• Title, Summary, Keyword: elastase

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Development of an Agar Diffusion Method to Measure Elastase Inhibition Activity Using Elastin-Congo Red

  • Jung Kyung-Hwan;Kim Hyun-Joo
    • Journal of Microbiology and Biotechnology
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    • v.16 no.8
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    • pp.1320-1324
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    • 2006
  • The pancreatic and neutrophil elastases are associated with several illnesses including lung and vascular diseases, various cancers, and pancreatitis. The development of a potent and specific inhibitor to the elastases could lead to new therapies. In this study, an agar diffusion method was modified to include a substrate-dye conjugate (Elastin-Congo red) as a substrate of elastase and an indicator of elastase inhibitory activity. The Elastin-Congo red agar plates consisted of 0.1 % Elastin-Congo red and 2.5% agar. The elastase and elastase inhibitors were simultaneously loaded into wells, ultimately resulting in halo formations in which the halo diameter decreased as the concentration of elastase inhibitor increased. The concentration of elastase inhibitor in the samples, therefore, was inversely proportional to the halo diameters. This simplified method provided an excellent correlation with the standard microplate technique, which uses a chromogenic substrate. The concentration of elastase inhibitor obtained from the culture supernatant of a recombinant elastase inhibitor produced by the yeast Pichia pastoris was easily determined. This study has established a simple modified and inexpensive agar diffusion method that is potentially useful for the identification, quantification, and screening of new elastase inhibitors.

Protective Effects of Gamipalmi-hwan on Elastase-induced Apoptosis of A549 Cells (가미팔미환(加味八味丸)의 elastase 유도성 A549 세포사멸에 대한 보호효과)

  • Oh, Ji-Seok;Park, Yang-Chun
    • The Journal of Korean Medicine
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    • v.31 no.2
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    • pp.137-148
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    • 2010
  • Objective: This study aimed to evaluate the protective effects of Gamipalmi-hwan (GPH) on elastase-induced lung cell injury. Materials and Methods: As an in vitro model of emphysema, the current study was performed to investigate potential activity of GPH in regulating injury responses of A549 human type II cell line mediated by elastase treatment. Results: GPH treatment increased the number of A549 cells which was reduced by elastase digestion. Elastin protein level, which was reduced by elastase treatment, was increased by GPH treatment. Labeling intensity with caspase 3 protein in elastase-treated cells was reduced by GPH treatment. Both Erk1/2 and Cdc2 protein levels, which were decreased by elastase treatment, were increased to a level similar to that of the normal cells. mRNA levels encoding IL-$1{\beta}$ and TNF-$\alpha$ were increased by elastase and then down-regulated by GPH. Conclusion: The present data suggest that A549 cells are subjected to inflammatory damage by elastase and can be recovered by GPH treatment. Further studies examining the protective activity of GPH in elastase-treated lung tissue would be useful for therapeutic strategies of emphysema treatment.

Screening and Extraction Condition of Anti-skin Aging Elastase Inhibitor from Medicinal Plants (각종 약용 식물로부터 피부노화 억제 관련 elastase 저해물질의 탐색 및 추출조건)

  • Kwak, Yoon-Jin;Lee, Dae-Hyoung;Kim, Na-Mi;Lee, Jong-Soo
    • Korean Journal of Medicinal Crop Science
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    • v.13 no.6
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    • pp.213-216
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    • 2005
  • To develop a new anti-skin aging cosmetics or functional foods by using elastase inhibitor, a potent elastase inhibitor was screened from various extracts of medicinal plants and its optimal extraction condition was investigated. Methanol extracts of Rubi fructus showed the highest elastase inhibitory activity of 85%. The elastase inhibitor of Rubi fructus was maximally extracted when it was treated with 80% methanol at $50^{\circ}C$ for 12hr and its elastase inhibitory activity $(IC_{50})$ was 0.52 mg.

