• Title, Summary, Keyword: zymography

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Screening of Mushrooms Having Fibrinolytic Activity (혈전용해능을 갖는 버섯류의 탐색)

  • Choi, Nack-Shick;Seo, Sung-Yum;Kim, Seung-Ho
    • Korean Journal of Food Science and Technology
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    • v.31 no.2
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    • pp.553-557
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    • 1999
  • Five fungi (mushrooms), Daedaleopsis styracina, Trichaptum abietium, Coriolus versicolor, Pisolithus tinctorius and Tricholomopsis decora, were screened and examined the fibrinolytic activity and specificity. The extracts of mushrooms showed a level of fibrinolytic activity that was about 3-4 times higher than that of plasmin 1.0 unit. In particular, Pisolithus tinctorius of them showed the greatest enzyme activity (4.71 plasmin unit/mL) by fibrin plate assay, and the highest specificity (1.32 plasmin unit/mL) using chromogenic substrate (N-p-Tosyl-Gly-Pro-Lys p-nitroanilide) by Tricholomopsis decora. And the same molecular mass 54 and 61kDa showing the fibrinolytic activity obtained from all fruiting bodies were confirmed, and it was found that Trichaptum abietium and Tricholomopsis decora have a strong fibrinolytic enzyme with an apparent size of 100 kDa and 84 kDa, respectively on SDS-fibrin zymography activity assay.

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A study on separation and characterization of matrix metalloproteinase-9 inhibitors from natural plants (천연 식물 추출물에서 Matrix Metalloproteinase-9 활성 억제제의 분리 및 특성화에 관한 연구)

  • Hur, Yong-Chul;Park, Sung-Woo;Kim, Tai-Jin
    • Analytical Science and Technology
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    • v.18 no.3
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    • pp.188-193
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    • 2005
  • Three different oriental natural plants (Angelicae Dahuricae Radix, Sanguisorba Officinalis L, Euonymus alatus) were extracted with 70% methanol under refluxing for 4 hr in order to investigate their inhibitory effect on Matrix Metalloproteinase-9 (MMP-9) by a modified gelatin zymography, where only euonymus alatus showed the inhibitory effect on the activity of Matrix MMP-9. The fraction was collected by using the mixture of ethyl acetate and hexane on silica gel column. Seven portions were obtained and three fractions of them (first, third and forth) showed inhibitory effect on the zymography. To verify the effect of these substances on cells, human hepatoma, Hep3B cells as a cancer model, and Chang liver cells as a normal model were selected. In order to examine the cell viability, $1{\mu}g/mL$ of each extract was treated on cells. Most of the methanol soluble fractions showed negligible toxicity on human liver cell line.

The Effect of Sodium Chloride on the Serine-type Fibrinolytic Enzymes and the Thermostability of Extracellular Protease from Bacillus amyloliquefaciens DJ-4

  • Choi, Nack-Shick;Kim, Seung-Ho
    • BMB Reports
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    • v.34 no.2
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    • pp.134-138
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    • 2001
  • By adding sodium chloride (2.5%) into a Bacillus amyloliquefaciens DJ-4 culture broth, two serine-type fibrinolytic proteases with a molecular weight of 29 (subtilisin DJ-4) and 38-kDa were stimulated on the SDS-fibrin zymogram or inhibitor gels. B. amyloliquefaciens DJ-4 showed the highest proteolytic activity (5.52 plasmin NIH unit/ml) on the fibrin plate based on the molar ratio when cells were subjected to the 2.5% NaCl. Using a fibrin plate, the secreted protease from this strain in the presence of 5% NaCl showed that about 49% of the enzyme's activity remained after incubation at $60^{\circ}C$ for 30 min, but as the salt concentration was increased (10% NaCl) the activity nearly disappeared (0.14 plasmin NIH unit/ml). However, through a fibrin zymography assay, three fibrinolytic enzymes (38, 53 and 80-kDa) from the cells in the presence of 10% NaCl were detected. Also, two salt-activated serine-type fibrinolytic professes (29 and 38kDa) showed thermostability from 65 to $70^{\circ}C$ for 30 min. Furthermore, these professes also showed stability, pH 6-11. In particular, 29-kDa (subtilisin DJ-4) was very stable in the pH range of 4-11 at $4^{\circ}C$ for 48 h.

