triton X-100 increased both high affinity (from $0.33{\pm}0.04\;to\;1.67{\pm}0.05$ pmoles/mg protein) and low affinity (from $1.92{\pm}0.16\;to\;7.57{\pm}0.50$ pmoles/mg protein) TNF binding without affecting the affinities for TNF, suggesting the presence of a large latent pool of TNF receptors. Affinity labeling of receptors whether from PM or solubilized PM resulted in cross-linking of $^{125}I$-TNF into $M_r$ 130 kDa, 90 kDa and 66kDa complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, $^{125}I$-TNF binding to control and TNF-pretreated membranes were assayed. Specific binding was increased by pretreatment with TNF (P<0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF. As a next step, the post-receptor events induced by TNF were examined. Although the signal transduction pathways for TNF have not been delineated clearly, the actions of many other hormones are mediated by the reversible phosphorylation of specific enzymes or target proteins. The present study demonstrated that TNF induces phosphorylation of 28 kDa protein (p28). Two dimensional soidum dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) resolved the 28kDa phosphoprotein into two isoforms having pIs of 6.2 and 6.1. The pIs and relative molecular weight of p28 were consistent with those of a previously characterized mRNA cap binding protein. mRNA cap binding proteins are a class of translation initiation factors that recognize the 7-methylguanosine cap structure found on the 5' end of eukaryotic mRNAs. In vitro, these proteins are defined by their specific elution from affinity columns composed of 7-methylguanosine 5'-triphosphate($m^7$GTP)-Sepharose. Affinity purification of mRNA cap binding proteins from control and TNF treated ME-180 cells proved that TNF rapidly stimulates phosphorylation of an mRNA cap binding protein. Phosphorylation occurred in several cell types that are important in vitro models of TNF action. The mRNA cap binding protein phosphorylated in response to TNF treatment was purifice, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor-4E(eIF-4E). These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the diverse cellular actions of TNF.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.275-281
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• 2004

Our previous research has demonstrated that the bacterium, Pseudomonas sp. NFQ-l capable of utilizing quin­oline (2,3-benzopyridine) as the sole source of carbon, nitrogen, and energy was isolated and characterized [Yoon et ai. (2003) Kor. J. Biotechnol. Bioeng. 18(3):174-179]. In this study, we have found that Pseudomonas sp. NFQ-l could degrade quinoline as well as benzoate, and extended this work to characterize the catechol 1,2­dioxygenase (C1,2O) purified from the bacterium cultured in benzoate media. Initially, C1,2O has been purified by ammonium sulfate precipitation, gel permeation chromatography, and Source 15Q. After Source 15Q, puri­fication fold was increased to approximately 14.21 unit/mg. Molecular weight of C1,2O was about 33 kDa. Physicochemical characteristics (e.g., substrate specificity, Km, Vmax, pH, temperature and effect of inhibitors) of purified C1,2O were examined. C1,2O demonstrated the activity for catechol, 4-methylcatechol and 3-meth­ylcatechol as a substrate, respectively. The Km and Vmax value of C1,2O for catechol was 38.54 ${\mu}M$ and $25.10\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}.$ The optimal temperature of C1,2O was $30^{\circ}C$ and the optimal pH was approximately 8.5. Metal ions such as $Ag^+,\;Hg^+,\;Ca^{2+},\;and\;Cu^{2+}$ show the inhibitory effect on the activity of C1,2O. N-terminal amino sequence of C1,2O was analyzed as ^1TVKISQSASIQKFFEEA^{17}.$ In this work, we found that the amino acid sequence of NFQ-l showed the sequence homology of 82, 71, 59 and $53\%$ compared with C1,2O from Pseudomonas aeruginosa PA0l, Pseudomonas arvilla C-1., P. putida KT2440 and Pseudomonas sp. CA10, respectively.

• The Korean Journal of Pain
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• v.1 no.1
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• pp.64-73
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• 1988

