• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.245-253
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• 2014

A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.612-619
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• 2000

To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.15-22
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• 2009

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide(KCN)/0.1 mM iodoacetic acid(IAA), and membrane transport function and cell viability were evaluated by measuring $Na^+$-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in $Na^+$-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited $Na^+$-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a $Na^+$ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in $Na^+$-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers(dimethylthiourea and thiourea), and amino acids(glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase $A_2(cPLA_2)$. The ATP depletion-dependent arachidonic acid release was inhibited by $cPLA_2$ specific inhibitor $AACOCF_3$. ATP depletion-induced alterations in $Na^+$-dependent phosphate uptake and cell viability were prevented by $AACOCF_3$. Inhibition of $Na^+$-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and $cPLA_2$ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

• YAKHAK HOEJI
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• v.59 no.3
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• pp.85-91
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• 2015

Highly active antiretroviral therapy (HAART) has reduced AIDS-related morbidity and mortality; however, it has been associated with metabolic abnormalities including dyslipidemia and dysglycemia depending on the regimens used. The aims of this study were to analyze the prescription patterns of antiretroviral agents and to examine the prevalence of lipid abnormalities among the prescriptions of HAART. The electronic medical records (EMR) of HIV patients were retrospectively reviewed from January 2007 to September 2012 based on our inclusion criteria. The patients who had taken HAART for at least 3 months were included in this study. The lipid profiles of patients on antiretrovirals (ARTs) were collected from his or her laboratory data, and dyslipidemia was defined as total cholesterol (TC) ${\geq}240mg/dL$ and triglycerides (TG) >200 mg/dL. Eighty-four prescriptions were discovered during the study period. Twenty-three prescriptions were the combination of two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI). Fifty-three prescriptions were the combination of two NRTIs and one protease inhibitor (PI) and thirty-nine prescriptions of them included a PI booster. Eight prescriptions were the combination of two NRTIs and one integrase inhibitor. The Incidence of hypertriglyceridemia among the patients receiving HAART was totally about 41.7% (2NRTIs+PI regimen vs. 2NRTIs+NNRTI regimen vs. 2 NRTIs+integrase inhibitor regimen, 52% vs. 12.5% vs. 25%), but there was no incidence of hypercholesterolemia. This study investigated that the prescription medication patterns and dyslipidemia associated with lipid abnormalities among HIV patients receiving HAART. The types of HAART prescription regimens had an effect on the occurrence of hypertriglycemia. Further studies related to metabolic abnormalities and adverse effects of HIV patients on ARTs are needed in the near future.

• Journal of Sasang Constitutional Medicine
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• v.19 no.1
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• pp.160-170
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• 2007

1. Objectives This study was performed to find the activities and characteristics of purified thrombolytic enzymes from Holotrichia extracts. 2. Methods In the first time, a coarse enzyme fluid was made by using the freedried Holotrichia extracts. After manufacturing total soluble proteins and purifing enzymes, it was evauluated the activities and characteristics of this enzyme's dissolving capability to fibrin and thrombus. This study was taken using azocasein assay, fibrin-plate method, native-PAGE and fibrin zymography. 3. Results A soluble proteins were efficiently extracted form freezedried Holotrichia extracts. And, this purified enzyme had a ten times fibrinolytic capability compare with ustulation Holotrichia sample. In native PAGE and fibrin zymography, Holotrichia extracts showed the respectable fibrinolytic activity. Also, It had higher thrombolytic activities compared with general thrombolytic enzyme 'plasmin'. In experiment of various protease inhibitors of the purified enzyme from Holotrichia extracts on the azocaseinolytic activity, the enzyme was strongly inhibited by EDTA ${\cdot}$ EGTA, and weakly by APMSF ${\cdot}$ PMSF ${\cdot}$ TPCK. 4. Conclusion Holotrichia extracts has the thrombolytic activities, and it will operate directly th fibrin-clot and thrombus.

• Biomolecules & Therapeutics
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• v.22 no.4
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• pp.321-327
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• 2014

Collagen pentapeptide (Lys-Thr-Thr-Lys-Ser, KTTKS) and its palmitoylated derivative (pal-KTTKS) have received a great deal of attention as cosmeceutical ingredients for their anti-wrinkle effects. The objective of this study was to evaluate stability and permeability of KTTKS and pal-KTTKS in hairless mouse skin. In this study, a liquid chromatography-tandem mass spectrometric method was developed for the quantification of pal-KTTKS, and used for stability and permeability studies. Stability studies were performed using skin extracts and homogenates. Both KTTKS and pal-KTTKS were rapidly degraded, but pal-KTTKS was more stable than KTTKS. When protease inhibitors were added, the stability of both compounds (KTTKS and pal-KTTKS) improved significantly. In the skin permeation study, neither KTTKS nor pal-KTTKS was detected in the receptor solution, which indicates that neither compound could permeate through the full-thickness hairless mouse skin in the experimental conditions of this study. While KTTKS was not detected in any of the skin layers (the stratum corneum, epidermis, and dermis), pal-KTTKS was observed in all skin layers: $4.2{\pm}0.7{\mu}g/cm^2$ in the stratum corneum, $2.8{\pm}0.5{\mu}g/cm^2$ in the epidermis, and $0.3{\pm}0.1{\mu}g/cm^2$ in the dermis. In conclusion, this study indicated that pal-KTTKS had greater stability and permeability than that of un-modified KTTKS, and may be a useful anti-wrinkle and anti-aging cosmeceutical agent.