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Miniscale Identification and Characterization of Subtilisins from Bacillus sp. Strains

  • CHOI NACK-SHICK;JU SUNG-KYU;LEE TAE YOUNG;YOON KAB-SEOG;CHANG KYU-TAE;MAENG PIL JAE;KIM SEUNG-HO
    • Journal of Microbiology and Biotechnology
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    • v.15 no.3
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    • pp.537-543
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    • 2005
  • Subtilisin (EC 3.4.21.14) is the major extracellular alkaline serine protease of Bacillus species. Previously, we found that subtilisins did not migrate in the electrophoretic field in the Laemmili buffer system due to their high pI values (over 8.8); however, it formed a 'binding mode' at the top of the separating gel [5]. Utilizing this characteristic, four subtilisins from Bacillus sp. strains (e.g., B. subtilis 168, B. subtilis KCTC 1021, B. amyloliquefaciens KCTC 3002, and Bacillus sp. DJ-1 and DJ-4) were easily and quickly identified by an over-running electrophoretic technique with a miniscale culture supernatant (less than 20 ml) without any column chromatographic steps. Two subtilisins (DJ-l and a recombinant version) from Bacillus sp. DJ-l were characterized, and the enzymatic properties were determined by SDS-fibrin zymography and densitometric analysis. Based on this observation, the recombinant pro-subtilisin DJ-l showed the same 'binding mode,' similar to native subtilisin DJ-l. On the other hand, mature subtilisin DJ -1 without pro-peptide showed no enzymatic activity.

Cytochalasin D-induced Matrix Metalloproteinase-2 Regulates Articular Chondrocytes Dedifferentiation

  • Choi, In-Kyu;Yu, Seon-Mi;Kim, Song-Ja
    • Biomedical Science Letters
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    • v.14 no.3
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    • pp.179-186
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    • 2008
  • Matrix metalloproteinases (MMPs), also designated matrixins, hydrolyze components of the extracellular matrix. These proteinases playa central role in many biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration. In previous data, disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, cyclooxygenase (COX)-2 expression, and prostaglandin $E_2$ production in chondrocytes cultured on plastic or during cartilage explants culture. In this study, we investigated the effects of the actin cytoskeleton architecture on MMP-2 expression and dedifferentiation by CD in rabbit articular chondrocytes. Rabbit articular chondrocytes were prepared from cartilage slices of 2-weeks-old New Zealand white rabbits by enzymatic digestion. CD was used as a disruptor of actin cytoskeleton. In this experiments measuring CD dose response, primary chondrocytes were treated with various concentrations of CD for 24h. The actin disruption was determined by immunostaining. MMP-2 expression levels were determined by immunoblot analysis and Reverse transcriptase-Polymerase chain reaction (RT-PCR) and MMP-2 activity was determined by gelatin zymography. We found that cell morphological change and up-regulation of MMP-2 expression by CD as determined via immunostaining, gelatin zymography and immunoblotting. Moreover, CD induced MMP-2 transcription was detected by RT-PCR. Also, CD-induced type II collagen expression was inhibited by MMP-2 inhibitor I treatment. Our results indicate that CD up-regulated MMP-2 activation causes dedifferentiation of articular chondrocyte.