Recent studios have shown that narcotic drags produce an unusually intense, prolonged and segmental analgesic action in man whoa injected into the spinal subarachnoid or epidural space (Wang et al, 1979; Behar et al, 1979; Cousins et al, 1979; Magora et a., 1980, Johnston and McCaughey, 1980). Since 1960, many investigators claimed that low molecular weight(LMW) dextran increased the clinical duration of lidocaine(Loder, 1960; Loder, 1962), tetracaine (Chinn and Wirjoatmadja, 1967) and bupivacaine(Kaplan et al, 1975) in man but the mechanism of the action of dextran was unclear. But Curtiss and Scurlock(1979), and Buckled and Fink(1979) claimed that LMW dextran has no effect on the duration of action of bupivacaine in animal studies. The present study was performed to evaluate the clinical efficacy of analgesia by the thoracic epidural injection of morphine and bupivacaine mixture for the relief of pain due to fractured or contused ribs, to evaluate the duration of analgesic effect by the use of the above mixture in a hypertonic solution(dextran 70 or 50% dextrose in water) and to observe the possibility of improvement in the lung function after the pain block. The complications following the pain block were also observed. The 50 single thoracic epidural injections of the mixture were divided into three groups : Group 1(n=15) served as a control group and drags used for the relief of pain were as follows(Mean$\pm$S.D.): morphine($2.13{\pm}1.64\;mg$), 0.5% bupivacaine($3.10{\pm}1.04\;ml$) and 0.9% saline($3.64{\pm}1.11\;ml$). Group 2(n=16) serves as an experimental group and drugs were as follows(Mean$\pm$S.D.): morphine($2.13{\pm}0.72\;mg$), 0.5% bupivacaine($3.06{\pm}0.77\;ml$) and dextran 70($3.75{\pm}1.29\;ml$). Group 3 (n=19) served as an experimental group and drags were as follows(Mean$\pm$S.D.) : morphine($2.42{\pm}0.51\;mg$), 0.5% bupivacaine($3.21{\pm}0.71\;ml$) and 50% dextrose in water($3.58{\pm}1.11\;ml$). The results are were follows: 1) The Dumber of patients who obtained excellent and good analgesic effects following the block were greater in the experimental Croup 2(94%) and Group 3 (90%) than theme of the control Group 1 (80%). 2) The duration of pain relief which lasted more than 3 days after the epidural block was longer in the experimental Group 2 (81%) and Group 3 (75%) than those of the control Croup 1(67%). 3) The pulmonary reserve(FVC%+FEV 1.0%) of 27 cases who were treated by the pain block between 1 and 31 drys following the chest injury was increased to about 13% than those before the block, and that of 13 cases between 32 and 82 days following the chest injury was decreased to about 4% than those before the block. 4) Of the complications following the pain block, there were 5 cased(10%) of nausea within 2 hours following the block, 4 cases(8%) of vomiting after 2 hours following the block, 10 cases(20%) of pruritus after 3~4 hours following the block, 17 cases(34%) of transient urinary retention which tasted 8 to 19 hours, 3 cases(6%) of headache within 2 hoers following the block and 2 cases(4%) of dural puncture. In conclusion, it is suggested that the clinical duration of analgesic effect produced by morphine and bupivacaine mixture can be prolonged by addition of the hypertonic solution to the mixture.

• Applied Biological Chemistry
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• v.37 no.2
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• pp.65-71
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• 1994

Chungkook-Jang was produced by fermenting Bacillus licheniformis CN-115. The changes of chemical composition, enzyme activity, and amino acids during the fermentation were investigated. The proximate composition was shown irregular fluctuation phenomenon during the fermentation, but only the moisture tended some reducing during the fermentation just after steaming. The content of amino nitrogen was increased radically after the 36 hours of fermentation and became the highest level at 18.072 mg/g at the 60 hours of it. In accordance with the fermentation of Chungkook-Jang, pH got to the 8.39 at 60 hours with increasing, protease activity was increased according to the fermentation and acid and neutral protease activity was reduced after being reached at the highest activity at 48 hours. The most suitable pH was 6.5 and temperature was $35^{\circ}C$ for dissolution-activated of protein in the process of fermentation of Chungkook-Jang. The content of water soluble protein and the content of salt soluble protein were increased at continuously according to the fermentation time of Chungkook-Jang the largest quantity. The molecular weight of water soluble protein of Chungkook-Jang fermented for 48 hours was about 19,000. The amino acids of water soluble protein just after steaming were totally 16 kinds and proline was amino acid and them was in series by glutamic acid and serine in that ordered. The amino acids salt soluble protein, just after steaming were totally 16 kinds and was the largest quantity phenylalanine, glutamic acid and aspartic acid and aspartic acid in that order.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.439-446
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• 2005

