• The Korean Journal of Pharmacology
• /
• v.32 no.3
• /
• pp.425-431
• /
• 1996

Human neutrophil elastases (HNElastase, EC 3.4.21.37), a causative factor of inflammatory diseases, are regulated by plasma proteinase inhibitors, alpha-proteinase inhibitor and ${\alpha}_2-macroglobulin$. Under certain pathological conditions, however, released enzymes or abnormal function of inhibitors may cause various inflammatory disease. NSAIDs have been clinically applied for treatment of inflammatory diseases. Inhibition of cyclooxygenase is a known mechanism of action of NSAIDs in the treatment of inflammatory disease. In in vitro experiments, HNElastase was inhibited by naproxen, phenylbutazone, and oxyphenbutazone, but ibuprofen, ketoprofen, aspirin, salicylic acid, and tolmetin did not inhibit elastase. HNElastase was also inhibited by chelating agents, EDTA & EGTA, and tetracyclines. Removal of divalent metal ions by EDTA caused inhibition of elastase, and reconstitution of the metal ions recovered the enzyme activity to a certain level. Frequencies and contours in the Raman spectra of various conditions of human neutrophil elastase undergo drastic changes upon partial removal and/or reconstitution of calcium and zinc ions. The metal ion content dependent activities and change of the contour of the Raman spectrogram suggest us that the mechanism of action of a chelator or chelator-like agents on neutrophil elastase may be related to the conformational change at/or near the active site, especially -C=O radical or -COOH radical.

• Clinical and Experimental Reproductive Medicine
• /
• v.34 no.1
• /
• pp.19-31
• /
• 2007

Objective: Pathogenesis of the endometriosis is very complex and the etiology is still unclear. Our hypothesis is that there may be some difference in gene expression patterns between eutopic endometriums with or without endometriosis. In this study, we analyzed the difference of gene expression profile with cDNA microarray. Methods: Endometrial tissues were gathered from patients with endometriosis or other benign gynecologic diseases. cDNA microarray technique was applied to screen the different gene expression profiles from early and late secretory phase endometria of those two groups. Each three mRNA samples isolated from early and late secretory phase of endometrial tissues of control were pooled and used as master controls and labeled with Cy3-dUTP. Then the differences of gene expression pattern were screened by comparing eutopic endometria with endometriosis, which were labeled with Cy5-dUTP. Fluorescent labeled probes were hybridized on a microarray of 4,800 human genes. Results: Twelve genes were consistently over-expressed in the endometrium of endometriosis such as ATP synthase H transporting F1 (ATP5B), eukaryotic translation elongation factor 1, isocitrate dehydrogenase 1 (NADP+), mitochondrial ribosomal protein L3, ATP synthase H+ transporting (ATP5C1) and TNF alpha factor. Eleven genes were consistently down-regulated in the endometriosis samples. Many extracellular matrix protein genes (decorin, lumican, EGF-containing fibulin-like extracellular matrix protein 1, fibulin 5, and matrix Gla protein) and protease/protease inhibitors (serine proteinase inhibitor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1), and insulin like growth factor II associated protein were included. Expression patterns of selected eight genes from the cDNA microarray were confirmed by quantitative RT-PCR or real time RT-PCR. Conclusion: The result of this analysis supports the hypothesis that the endometrium from patients with endometriosis has distinct gene expression profile from control endometrium without endometriosis.

• Archives of Pharmacal Research
• /
• v.28 no.7
• /
• pp.816-822
• /
• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1995.04a
• /
• pp.114-114
• /
• 1995