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Momordica cochinchinensis Seed Extracts Suppress Migration and Invasion of Human Breast Cancer ZR-75-30 Cells Via Down-regulating MMP-2 and MMP-9

  • Zheng, Lei;Zhang, Yan-Min;Zhan, Ying-Zhuan;Liu, Chang-Xiao
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.3
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    • pp.1105-1110
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    • 2014
  • Objective: Metastases and invasion are the main reasons for oncotherapy failure. Momordica cochinchinensis (Mu Bie Zi in Chinese) had been used for a variety of purposes, and shown anti-cancer action. In this article, we focused on effects on regulation of breast cancer cell ZR-75-30 metastases and invasion by extracts of Momordica cochinchinensis seeds (ESMCs). Methods: Effect of ESMCs on ZR-75-30 human breast cancer cells proliferation were evaluated by MTT assay and on invasion and migration by wound-healing and matrigel invasion chamber assays. Expression and protease activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blotting and gelatin zymography, respectively. Results: ESMC revealed strong growth inhibitory effects on ZR-75-30 cells, and effectively inhibited ZR-75-30 cell invasion in a dose-dependent manner. Western blot and gelatin zymography analysis showed that ESMC significantly inhibited the expression and secretion of MMP-2 and MMP-9 in ZR-75-30 cells. Conclusions: ESMC has the potential to suppress the migration and invasion of ZR-75-30 cancer cells, and it might prove to of interest in the development of novel inhibitors for breast cancer.

Steroidal Saponins from Paris polyphylla Suppress Adhesion, Migration and Invasion of Human Lung Cancer A549 Cells Via Down-Regulating MMP-2 and MMP-9

  • He, Hao;Zheng, Lei;Sun, Yan-Ping;Zhang, Guang-Wei;Yue, Zheng-Gang
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.24
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    • pp.10911-10916
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    • 2015
  • Background: Tumor metastases are the main reasons for oncotherapy failure. Paris polyphylla (Chinese name: Chonglou) has traditionally been used for its anti-cancer actions. In this article, we focus on the regulation of human lung cancer A549 cell metastases and invasion by Paris polyphylla steroidal saponins (PPSS). Materials and Methods: Cell viability was evaluated in A549 cells by MTT assay. Effects of PPSS on invasion and migration were investigated by wound-healing and matrigel invasion chamber assays. Adhesion to type IV collagen and laminin was evaluated by MTT assay. Expression and protease activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blotting and gelatin zymography, respectively. Results: PPSS exerted growth inhibitory effects on A549 cells, and effectively inhibited A549 cell adhesion, migration and invasion in a concentration-dependent manner. Western blotting and gelatin zymography analysis revealed that PPSS inhibited the expression and secretion of MMP-2 and MMP-9 in A549 cells. Conclusions: PPSS has the potential to suppress the migration, adhesion and invasion of A549 cells. PPSS could be a potential candidate for interventions against lung cancer metastases.

The Expression of MMPs and TIMPs in IPF and NSIP (IPF와 NSIP에서 MMPs와 TIMPs의 발현)

  • Kim, Yu Jin;Kim, Jung Ho;Jeon, Hyo Keun;Kim, Mi Kyeong;Jo, Young Chan;Kyung, Sun Yong;An, Chang Hyeok;Lee, Sang Pyo;Park, Jung Woong;Ha, Seung Yeon;Jeong, Sung Hwan
    • Tuberculosis and Respiratory Diseases
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    • v.61 no.5
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    • pp.447-455
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    • 2006
  • Background: MMPs and TIMPs are important factors for abnormal remodeling the pulmonary parenchyme in idiopathic interstitial pneumonia(IIP) This study evaluated the expression of MMPs and TIMPs in the tissue of IPF, NSIP and normal control subjects. Method: The MMP-2 and -9 activity in the lung tissue was studied by gelatin zymography, and the expression of MMP-1, -2, -9, TIMP-1 and -2 in the lung tissue was measured by immunohistochemistry. Thirty five patients, who were diagnosed with IIP (UIP ; 22, NSIP ; 13), were enrolled in the immunohistochemical study. Thirteen patients with IIP (UIP ; 9, NSIP ; 4) and five patients with lung cancer were enrolled in the zymographic assay. Results: (1) The immunohistochemistry for MMP-1,-2,-9, TIMP-1 and-2 ; MMP-1,-9 and TIMP-2 were stained stronger in the UIP subjects than NSIP and the normal control. TIMP-2 was strongly stained in the UIP tissue. particularly the fibroblasts in the fibroblastic foci. (2) Zymography for MMP-2 and MMP-9 revealed MMP-2 to have prominent expression in the UIP tissue than in the NSIP tissue. Conclusions: These results suggest that the overexpression of the TIMPs and gelatinases in UIP might be important factors in the irreversible fibrosis of the lung parenchyme.