Ferritin is known to be the principle iron-storage protein in a wide variety of rganisms. The electro­phoretic mobility and immunological cross-reactivity of dog splenic ferritin were compared with those of horse, bovine, and pig splenic ferritin after isolation using heat treatment, salting out, column chromatography, and ultrafiltration. These isolation methods allowed the recovery of $\~84{\mu}g$ of the ferritin per g of spleen. The iron content in the dog ferritin was $22.7\%$, which appeared to be higher than those in the other mammalian ferritins tested. The electrophoretic mobility of the dog ferritin under nondenaturing conditions was similar to its bovine counterpart, whereas it was more identical to pig and horse ferritins on an SDS-polyacrylamide gel. The molecular weight of the dog ferritin subunit was 19.5 kDa on an SDS-polyacrylarnide gel, and the subunit was unable to bind with iron. The polyclonal anti-dog ferritin raised in rats was able to cross-react with the pig, bovine, and horse ferritins, upon Ouchterlony double immunodiffusiion. A Western blot analysis also revealed that the anti-dog ferritin, which specifically bound with the dog ferritin subunit, could also recognize the horse, bovine, and pig ferritin subunits and the maximum cross-reactivity was exhibited with the pig ferritin subunit, indicating that the dog ferritin is immunochemically more similar to the pig ferritin than its other mammalian counterparts. Accordingly, these results elucidate the biochemical and immunochemical characteristics of dog ferritin that might have a potential to be applied as an oral iron supplement to treat iron deficiency anemia.

• Applied Biological Chemistry
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• v.45 no.2
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• pp.101-107
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• 2002

The essential oils isolated from leaves, flowers, stems, and fruits of Vitex rotundifolia by steam distillation and extraction (SDE) method were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). A total of 76 components detected by GC, 42 components were identified positively by GC-MS and GC co-injection with authentic standards, and 34 components were identified tentatively by mass spectral data only. They included 16 monoterpene hydrocarbons, 30 oxygenated hydrocarbons, 10 sesquiterpene hydrocarbons, 8 oxygenated sesquiterpenes, 3 diterpenes, and 9 miscellaneous components. The major components in the oil from the leaves were ${\alpha}-pinene$ (30.25%), 1,8-cineole (19.89%), sabinene (9.56%), ${\alpha}-terpineol$ (7.94%), ${\beta}-pinene$ (5.69%), and terpinen-4-ol (2.37%), and those in the flower oil were ${\alpha}-pinene$ (25.47%), 1,8-cineole (7.69%), manoyl oxide (6.21%), ${\beta}-pinene$ (4.20%), ${\alpha}-te.pineol$ (3.76%), and sabinene (2.78%). The major components in the oil from the stems were ${\alpha}-pinene$ (13.24%), ${\alpha}-terpineol$ (10.64%), 1,8-cineole (4.40%), manoyl oxide (4.02%), ${\beta}-pinene$ (2.39%), and terpinen-4-ol (2.21%) while those in the oil from the fruits were ${\alpha}-pinene$ (20.24%), 1,8-cineole (11.47%), ${\beta}P-pinene$ (9.79%), ${\alpha}-terpineol$ (7.08%), sabinene (3.68%), and limonene (2.77%). The percentage composition of monoterpenes in the oils from the leaves and the fruits were higher than in those from the flowers and the stems, whereas the oil from the flowers and the stems were characterized by a large content of sesquiterpenes, diterpenes and other unknown high molecular weight components.

• Applied Chemistry for Engineering
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• v.5 no.4
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• pp.681-697
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• 1994

The enzymatic hydrolysate of gelatin extracted from fish skin was fractionated and recycled through the membrane reactor according to the molecular weight for the purpose of using as functional material. In addition, the enzymatic hydrolysis conditions of gelatin, enzyme stability by membrane and mechanical shear, and effect on the long-term operational stability of the recycle membrane reactor were investigated. Using the pH-drop technique, Alcalase, pronase E and collagenase were identified as the most suitable enzymes for the hydrolysis of fish skin gelatin. The optimum hydrolysis conditions in the 1st-step membrane reactor(1st-SMR) by Alcalase were enzyme concentration 0.2mg/ml, substrate-to-enzyme ratio(S/E) 50(w/w), $50^{\circ}C$, pH 8.0, reaction volume 600ml and flow rate 6.14ml/min. In the 2nd-SMR by pronase E were enzyme concentration 0.3mg/ml, S/E 33(w/w), $50^{\circ}C$, pH 8.0, reaction volume 600ml and flow rate 6.14ml/min. In the case of 3rd-SMR, enzyme concentration 0.1mg/ml, S/E 100(w/w), $37^{\circ}C$, pH 7.5, reaction volume 600ml and flow rate 10ml/min. Decreased enzyme activities by mechanical shear and membrane were 30% and 15% in the 1st-SMR, were 14% and 5% in the 2nd-SMR, and 18% and 8% in the 3rd-SMR, respectively. Under the optimum conditions, the degree of hydrolysis in the 1st, 2nd and 3rd-SMR were 3.5%(Kjeldahl method, 87%), 3.1%(77%) and 2.7%(70%), respectively. The productivity of hydrolysate in the continuous three-step membrane reactor was 430mg per enzyme(mg) for 10 times of volume replacements.

