• 제목/요약/키워드: zymography

검색결과 233건 처리시간 0.025초

Expression of Alpha-Amylase Gene from Bacillus licheniformis in Lactobacillus brevis 2.14

  • Lee, Kang-Wook;Park, Ji-Yeong;Kim, Gyoung-Min;Kwon, Gun-Hee;Park, Jae-Yong;Lee, Mee-Ryung;Chun, Ji-Yeon;Kim, Jeong-Hwan
    • Preventive Nutrition and Food Science
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    • 제13권3호
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    • pp.190-195
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    • 2008
  • The $\alpha$-amylase gene, amyL, from Bacillus licheniformis was expressed in Lactobacillus brevis 2.14 and Escherichia coli $DH5{\alpha}$ using two different shuttle vectors, pCW4 and pSJE. E. coli transformants (TFs) harboring either $pCW4T{\alpha}$ or $pSJET{\alpha}$ produced active $\alpha$-amylase but L. brevis TFs did not, as determined by enzyme assays and zymography. But amyL transcripts were synthesized in L. brevis TFs. In terms of plasmid stability, pSJE, a theta-type replicon, was more stable than pCW4, an RCR (rolling circle replication) plasmid, in L. brevis without antibiotic selection.

Mangiferin inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 expression and cellular invasion by suppressing nuclear factor-κB activity

  • Dilshara, Matharage Gayani;Kang, Chang-Hee;Choi, Yung Hyun;Kim, Gi-Young
    • BMB Reports
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    • 제48권10호
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    • pp.559-564
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    • 2015
  • We investigated the effects of mangiferin on the expression and activity of metalloproteinase (MMP)-9 and the invasion of tumor necrosis factor (TNF)-$\alpha$-stimulated human LNCaP prostate carcinoma cells. Reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that mangiferin significantly reversed TNF-$\alpha$-induced mRNA and protein expression of MMP-9 expression. Zymography data confirmed that stimulation of cells with TNF-$\alpha$ significantly increased MMP-9 activity. However, mangiferin substantially reduced the TNF-$\alpha$-induced activity of MMP-9. Additionally, a matrigel invasion assay showed that mangiferin significantly reduced TNF-$\alpha$-induced invasion of LNCaP cells. Compared to untreated controls, TNF-$\alpha$-stimulated LNCaP cells showed a significant increase in nuclear factor-${\kappa}B$ (NF-${\kappa}B$) luciferase activity. However, mangiferin treatment markedly decreased TNF-$\alpha$-induced NF-${\kappa}B$ luciferase activity. Furthermore, mangiferin suppressed nuclear translocation of the NF-${\kappa}B$ subunits p65 and p50. Collectively, our results indicate that mangiferin is a potential anti-invasive agent that acts by suppressing NF-${\kappa}B$-mediated MMP-9 expression.

Neovastat(AE-941) inhibits the airway inflammation and hyperresponsiveness in a murine model of asthma

  • Lee, Sook-Young;Paik, Soon-Young;Chung, Su-Mi
    • Journal of Microbiology
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    • 제43권1호
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    • pp.11-16
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    • 2005
  • Matrix metalloproteinase (MMP)-9 plays an important role in the pathogenesis of bronchial asthma. Neovastat, having significant antitumor and antimetastatic properties, is classified as a naturally occurring multifunctional antiangiogenic agent. We evaluated the therapeutic effect of Neovastat on airway inflammation in a mouse model of asthma. BALB/c mice were immunized subcutaneously with ovalbumin (OVA) on days 0, 7, 14, and 21 and challenged with inhaled OVA on days 26, 29, and 31. Neovastat was administrated by gavage (5 mg/kg body weight) three times with 12 h intervals, beginning 30 min before OVA inhalation. On day 32, mice were challenged with inhaled methacholine, and enhanced pause (Penh) was measured as an index of airway hyperresponsiveness. The severity of airway inflammation was determined by differential cell count of bronchoalveolar lavage (BAL) fluid. The MMP-9 concentration in BAL fluid samples was measured by ELISA, and MMP-9 activity was measured by zymography. The untreated asthma group showed an increased inflammatory cell count in BAL fluid and Penh value compared with the normal control group. Mice treated with Neovastat had significantly reduced Penh values and inflammatory cell counts in BAL fluid compared with untreated asthmatic mice. Furthermore, mice treated with Neovastat showed significantly reduced MMP-9 concentrations and activity in BAL fluid. These results demonstrate that Neovastat might have new therapeutic potential for airway asthmatic inflammation.