Antibiotics; Methicillin, Cefamandole and Oxytetracycline, Can Modulate the Activity of Human Neutrophil Elastases (Methicillin, Cefamandole, Oxytetracycline에 의한 사람 호중구 Elastase의 변화)

  • Ghim, Sa-Youl;Jeong, Hye-Young;Bae, Sung-Jun;Kang, Koo-Il
    • The Korean Journal of Pharmacology
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    • v.25 no.1
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    • pp.109-113
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    • 1989
  • Human neutrophil elastase (HNE, EC 3, 4 21, 11), a major causative factor in the induction of pulmonary emphysema, were purified by two steps of liquid chromatography. Purified elastases were cross-reacted with antibody to human neutrophil elastases. Methicillin and cefamandole, which are known as inhibitors of cell wall synthesis of microorganisms, could inhibit the activity of human neutrophil elastase up to 50% with 10mM of both agents and $IC_{50}$ of methicillin was 9.8 mM. Gentamicin, one of the aminoglycosides, also inhibits human neutrophil elastases up to 60% of original activity with 10 mM of this agent and $IC_{50}$ was 9.0 mM. We could demonstrate similar effects in oxytetracycline. 10 mM of oxytetracycline inhibited 95% of human neutrophil elastase and $IC_{50}$ was 0.3 mM. Overall, oxytetracycline, cefamandole and methicillin are strong inhibitors of human neutrophil elastase, and they could be a drug of cholice for the diseases which were known as pathogenesis related to elastase. We also suggest that the mechanism of action of these antibitics are different from the mechanism of antimicrobial effects like inhibition of both cell wall synthesis and protein synthesis.

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Screening System Establishment for Potential Anti-wrinkle Agents Using Human Fibroblast Elastase (엘라스타제를 이용한 주름개선 후보물질 검색 시스템의 구축)

  • Oh, Mi-Hee;Lee, Ju-Eun;Kim, Su-Yeon;Kim, So-Young;Park, Kyoung-Chan;Yun, Hye-Young;Baek, Kwang-Jin;Kwon, Nyoun-Soo;Kim, Dong-Seok
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.35 no.1
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    • pp.19-25
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    • 2009
  • It has been reported that not only collagen but also elastin contribute to inhibit skin wrinkle formation. Ultraviolet (UV) radiation induces photo-aging on human skin. Because UV radiation increases elastase activity, it is thought that increased elastase activity could be the major reason for skin elasticity reduction and wrinkle formation by UV. In the present study to standardize elastase activity measuring system, purified elastases from porcine pancrease and human neutrophil, and cell extracts of normal human primary fibroblasts, 3T3 mouse fibroblasts, and CCD-25Sk human fibroblasts were used as various enzyme sources. Furthermore, elastase activities were compared according to concentrations of enzyme and substrate and incubation time. Phosphoramidon was used as a positive control to inhibit elastase activities of normal human primary fibroblasts and CCD-25Sk fibroblasts. However, it had no influence on the activity of porcine pancreatic elastase. Therefore, it is suggested that elastase used for testing anti-wrinkle agents should be selected carefully.

The Relationship between Human Neutrophil Elastase and Coronary Arterial Dilatation in Kawasaki Disease (가와사끼병 환아에서의 혈장 및 호중구의 Elastase 활성도와 관상동맥 증대와의 관계)

  • Shim, Jun Yong;Choi, Hee Won;Hong, Ja Hyun;Lee, Jong Kyun;Lee, Hae Yong
    • Clinical and Experimental Pediatrics
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    • v.46 no.9
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    • pp.903-908
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    • 2003
  • Purpose : Kawasaki disease is notorious for coronary arterial complication which is usually developed as a febrile disease in early childhood. Increased polymorphonucleus(PMN) cell levels in acute phases may be associated with the pathophysiology of Kawasaki disease. We studied the relationship between coronary arterial dilatation and elastase activity which was excreted from PMN cell and roles as an important factor for vasculitis. Methods : Ten patients diagnosed with Kawasaki disease in Yonsei University Medical Center were examined between November, 2001 and January, 2002. In addition, 15 patients with other febrile diseases were also examined. Echocardiography was done in patients with Kawasaki disease on the first day of admission and four weeks after the onset of the disease. At each time, venous samples were drawn and separated into plasma and leukocytes. In patients with other febrile disease, samples were drawn on admission. Elastase activities in plasma and neutrophil extracts were measured. Results : The significant increased plasma elastase activity, $6.19{\pm}0.74U/mL$, found in Kawasaki disease patients compared with the other febrile disease patients, $4.86{\pm}1.17U/mL$(P<0.05). And there was no significance between the above two diseases in terms of the elastase activity in neutrophil extracts. The relationship between initial elastase activity and the coronary arterial complication which was shown in subacute phase wasn't significant. Conclusion : Plasma elastase activity was increased in Kawasaki disease significantly, but the initial plasma elastase activity in the acute phase could not reflect the range of coronary arterial complication.