• Journal of Life Science
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• v.27 no.7
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• pp.796-804
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• 2017

This study was carried out to evaluate the antioxidant effect of methanolic extract of Houttuynia cordata (HCME) and to identify a compound having antioxidant effect. The ethyl acetate fraction of HCME showed the highest antioxidant effect in organic solvent fractions. The fraction was then separated into 12 fractions by open column chromatography. Among these fractions, the fraction 10 (Fr. 10) with the highest antioxidant activity was isolated, and its antioxidant effect was evaluated by DPPH radical scavenging activity, reducing power, TBARS, cell viability, DNA oxidation and DCF fluorescence. The Fr. 10 at a $64{\mu}g/ml$ showed 60% of inhibitory effect similar to that of vitamin C at $10{\mu}g/ml$, compared with blank group. The Fr. 10 at $64{\mu}g/ml$ showed 264% of reducing power, compared with blank group. TBARS assay showed that the Fr. 10 at $64{\mu}g/ml$ had 35.5% of inhibitory effect similar to that of vitamin E at $1,000{\mu}g/ml$, compared with blank group. The Fr. 10 above $32{\mu}g/ml$ displayed cytotoxicity. However, it was observed that the Fr. 10, above $1{\mu}g/ml$ reduced DNA damage. DCF fluorescence assay showed that the Fr. 10 inhibited oxidative stress by $H_2O_2$ in a dose dependent manner. The compound of Fr. 10 was identified to be rutin whose molecular weight is 610 by the IR and LC-MS analyses. Therefore, these results suggest that the rutin of Fr. 10 could use as a natural antioxidant for development of cosmetics and functional foods.

• Journal of Life Science
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• v.22 no.11
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• pp.1487-1492
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• 2012

Peach (Prunus persica) has been recognized as a food allergen for over 20 years. However, there is little information about cross-reactivity with other foods. The aim of this study was to research cross-reactivity of Korean cherry and hot pepper on patients allergic to peach and its stability by digestive enzyme treatment. Peach, Korean cherry, and hot pepper proteins were extracted and separated by Tricine-SDS-PAGE analysis. The protein extracts had a wide range of molecular weight, from 3 kDa to more than 26 kDa, and displayed different patterns of protein bands on Tricine-SDS-PAGE. Peach allergic patients' sera were used to detect the allergenic protein in three samples. Three peach allergic patients' sera reacted strongly with 9 kDa protein of peach, which was the expected lipid transfer protein (LTP) as the major allergen of peach and was detected with anti-LTP1 polyclonal antibody. However, the reactivity of the 23 kDa protein in Korean cherry and hot pepper protein was stronger than that of the 9 kDa protein. The stability of protein extracts on digestive enzyme treatment was examined using simulated gastric fluids (SGF) and simulated intestinal fluids (SIF), in which digestive enzyme stability is one of the characteristics of allergen potentially causing food allergy. Findings confirmed that allergenic proteins in peach, Korean cherry, and hot pepper were not completely digested by SGF and SIF treatments from results of SDS-PAGE analysis. These results confirmed that Korean cherry and hot pepper might cause cross-reactivity in peach allergic patients, and its allergenic proteins have stability against digestive enzymes.

• Journal of Life Science
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• v.24 no.3
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• pp.242-251
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• 2014

The objective of this study was to isolate a photosensitizer from Pueraria thunbergiana leaves that induces apoptosis in SK-HEP-1 cells. Column chromatography and thin layer chromatography were used to isolate active compounds from extracts of P. thunbergiana leaves. The structures of the isolated compounds were determined by 1D-NMR, 2D-NMR, and FAB-mass spectroscopy. A substance, named M4-3, was purified from the leaves of P. thunbergiana using various chromatography methods, and the absorbance of the substance was measured. The absorbance was highest at 410 nm, suggesting that the M4-3 substance was a different compound from chlorophyll a and b, which absorb at 410, 502, 533, and 607 nm. Further analyses revealed that the M4-3 compound was a $13^2$-hydoxy pheophorbide, a methyl ester with a molecular weight of 662. M4-3 was identified as a derivative compound of pheophorbide, with a structure that magnesium comes away from the porphyrin ring. The results of the analysis of the cytotoxicity of the M4-3 substance against the SK-HEP-1 cells revealed that it inhibited rates of cell growth by 40% and 80% at a concentration of 0.04 ${\mu}M$ and 0.08 ${\mu}M$, respectively. The M4-3 compound was found to be a photosensitizer for cytotoxicity because it was appeared only in light condition as examining activity in different irradiation conditions (light condition and nonlight condition) under the same concentration. Analysis of morphological changes in the cells following cell death induced by exposure to the M4-3 substance reveled representative phenomena of apoptosis (nuclear condensation, vesicle formation, and fragmentation of DNA). The induction of apoptosis was attributed to the compound's photodynamic activity.