• Korean Journal of Food Science and Technology
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• v.34 no.1
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• pp.114-117
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• 2002

During the screening of inhibitors of caspase-3 induction in U937 human monocytic leukemia cells from natural sources, Forsythiae fructus, which showed a high level of inhibition, was selected. And then, the compound was purified from the methanol extract using silica gel column chromatography and HPLC. The inhibitor was identified as rengyolone, by spectroscophic methods of ESI-MS, $^1H-NMR$, $^{13}C-NMR$, DEPT, and HMBC. Rengyolone showed inhibitory activity of caspase-3 induction, a major protease of apoptosis cascade, with an $IC_{50}$ value of $6.25\;{\mu}g/mL$ after 7 h of treatment in U937 cells. It also showed inhibitory activity of caspace-1 induction, with an $IC_{50}$ value of $7.50\;{\mu}g/mL$ after 40 h of treatment in D10S cells. In addition, it showed protective effect against cell death with an $IC_{50}$ value of $11\;{\mu}g/mL$ on U937 cells induced by etoposide after 24 h of treatment, but did not show any cytotoxicity at the same condition without etoposide, a caspase 3 inducing agent.

• Journal of the Korean Society of Food Science and Nutrition
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• v.26 no.5
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• pp.775-783
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• 1997

Morphologically different 18 strains were isolated and examined for their abilities to inhibit ACE. Those strains were cultured in the medium containing 10% of soybean extract at 37$^{\circ}C$ for 48hr or fermented with boiled soybean at 3$0^{\circ}C$ for 60 days. The concentration of inhibitors to inhibit 50% of ACE activity, $IC_{50}$ was measured on the culture broth of each strain and also on the hot-water extract from 20, 40 and 60 day fermented Doenjang by each strain. As a result, SCB-3 which is isolated from Meju showed the highest ACE inhibitoryactivity on the cultured broth and 40 day matured Doenjang. Then, $IC_{50}$ of SCB-3 was 0.02 mg/ml and 0.26mg/ml respectively. SCB-3 was identified as a Bacillus subtilis based upon its morphological, biochemical and physiological properties. Changes in general components and ACE inhibitory activity of Doenjang fermented by SCB-3 were examined during 90 days. Total acidity of Doenjang was increased from 1.39% to 1.66% and pH was decreased from 6.02 to 5.79 after 90 days fermentation. Total sugar contents were decreased from 16.4% to 15.1% and reducing sugar contents was also decreased from 2.45% to 1.62%. Total nitrogen contents were nearly not changed, but amino nitrogen contents were drastically increased from 196mg% to 541mg%. The numbers of total microorganism were increased to 1.1$\times$$10^{8}$ cells/g after 45 days. Protease activity was increased to 622.1U/g after 75 days. The highest ACE inhibitory activity was shown in 60 day fermented Doenjang and $IC_{50}$ of the hot-water extract was 0.31mg/ml.

• Korean Journal of Food Science and Technology
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• v.35 no.4
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• pp.680-683
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• 2003

Through the screening of caspase-3 inducing inhibitors in U937 human monocytic leukemia cell from natural sources, Caesalpiniae sappan, which showed a high level of inhibition, was selected. The inhibition compound was purified from methanol extract by silica gel column chromatography and HPLC. The inhibitor was identified as brazilin by spectroscopic methods of ESI-MS, $^1H-NMR$, and $^{13}C-NMR$. Brazilin showed inhibitory activity of caspase-3 induction, a major protease of apoptosis cascade, with $IC_{50}$ value of $4.5\;{\mu}g/mL$ after 7 hr of treatment in U937 cells.

• The Journal of the Korean bone and joint tumor society
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• v.8 no.2
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• pp.48-53
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• 2002

Although direct skeletal muscle invasion by carcinoma is well recognized, distant metastasis to skeletal muscle is uncommon. Furthermore, multifocal skeletal muscle metastasis is a very exceptional event. Some factors such as variable intra-muscular blood flow, mechanical factors including turbulent blood flow and muscle contraction, intra-muscular acidic condition, lactic acid, protease inhibitors in the extra-cellular matrix were proposed as causes of the rarity of distant metastasis to skeletal muscle. We report here a case of a 67 year old male who had multifocal skeletal muscle metastasis from the transitional cell carcinoma of left kidney.