Use of Zymography for Identification of the Same Clone in a Clone Bank of Pinus densiflora Sieb. et Zucc (Isoperoxidase 변이형(變異型)에 의(依)한 소나무 Clone 감별(鑑別))

  • Park, Young-Goo;Choi, Jung Suk
    • Journal of Korean Society of Forest Science
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    • v.18 no.1
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    • pp.17-22
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    • 1973
  • Using of zymography for identification of same clone of Pinus densiflora, the two year old needle leaves of 48 ramtes including 8 clones (6 ramets per clone) were collected in the clone bank which has been established on the near campus of Institute of Forest Genetics, Suwon, in 1962. All 8 bands are named from cathod to anode, G, H, K, M, N, Q, S and Y. Only CB-1 clone shows all bands, while KW-3 clone reveals only 5 bands. Other 6 clones were found 7 bands but the occurred frequencies of those bands are variable among those clones. Though the grafting stock are used various individuals grown seed propagation of P. densiflora, moreover, three stocks have been different species and that one has been P. rigida and two individuals have been P. koraiensis, the zymograms of the ramets belonging to the same clone reveals the identified patterns. The results show that the stocks for grafting have not been affected on the isoperoxidase patterns of their scions in P. densiflora. Among 48 ramets of 8 clones, 4 ramets are found the different isoperoxidase patterns from that of the remained rametes within same clone. Thus, it is conluded that zymography is useful for testing genuineness of the grafted clones of P. densiflora.

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Gelatinases of Extracellular Matrix of Human Oocyte-Cumulus Complex (사람 난자-난구 복합체 ECM의 Gelatinase)

  • 이인선;나경아;김해권
    • Development and Reproduction
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    • v.5 no.2
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    • pp.123-129
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    • 2001
  • When mammalian oocytes undergo maturation, cumulus cells surrounding the oocyte exhibit remodeling of their structure known as cumulus expansion. Many molecules including hyaluronic acid participate in this remodeling. The present study aimed to investigate a possible existence of matrix metalloproteinases(MMPs) in the extracellular matrix(ECM) of human oocyte-cumulus complex. ECM was extracted from the human oocyte-cumulus complex. Gelatin gel zymogram of ECM exhibited 7 gelatinases having molecular weight of 300kDa, 240kDa, 200kDa, 180kDa, 116kDa, 97kDa, and 84kDa. This gelatinase profile was very different from that of ovarian mural granulosa cell extract or white blood cell extract, indicating that the oocyte-cumulus complex donating ECM was free from other than cumulus cells. When ethylenediaminetetraacetic acid or 1', 10'-phenanthroline was added to the reaction buffer during zymographic development, almost gelatinase activities were abolished, suggesting that they were MMPs. Following incubation of ECM in the presence of aminophenylmercuric acetate, an activator of proMMPs, 4 gelatinases of 240kDa, 180kDa, 97kDa, and 84kDa disappeared with the concomitant appearance of 80kDa, 65kDa, and 60kDa gelatinases. Based upon these observation, it is suggested that ECM of the human oocyte-cumulus complex consists of gelatinases, presumed to be MMP-2 and MMP-9 isoforms.

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