• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.798-803
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• 1999

Prolyl endopeptidase [PEP; EC 3.4.21.26], a serine protease which is known to cleave peptide bonds on the carboxy side of a proline residue, plays an important role in the degradation of proline-containing neuropeptides that have been suggested to participate in learning and memory processes. An abnormal increase in the level of PEP, which can lead to generation of $A{\beta}$, is also suggested to be involved in Alzheimer's type senile dementia. In the course of screening PEP inhibitors from Basidiomycetes, the mushroom Polyozellus multiplex exhibited a high inhibitory activity against PEP. Two active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification, using silica gel, Sephadex LH-20, and Lobar RP-18 chromatography. The chemical structures of these compounds were identified as thelephoric acid and 12-acety1-2,3,7,8-tetrahydroxy-[12H]-12-hydroxymethylbenzobis[I.2b,3.4b'] benzofuran-11-one (kynapcin-9) by spectral data including UV, IR, MS, HR-MS, $^1H-,{\;}^{13}C-$, and 2D-NMR. The $IC_{50}$ values of the thelephoric acid and kynapcin-9 were 0.157 ppm (446nM) and 0.087 ppm (212nM) and their inhibitor constants ($K_i$) were 0.73ppm ($2.09{\;}\mu\textrm{m}$) and 0.060 ppm (146 nM), respectively. Furthermore, they were non-competitive with a substrate in Dixon plots.

• Tuberculosis and Respiratory Diseases
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• v.58 no.1
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• pp.43-53
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• 2005

Emphysema is characterized by air space enlargement and alveolar destruction. The mechanism responsible for the development of emphysema was thought to be protease/antiprotease imbalance and oxidative stress. A very recent study shows that alveolar cell apoptosis causes lung destruction and emphysematous changes. Thus, this study was performed to support the evidence for the role of apoptosis in the development of emphysema by characterizing cigarette smoke extract (CSE)-induced apoptosis in A549 (type II pneumocyte) lung epithelial cells. CSE induced apoptosis at low concentration (10% or less) and both apoptosis and necrosis at high concentration (20%). Apoptosis was demonstrated by DNA fragmentation using FACScan for subG1 fraction. Discrimination between apoptosis and necrosis was done by morphologic analysis using fluorescent microscopy with Hoecst 33342/propium iodide double staing and electron microscopy. Cytochrome c release was confirmed by using immunofluorescence with monoclonal anti-cytochrome c antibody. However, CSE-induced cell death did not show the activation of caspase 3 and was not blocked by caspase inhibitors. This suggests that CSE-induced apoptosis might be caspase-independent apoptosis. CSE-induced cell death was near completely blocked by N-acetylcystein and bcl-2 overexpression protected CSE-induced cell death. This results suggests that CSE might induce apoptosis through intracellular oxidative stress. CSE also activated p53 and functional knock-out of p53 using stable overexpression of HPV-E6 protein inhibited CSE-induced cell death. The characterization of CSE-induced cell death in lung epithelial cells could support the role of lung cell apoptosis in the pathogenesis of emphysema.

• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.4
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• pp.458-464
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• 1993

In the present paper enzymatic properties of the trypsins from the four dark fleshed fish were compared with each other and thermal stabilities of the enzymes were also investigated. The trypsins from the dark fleshed fish showed their activity only in BA-p-NA substrate of the amide substrates such as BA-p-NA and SP-p-NA, and BAEE and TAME of the ester substrate such as ATEE, BAEE, BTEE, and TAME. The enzymes were strongly inhibited by the serine protease inhibitors such as antipain, leupeptin, TLCK, DFP and SBTI, and were also inhibited by such metal ions as Cu$^{2＋}$ and Hg$^{2＋}$, but fairly activated by $Mg^{2＋}$. Denaturation constants of the enzymes were 13.4$\times$10$^{-4}$ sec$^{-1}$ for anchovy trypsin, 47.18$\times$10$^{-4}$ sec$^{-1}$ for mackerel trypsin A, 34.06$\times$10$^{-4}$ sec$^{-1}$ mackerel trypsin B, 42.28$\times$10$^{-4}$ sec$^{-1}$ for yellowfin tuna trypsin and 16.6$\times$10$^{-4}$ sec$^{-1}$ for albacore trypsin at 55$^{\circ}C$. The activation energies of the trypsins at a temperature range of 3$0^{\circ}C$ to 5$0^{\circ}C$ were estimated to be 13.91 ㎉/mole for anchovy trypsin, 11.61㎉/mo1e and 8.43㎉/mole for mackerel trypsin A and for mackerel typsin B, 4.35㎉/mole for yellowfin tuna trypsin, and 3.76㎉/mole for albacore trypsin.