Expression of Starch-degrading Genes in Escherichia Coli and Kactococcus Lactis

  • Jeong, Jong-Jin;Kim, Tea-Youn;Moon, Gi-Seong;Lee, Hyo-Jeong;Kim, Jong-Sang;Kim, Jeong -Hwan
    • Preventive Nutrition and Food Science
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    • 제3권1호
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    • pp.98-104
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    • 1998
  • As an efffort ot construct LAB (latice acid bacteria), capable of utilizing starch as fermentation substrate without the aid of externally supplied enzymes, plasmid vectors containing the amyL($\alpha$-amylase/pullulansase gene) from Clostridium thermophydrosulfuricum, and glucoamylase cDNA from Asperigillus shirousamii were constructed and introduced itno E. coli and L. lactis. For expression in procaryotes , 1.9kb glucoamylase cDNA encoding the mature form of enzyme was PCR amplified and translationaly fused to a PCR amplified 260 bp fragment containing the promotor and secretion signals of amyl in the same reading frame. The production of $\alpha$-amylase, Apu, and glucoamlase in E. coli and L. lactis was confirmed by enzyme assay and zymography . Enzymeswere detected in both cellpellets and supernatants, indicating theworking of scretion signals in heterologous hosts. The efficiencies of secretion were varibale depending on the gene and host. The highest $\alpha$- amylase acitivity observed was 1.1 units and most activiity was detected from thecell pellets. The degree of gene expression in both hosts and the effect on the growth of hosts were examined.

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Effect of capsaicin on melanoma growth and metastasis

  • Kim, Ok-Hee;Jun, Hye-Seung;Han, Beom-Seok;Song, Chi-Won;Lee, Chang ki;Park, Mi-Sun;Eom, Mi-Ok;Jee, Seung-Wan;Ryeom, Tai-Kyung
    • 한국독성학회:학술대회논문집
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    • 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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    • pp.143-143
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    • 2002
  • Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a pungent ingredient of hot chili peppers, has been reported to possess substantial anticarcinogenic and antimutagenic activities. In our previous study, we found that capsaicin (100 ${\mu}$M) induced significant inhibition of matrix metalloproteinase-2 activity by gelatin zymography, and that capsaicin (i.p., 2.5mg/kg) inhibited development of lung colonization (58%) in experimental lung metastasis assay.(omitted)

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Heterologous Gene Expression of aprE2 Encoding a 29 kDa Fibrinolytic Enzyme from Bacillus subtilis in Bacillus licheniformis ATCC 10716

  • Kwon, Gun-Hee;Jeong, Woo-Ju;Lee, Ae-Ran;Park, Jae-Yong;Cha, Jae-Ho;Song, Young-Sun;Kim, Jeong-Hwan
    • Food Science and Biotechnology
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    • 제17권6호
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    • pp.1372-1375
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    • 2008
  • The aprE2 gene from Bacillus subtilis CH3-5 was expressed in Bacillus licheniformis ATCC 10716 using a Bacillus-Escherichai coli shuttle vector, pHY300PLK. The fibrinolytic activity of transformant (TF) increased significantly compared to B. licheniformis 10716 control cell. During the 100 hr incubation in Luria-Bertaini broth at $37^{\circ}C$, fibrinolytic activity of B. licheniformis TF increased rapidly at the late growth stage, after 52 hr of incubation, which was confirmed by zymography using a fibrin gel. pHY3-5 was stably maintained in B. licheniformis without tetracycline (Tc) in the media, 60.9% of cells still maintained pHY3-5 after 100 hr of cultivation.

ESI/MS/MS를 이용한 혈장 중 카르니틴 정량분석 (Quantitation of L-carnitine in plasma by electrospray ionization tandem mass spectrometry)

  • 강승우;김호현;이경률;윤혜란
    • 분석과학
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    • 제18권2호
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    • pp.163-167
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    • 2005
  • 본 연구에서는 ESI/MS/MS를 이용하여 혈장에서 카르니틴을 신속하게 정량하는 분석방법을 개발하였다. 분석방법은 free carnitine (FC)는 유도체화 없이 혈액여지를 80% 메탄올로 추출하여 분석하였고, Total carnitine (TC)은 가수분해 한 후 추출하여 multiple reaction monitoring (MRM) scan mode로 분석하였다. Acyl carnitine (AC)는 TC에서 FC를 뺀 값을 사용하였다. 카르니틴의 분석에서 상관계수 ($r^2$)는 0.9995, 회수율은 97%, 재현성은 변동계수가 10% 이하, 검출한계는 $0.0016{\mu}mol/L$ 였다. 이 방법은 기존의 액체 크로마토그래피 보다 전처리가 간다하고 짧은 분석시간과 좋은 감도와 재현성을 보여 주었다. 정상인은 AC가 낮고 FC는 높은 패턴을 보였고, 유전성대사질환 환자는 FC는 낮고 AC가 높은 패턴으로 나타나 카르니틴의 3분획 분석은 유전성대사질환 환자의 확진 및 모니터링에 유용함을 보여주었다.