이 연구에서는 혈전증 치료에 사용되는 혈전 응해제를 국내 독사독으로부터 개발하기 위한 실험을 실시하였다. 그 내용을 요약하면 다음과 같다. 국내에 서식하는 독사인 Agkistrodon blomoffi brevicaudus, Agkistrodon caliginosus와 Agkistrodon saxatilis에서 각각 사독을 채취하여 fibrin plate 방법으로 fibrin 분해능을 조사하여 Agkistrodon blomoffi brevicaudus의 독이 분해능이 가장 우수함을 밝혔다. 이와 같은 사실에 기초하여 A. blomoffi brevicaudus의 독으로부터 p-Aminobenzamidine affinity chromatography와 DEAE ion-exchange chromatography를 이용하여 분자량이 50.8 kDa인 황성 단백질을 정제하였다. 위에서와 같은 방법으로 정제한 단백질은 fibrin 분해능이 우수하고 fibrinogen의 ${\gamma}$ chain은 분해하지 않으나 B$\beta$ chain을 $A\alpha$ chain에 비하여 보다 선택적으로 분해하는 단백분해 효소임을 증명하였다. 이 정제 효소의 Fibrin에 대한 분해능은 266$\mu\textrm{g}$/${\mu}\ell$의 농도에서 Plasmin 1.0 unit (=3.0 WHO unit)보다 높게 나타났다. 정제된 효소는 chromogenic substrate인 N-Benzoyl-Phe-Val-Arg-pNA와 N-p-Tosyl- Gly-Pro-Arg-pNA의 arginine carboxyl side를 분해하고 pH 7.5에서 최대 활성을 보이며, Vmax는 5.46 umo1/1ㆍmin이고, Km 값은 0.20mM이며, 그리고 Cu$^{2+}$, $Zn^{2+}$, soybean trypsin inhibtor에 의해 25～50% 정도, serine proteinase inhibitor인 phenylmethylsulfonyl floride에 의해 80%정도 활성이 억제되는 특성이 있음을 규명하였다.

• Development and Reproduction
• /
• v.7 no.2
• /
• pp.113-118
• /
• 2003

Follicular fluid(FF) of mammalian Graafian follicles contains various kinds of proteins and proteinases that are believed to play important roles during follicular growth oocyte maturation and ovulation of mature oocytes. Previous studies of human FF(hFF) demonstrated the presence of many serine/threonine proteinases and matrix metalloproteinases such as gelatinases, however, little is known about the caseinases. Present study was aimed to examine the presence and the property of caseinolytic enzyme in hFF. Using casein zymographic method, it was found that hFF, human adult serum and cord serum exhibited one intense 80 kDa and another weak 78 kDa bands having caseinolytic activity. When inhibitors were added to the zymographic substrate buffer, caseinolytic activity of both 80 kDa and 78 kDa proteins were inhibited by othylenediarnine tetraacetic acid(EDTA) or soybean trypsin inhibitor(SBTI), but not by E-64, phenylmethylsulfonyl fluoride(PMSF) or 1,10-phenanthroline. Thus both enzymes appear to belong to a family of trypsin-like enzyme. Addition of EDTA to the zymographic substrate buffer almost abolished the caseinolytic activity of both enzymes. However, further addition of a divalent metal ion such as CaC $l_2$, MgC $l_2$, MnC $l_2$ or ZnC $l_2$ to the same buffer fully restored the enzyme activity at 5 mM concentration despite the presence of EDTA. Based upon these observations, 80 kDa and 78 kDa caseinolytic enzymes are present in human follicular fluid and they appear to be trypsin-like enzymes of which caseinolytic activity needs the presence of $Ca^{++}$, aM $g^{++}$, M $n^{++}$ or Z $n^{++}$././././.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.26 no.1
• /
• pp.163-186
• /
• 2000

We have previously screened 150 medicinal plants on the inhibition of elastase and found a significant inhibitory effects of the extracts of Areca catechu L. on the aging and inflammation against the skin tissues. To isolate and identify the compounds having biological activity, we was further purified by each of the solvent fractions, silica gel column chromatography, preparative TLC and reversed-Phase HPLC. Peak in HPLC, which coincided with the inhibitory activity against elastase, was identified as Phenolic substance using various colorimetric methods, UV, and IR. $IC_{50}$/ values of phenolic substance purified from Areca catechu were 26.9 $\mu\textrm{g}$/$m\ell$ for porcine pancreatic elastase (PPE) and 60.8 $\mu\textrm{g}$/$m\ell$ for human neutrophil elastase (HNE). This Phenolic substance showed more potent activity than those of reference compounds, oleanolic acid (76.5 $\mu\textrm{g}$/$m\ell$ for PPE, 219.2 $\mu\textrm{g}$/$m\ell$ for HNE) and ursolic acid (31.0 $\mu\textrm{g}$/$m\ell$ for PPE, 118.6 $\mu\textrm{g}$/$m\ell$ for HNE). According to the Lineweaver-Burk Plots, the inhibition against both PPE and HNE by this phenolic substance was competitive with substrate. Phenolic substance from Areca catechu exhibited high free radical scavenging effect ($SC_{50}$/ : 6 $\mu\textrm{g}$/$m\ell$) and inhibited effectively hyaluronidase activity ($IC_{50}$/: 210 $\mu\textrm{g}$/$m\ell$). These results suggest that the Phenolic substance Purified from Areca catechu showed anti-aging effect by protecting connective tissue proteins.