• Journal of Microbiology and Biotechnology
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• v.30 no.4
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• pp.571-582
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• 2020

Quorum sensing (QS)-mediated infections cause severe diseases in human beings. The control of infectious diseases by inhibiting QS using antipathogenic drugs is a promising approach as antibiotics are proving inefficient in treating these diseases. Marine fungal (Pestalotiopsis sydowiana PPR) extract was found to possess effective antipathogenic characteristics. The minimum inhibitory concentration (MIC) of the fungal extract against test pathogen Pseudomonas aeruginosa PAO1 was 1,000 ㎍/ml. Sub-MIC concentrations (250 and 500 ㎍/ml) of fungal extract reduced QS-regulated virulence phenotypes such as the production of pyocyanin, chitinase, protease, elastase, and staphylolytic activity in P. aeruginosa PAO1 by 84.15%, 73.15%, 67.37%, 62.37%, and 33.65%, respectively. Moreover, it also reduced the production of exopolysaccharides (74.99%), rhamnolipids (68.01%), and alginate (54.98%), and inhibited the biofilm formation of the bacteria by 90.54%. In silico analysis revealed that the metabolite of P. sydowiana PPR binds to the bacterial QS receptor proteins (LasR and RhlR) similar to their respective natural signaling molecules. Cyclo(-Leu-Pro) (CLP) and 4-Hydroxyphenylacetamide (4-HPA) were identified as potent bioactive compounds among the metabolites of P. sydowiana PPR using in silico approaches. The MIC values of CLP and 4-HPA against P. aeruginosa PAO1 were determined as 250 and 125 ㎍/ml, respectively. All the antivirulence assays were conducted at sub-MIC concentrations of CLP (125 ㎍/ml) and 4-HPA (62.5 ㎍/ml), which resulted in marked reduction in all the investigated virulence factors. This was further supported by gene expression studies. The findings suggest that the metabolites of P. sydowiana PPR can be employed as promising QS inhibitors that target pathogenic bacteria.

• Microbiology and Biotechnology Letters
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• v.37 no.3
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• pp.183-188
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• 2009

Seven isolates showing strong proteolytic activity, KYC 1028, 1100, 1134, 1139, 1151, 1159, and 1182, were collected. Out of them, the broth of KYC 1134 and KYC 1139 showed the high proteolytic activity measured by azocazein. To determine 16S rDNA sequences for identification, 16S rDNA of seven isolates were amplified and compared with the 16S rDNA sequences of other myxobacteria at NCBI. It is evident from the phylo-genetic tree that the isolates belong to the genus Myxococcus. Sharing high percentage similarity values with myxobacteria, the 16S rDNA sequences were involved in two species, Myxococcus macrospores and M. Fulvus. Biochemical characteristics of KYC 1134 broth, which showed the highest proteolytic activity, showed increased activity 8 times to seven days after culture, and protein production were increased gradually and stopped at five days. The broth had optimal temperature at $60^{\circ}C$ for proteolytic activity, and stability of pH was ranged from pH 5 to 10, at $50^{\circ}C$ and 60, respectively. To classify proteases being in the broth, ten inhibitors were determined and only bestatin showed 27% inhibition effect. The inhibition result demonstrates that the broth contains kinds of amino peptidases and other exopeptidases.

• Microbiology and Biotechnology Letters
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• v.35 no.4
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• pp.304-309
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• 2007

Soybean is useful source of protein, especially in Asia. But soybean needs heat inactivation or fermentation process before consumption, since it contains the toxic lectin and various protease inhibitors. Therefore, production of soybean boiling-waste liquor (SBWL) as a byproduct is inevitable. In this study, the chemical composition of SBWL and the optimization of culture conditions for Bacillus pumilus JB-1, a selected strain for functional chungkuk-jang fermentation, using SBWL were investigated. The SBWL contains 88% water, 9.5% free sugar, 1.6% crude protein, 0.3% crude fat, 0.1% crude fiber and 2.1% ash, respectively. The contents of total polyphenol, total flavonoids and free amino acid in SBWL were 55%, 76%, and 30% of those of raw soybean, respectively. Culture conditions for B. pumilus JB-1 in SBWL were optimized. The 1/10-diluted, 0.1 % of $(NH_4)_2SO_4$ added SBWL without pH adjustment and carbon source addition was cultured at $37^{\circ}C$ for 48 h with agitation (120 rpm). The 0.5% of inoculation was enough. The large scale fermentation in 5-L jar fermentor showed that the SBWL is a good resource for production of chungkuk-jang starter and functional ingredients.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9511-9515
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• 2014

Cholangiocarcinoma (CCA) is a cancer of the bile duct epithelial cells. The highest incidence rate of CCA with a poor prognosis and poor response to chemotherapy is found in Southeast Asian countries, especially in northeastern Thailand and Lao PDR. Cathepsin B is a lysosomal cysteine protease which is regulated by cysteine proteinase inhibitors such as cystatin C. Elevation of cathepsin B levels in biological fluid has been observed in patients with inflammatory diseases and many cancers. We aimed to investigate the serum cathepsin B and cystatin C levels of CCA patients to evaluate the feasibility of using cathepsin B and cystatin C as markers for the diagnosis of CCA. Fifty-six sera from CCA patients, 17 with benign biliary diseases (BBD) and 13 from controls were collected and the cathepsin B and cystatin C levels were determined. In addition, cathepsin B expression was investigated immunohistochemically for 9 matched-pairs of cancerous and adjacent tissues of CCA patients. Serum cathepsin B, but not cystatin C, was significantly higher in CCA and BBD patient groups compared to that in the control group. Consistently, all cancerous tissues strongly expressed cathepsin B while adjacent tissues were negative in 7 out of 9 cases. In contrast, serum cystatin C levels were comparable between CCA and control groups, although serum cystatin C levels in the BBD group was higher than that in the control or CCA groups. When the serum cathepsin B to cystatin C ratio was calculated, that of the CCA group was significantly higher than that of the control group, and, although statistically not significant, the ratio of CCA group showed a trend to be higher than that of the BBD group. Thus, the cathepsin B to cystatin C ratio might be used as an alternative marker for aiding diagnosis of CCA.