Multiple Chromosomal Integration of a Bacillus Ya-B Alkaline Elastase Gene (고초균(Bacillus) 염색체상에서 외래 유전자 Alkaline Elastase Gene의 증폭)

  • 김병문;정봉현
    • Microbiology and Biotechnology Letters
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    • v.23 no.5
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    • pp.544-549
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    • 1995
  • The alkaline elastase is an extracellular serine protease of the alkalophilic Bacillus strain Ya-B. To increase the gene copy number and the production level of the alkaline elastase Ya-B, we designed, on the B. subtilis chromosome, a gene amplification of the 10.6 kb repeating unit containing amyE, aleE (alkaline elastase Ya-B gene) and tmrB. The aleE was inserted between amyE and tmrB, and B. subtilis APT119 strain was transformed with this amyE-aleE-tmrB-junction region fragment. As a result, we succeeded in obtaining tunicamycin-resistant (Tm$^{r}$) transformants (Tf-1, Tf-2) in which the designed gene amplification of 10.6 kb occurred in chromosome. The transformants showed high productivity of $\alpha $-amylase and alkaline elastase Ya-B. The copy number of the repeating unit (amyE-aleE-tmrB) was estimated to be 25, but plasmid vector (pUC19) was not integrated. The amplified aleE of chromosome was more stable than that of plasmid in absence of antibiotics.

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Molecular Pharmacological Interaction of Phenylbutazone to Human Neutrophil Elastase

  • Kang, Koo-Il
    • The Korean Journal of Physiology and Pharmacology
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    • v.2 no.3
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    • pp.385-393
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    • 1998
  • Human neutrophil elastase (HNElastase, EC 3.4.21.37), a causative factor of inflammatory diseases, was purified by Ultrogel AcA54 gel filtration and CM-Sephadex ion exchange chromatography. HNElastase was inhibited by phenylbutazone in a concentration dependent manner up to 0.4 mM, but as the concentration increased, the inhibitory effect gradually diminished. Binding of phenylbutazone to the human neutrophil elastase caused strong Raman shifts at 200, 440, and 1194 $cm^{-1}$. The peak at 1194 $cm^{-1}$ might be evidence of the presence $of\;-N=N-{\Phi}$ radical. The core area of the elastase, according to the visual molecular model of human neutrophil elastase, was structurally stable. A deeply situated active center was at the core area surrounded by hydrophobic amino acids. Directly neighboring the active site was one positively charged atom and two atoms carrying a negative charge, which enabled the enzyme and the drug to form a strong interaction. Phenylbutazone may form a binding, similar to a key & lock system to the atoms carrying opposite charges near the active site of the enzyme molecule. Furthermore, the hydrophobicity of the surrounding amino acid near the active site seemed to enhance the binding strength of phenylbutazone. Binding of phenylbutazone near the active site may cause masking of the active site, preventing the substrate from approaching the active site and inhibiting elastase activity.

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Effects of Astragali Radix Extracts on the Elastase Activity and DPPH and NO Scavenging Activities (황기(黃芪)의 elastase 활성과 DPPH, NO 소거능에 미치는 영향)

  • Mou, Jong-Cheng;Lee, Se-Na;Kim, Myung-Gyou;Kim, Myoung-Hee;Kim, Hyung-Jun;Jo, Hak-Jun;Leem, Kang-Hyun
    • The Korea Journal of Herbology
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    • v.26 no.1
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    • pp.59-63
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    • 2011
  • Objectives : Elastic fibers are found in the skin, lungs, arteries, veins and other structures. The defects of elastic matrix aggravate hypertension which is associated with alteration in the great arteries, arteries, and arterioles. The elastase inhibitors were undergoing in clinical studies about emphysema and pulmonary hypertension. This study was designed to investigate the effect of Astragali Radix extracts (AR) on elastase activity and anti-oxidative effects. Methods : The elastase inhibitory activity and DPPH (1,1-diphenyl-2-picrylhydrazyl) and NO free radical scavenging activities of AR were measured. Results : The elastase activity was significantly inhibited by AR. The significant DPPH and NO free radical scavenging activities were observed in AR as well. Conclusion : AR showed the anti-elastase effects and anti-oxidative activities in vitro. These results suggest that AR may be a possible drug for the treatment of pulmonary emphysema and pulmonary hypertension.

Isolation and Characterization of Elastase Inhibitor from Areca catechu (빈랑으로부터 Elastase 저해물질의 분리 및 특성조사)

  • 조중제;이건국;조병기;최정도
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.26 no.1
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    • pp.163-186
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    • 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.

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