Characterization of Aspergillus niger Mutants Deficient of a Protease

  • Chung, Hea-Jong;Park, Seung-Moon;Kim, Dae-Hyuk
    • Mycobiology
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    • 제30권3호
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    • pp.160-165
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    • 2002
  • Aspergillus niger has been used as a host to express many heterologous proteins. It has been known that the presence of an- abundant protease is a limiting factor to express a heterologous protein. The protease deficient mutant of A. niger was obtained using UV-irradiation. A total of $1{\times}10^5$ spores were irradiated with $10{\sim}20%$ survival dose of UV, 600 $J/m^2$ at 280 nm, and the resulting spores were screened on the casein-gelatin plates. Ten putative protease deficient mutants showing the reduced halo area around colonies were further analyzed to differentiate the protease deficient mutant from other mutant types. Among ten putative mutants, seven mutants showed significant growth defect on nutrient rich medium and two mutants appeared to be the secretory mutants, which resulted in the impaired secretion of extracellular proteins including proteases. A mutant $pro^--20$ showed reduced halo zone without any notable changes in growth rate. In addition, the starchdegrading and glucose oxidase activities in the culture filtrate of $pro^--20$ mutant showed the similar range as that of the parental strain, which suggested that the $pro^--20$ mutant ought to be the protease deficient mutant rather than a secretory mutant. The reduced proteolytic activity of the $pro^--20$ was demonstrated using SDS-fibrin zymography gel. The reduced extracellular proteolysis was quantified by casein degradation assay and, comparing with the parental strain, less than 30% residual extracellular protease activity was detected in the culture filtrate of the $pro^--20$ mutant. The bio-activity of an exogenously supplemented hGM-CSF(human Granulocyte-Macrophage Colony Stimulating Factor) in the culture filtrate of $pro^--20$ mutant was detected until eight times more diluted preparations than that of the parental strain.

Detection of Matrix Metalloprotease-9 and Analysis of Protein Patterns in Bovine Vaginal Mucus during Estrus and Pregnancy

  • Kim, Sang-Hwan;Baek, Jun-Seok;Lee, Ho-Jun;Min, Kwan-Sik;Lee, Deuk-Hwan;Yoon, Jong-Taek
    • 한국수정란이식학회지
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    • 제27권2호
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    • pp.93-100
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    • 2012
  • To investigate the biochemical nature of changes in vaginal physiology during estrus and pregnancy, we examined the cytology and viscosity, and monitored the protein expression profile in vaginal mucus during estrus and pregnancy. The viscosity progressively decreased from estrus to pregnancy. Cell type analysis revealed that white blood cells progressively increased from estrus to pregnancy, while red blood cells progressively decreased during pregnancy. The cornification index (CI) was higher in estrus than in pregnancy. Protein mass spectrumetry identified the presence of ribosome-binding protein 1, GRIP 1 (Glutamate receptor-interacting protein 1)-associated protein 1, DUF729 (Domain of unknown function729) domain-containing protein 1, prolactin precursor, dihydrofolatereductase, and MMP (Matrix metalloprotease)-9 in vaginal mucus. MMP-2 and MMP-9 proteins in the vaginal mucus were active throughout estrus and gestation, as measured by a gelatinase assay, but most abundant in the vaginal mucus on day 0 of estrus. Results from ELISA of MMP-2 and MMP-9 were in accordance with the gelatinase assay. In light of the crucial role of metalloproteinases in extracellular matrix remodeling, the level of MMP-9 in vaginal mucus might be useful as an indicator of estrus and pregnancy to increase the efficiency of reproduction.

Inhibition of MMP-2 and MMP-9 Activities by Limonium tetragonum Extract

  • Bae, Min-Joo;Karadeniz, Fatih;Lee, Seul-Gi;Seo, Youngwan;Kong, Chang-Suk
    • Preventive Nutrition and Food Science
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    • 제21권1호
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    • pp.38-43
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    • 2016
  • Matrix metalloproteinases (MMPs) are crucial extracellular matrices degrading enzymes that take important roles in metastasis of cancer progression as well as other significant conditions such as oxidative stress and hepatic fibrosis. Natural products are on the rise for their potential to provide remarkable health benefits. In this context, halophytes have been of interest in the nutraceutical field with reported instances of isolation of bioactive compounds. In this study, Limonium tetragonum, an edible halophyte, was studied for its ability to inhibit MMP-2 and -9 using HT1080 fibrosarcoma cells. Results showed that L. tetragonum extract was able to inhibit the enzymatic activity and mRNA expression of MMP-2 and -9 according to gelatin zymography and RT-PCR assays, respectively, but it was not able to significantly change the MMP pathway related factors such as tissue inhibitors of metalloproteinases. Also, Mitogen-activated protein kinases pathway-related protein levels and their phosphorylation were assayed. While the phosphorylated p38 levels were decreased, extracellular signal-regulated kinase and c-Jun N-terminal kinase were not affected by L. tetragonum treatment. In conclusion, it was suggested that L. tetragonum contains substances acting as MMP inhibitors on enzymatic activity rather than intracellular pathway intervention, which could be useful for further utilization of L. tetragonum as a source for anti-MMP agents.