• Korean journal of applied entomology
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• v.53 no.4
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• pp.331-338
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• 2014

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus (CpBV). Our previous study indicated that a transient expression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. To directly demonstrate the protein function, this study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to determine its inhibitory activity against cysteine protease and to assess its physiological alteration in insect immune and development. The open reading frame of CpBV-CST1 encodes a polypeptide of 138 amino acids (${\approx}15kDa$). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was over-expressed by 0.5 mM IPTG for 4 h. In biological activity assay, the purified rCpBV-CST1 showed a significant inhibition against papain activity. It inhibited a cellular immune response of hemocyte nodule formation in the beet armyworm, Spodoptera exigua. Moreover, its oral administration retarded larval development of the diamondback moth, Plutella xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 may be applied to control insect pest populations.

• Korean Journal of Food Science and Technology
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• v.34 no.3
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• pp.498-504
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• 2002

Isoflavone (daidzein and genistein) contents, and antioxidative and fibrinolytic activities of seven commercial Korean cooking-with-rice soybeans, including Seonbikong, Chungtae, Gangnamkong, Whangtae, Geodoo, Seolitae, and Wooltalikong were investigated. Daidzein and genistein were not found in Selitae, nor was didzein in Gangnamkong and Geodoo. Total daidzein and genistein levels in Chungtae and Whangtae were 500 and 1550 mg per kg, respectively. Wooltalikong, Whangtae, and Gangnamkong had very high electron donation abilities, over 90%, but Seonbikong and Chungtae showed significantly lower activities. SOD-like activities were also the highest in Gangnamkong and Wooltalikong. Fibrinolytic activities in Seonbikong, Whangtae, and Gangnamkong were similarly strong. Fibrinolytic substances purified from protease inhibitors or activated under various pH or heat treatment conditions, were different among the soybean varieties. This study revealed that, although several cooking-with-rice soybeans were poor in isoflavones, Wooltalikong and Gangnamkong could be good sources for functional products due to their strong antioxidative activities, and heat- and acid-resistant proteolytic abilities.

• Tuberculosis and Respiratory Diseases
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• v.53 no.4
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• pp.420-430
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• 2002

Background : Excessive extracellular matrix (ECM) deposition by airway inflammation is presumed to play an important role in the pathogenesis of worsening airflow obstruction (Ed- acceptable three-word noun) seen during acute exacerbations of chronic bronchitis. Although many proteases can cleave ECM molecules, matrix metalloproteinases (MMPs) and their inhibitors are likely to be the physiologically relevant mediators of ECM degradation. Objectives ; The purpose of this study was to demonstrate that antibiotic treatment can change airway MMPs and TIMP-1 concentrations/levels by controlling airway inflammation in acute exacerbation of chronic bronchitis. Methods : We studied 40 patients, all of whom had an acute exacerbation of chronic bronchitis. The patients were treated with two different antibiotics, moxifloxacin and clarithromycin, in a double-blind manner for 7 days. Sputum samples were induced and collected before and after antibiotic therapy. We measured the sputum concentration of MMP-1,-9, TIMP-1, IL-8 and secretory leukocyte proteinase inhibitor (SLPI) in sputum supernatants by ELISA method. Results : There was no difference after antibiotic treatment in the sputum concentrations of MMP-1,-9, TIMP-1, IL-8 and SLPI between the patients treated with moxifloxacin and those treated with clarithromycin. But the sputum concentrations of TIMP-1, and SLPI, and the TIMP-1/MMP-1 ratio were significantly reduced by the antibiotic therapy. There were significant positive correlations between sputum TIMP-1 levels and IL-8 levels (p<0.01, r=0.751), and between the sputum TIMP-1/MMP-1 ratio and IL-8 levels (p<0.01, r=0.752). The sputum SLPI levels were significantly elevated by antibiotic treatment and were negatively correlated with sputum TIMP-1 levels (p<0.01, r=-0.496) and TIMP-1/MMP-1 levels (p<0.01, r=-0.456). Conclusion : The study shows that the worsening of airway inflammation in acute exacerbation of chronic bronchitis is associated with an imbalance between the concentrations/levels of TIMP-1 and MMPs. Antibiotic treatment can prevent progression of airway narrowing in acute exacerbation of chronic bronchitis by modulation of the protease and anti-protease imbalance.