Fibrin Zymography Research Articles

A Protease from Moringa oleifera Lam. Exhibits In-vitro Blood Clot Solubilization and Fibrin Hydrolysis.

Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. A fibrinolytic protease was isolated from fresh leaves of Moringa oleifera Lam. and characterized for its potential to solubilize blood clots and hydrolyse fibrin under in-vitro conditions. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 oC with 50µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. Km and Vmax of isolated protease were determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clots by 96h under in-vitro conditions. In-vitro fibrin hydrolysis and blood clot solubilization activities shown by an isolated protease from leaves of Moringa oleifera Lam. suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis.

In vitro assessment of thrombolytic potential of red and white ginger (Zingiber officinale)

Background: Cardiovascular diseases (CVDs) are a leading cause of death, and their pathogenesis is commonly attributed to thrombosis. Although existing medications are effective and fast-acting for thrombosis management, they tend to be expensive and cause severe side effects. Plant-based thrombolytic agents are actively sought after as inexpensive and safe alternatives for the treatment and prevention of thrombosis. Red ginger (Zingiber officinale var. rubrum) and white ginger (Zingiber officinale var. officinale) are widely used in foods and beverages and are believed to confer a wide variety of health benefits. Objective: This study aims to elucidate the thrombolytic and fibrinolytic potential of red and white ginger extracts in vitro.Methods: In this study, in vitro analyses were performed using erythrocyte liberation, euglobulin degradation, fibrin degradation, and fibrin zymography assays. The ability of crude enzyme extracts from both red (rgEx) and white ginger (wgEx) to degrade blood clots was analyzed using the erythrocytes liberation assay. Then, the thrombolytic and fibrinolytic activities of rgEx and wgEx were evaluated using euglobulin and fibrin degradation assays, both of which were visualized using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Finally, fibrinolytic enzymes were identified using a fibrin zymography assay. Results: Red and white ginger extracts were found to have strong thrombolytic properties via high total liberated erythrocyte count from the erythrocyte liberation assay. The ginger extract proteases can rapidly degrade euglobulin and fibrin, with their priority order beginning with A

Urokinase-type plasminogen activator promotes corneal epithelial migration and nerve regeneration

Urokinase-type plasminogen activator (uPA) is a serine protease that plays a central role in the pericellular fibrinolytic system, mediates the degradation of extracellular matrix proteins and activation of growth factors, and contributes to the regulation of various cellular processes including cell migration and adhesion, chemotaxis, and angiogenesis. The corneal epithelium responds rapidly to injury by initiating a wound healing process that involves cell migration, cell proliferation, and tissue remodeling. It is innervated by sensory nerve endings that play an important role in the maintenance of corneal epithelial homeostasis and in the wound healing response. We here investigated the role of uPA in corneal nerve regeneration and epithelial resurfacing after corneal injury with the use of uPA-deficient mice. Both the structure of the corneal epithelium and the pattern of corneal innervation in uPA−/− mice appeared indistinguishable from those in uPA+/+ mice. Whereas the cornea was completely resurfaced by 36–48 h after epithelial scraping in uPA+/+ mice, however, such resurfacing required at least 72 h in uPA−/− mice. Restoration of epithelial stratification was also impaired in the mutant mice. Fibrin zymography revealed that the expression of uPA increased after corneal epithelial scraping and returned to basal levels in association with completion of re-epithelialization in wild-type animals. Staining of corneal whole-mount preparations for βIII-tubulin also revealed that the regeneration of corneal nerves after injury was markedly delayed in uPA−/− mice compared with uPA+/+ mice. Our results thus demonstrate an important role for uPA in both corneal nerve regeneration and epithelial migration after epithelial debridement, and they may provide a basis for the development of new treatments for neurotrophic keratopathy.

Fibrinolytic activity and molecular identification of PB-12 isolate from Papuma Coastal at Jember regency

Background: Coastal areas have a high bacterial diversity, which can be used as a source of pharmaceutical substances, including the fibrinolytic enzyme. Fibrinolytic enzymes can lyse blood clots by degrading the fibrin. These enzymes are important for treating circulatory disorders, e.g., pulmonary embolism, deep vein thrombosis, and myocardial infarction. Objective: This study aimed to determine the fibrinolytic activity of crude protein extract and precipitate protein of fibrinolytic bacteria PB-12 isolated from Papuma coastal, Jember district, Indonesia. Method: The molecular weight of fibrinolytic enzymes was determined by fibrin zymography, while 16s rDNA sequencing was performed to identify the PB-12 isolate. Result: The fibrinolytic activity result showed CPE at 24 hours had the highest fibrinolytic activity with a lysis diameter of 5.23 ± 0.68 mm. The fibrinolytic activity of this bacterium was contributed with ~114 kDa protein. PB-12 was identified as Bacillus aryabhattai based on 16S rRNA gene sequence. Conclusion: This is the first to report Bacillus aryabhattai as a potential fibrinolytic enzyme producer.

Cost-effective fibrinolytic enzyme production by microalga Dunaliella tertiolecta using medium supplemented with corn steep liquor.

A fibrinolytic enzyme from the microalga Dunaliella tertiolecta was produced under mixotrophic conditions using different corn steep liquor (CSL) concentrations ( 0 ≤ CLS ≤ 0.75%), purified using a combination of salting out and ion-exchange chromatography, and then biochemical characterized. Cultivation of this microalga using 0.5% CSL led to the highest maximum cell concentration (1.960±0.010 mg L-1) and cell productivity (0.140g L-1 day-1), besides a high fibrinolytic activity of the extract obtained by the homogenization method (102 ±1 U mL-1). The enzyme extracted from the microalgal biomass was 5-fold purified with a 20% yield and was found to have a specific activity of 670 U mg-1. The enzyme, whose molecular weight determined by fibrin zymography was 10 kDa, was shown to be stable at pH 3.0-9.0 and up to 70°C with optimal pH and temperature values of 8.0 and 50°C, respectively. When compared to other fibrinolytic enzymes, this protease stood out for its high fibrinolytic activity, which was enhanced by Fe2+, inhibited by Zn2+, Cu2+, Mg2+, and Ca2+, and strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that it belongs to the serine metalloprotease family. Moreover, thanks to its thermal stability, the enzyme may be easily preserved and activated under high-temperature conditions.

Thrombolytic potential in bacteria isolated from fermented durian tempoyak

Abstract. Priskila C, Vidian V, Sanjaya A, Sugata M, Pinontoan R. 2022. Thrombolytic potential in bacteria isolated from fermented durian tempoyak. Biodiversitas 23: 5731-5737. Cardiovascular disease is one of the leading causes of death worldwide and is primarily caused by dysregulation in the blood clotting system leading to the formation of unwanted blood clots (thrombi). Thrombolytic therapy, which aims to break down unwanted blood clots, employs various agents possessing thrombolytic activity. However, the harmful side effects of conventional thrombolytic agents necessitate the exploration of new and safer agents with high thrombolytic activities. Thrombolytic agents can be obtained from various sources, including fermented foods. Tempoyak is a condiment made from fermented durian, which is found in some regions of Indonesia, and is reported to contain beneficial microorganisms. The aim of this study was to isolate and identify thrombolytic agents from tempoyak. After several pure microbial colonies were successfully isolated from tempoyak, they were screened for proteolytic activity on skim milk agar medium. The isolated colonies with proteolytic activity were then subjected to several tests, including the whole blood clot lysis test, euglobulin clot lysis test, fibrin plate method, and zymography. Subsequently, the isolates with thrombolytic and fibrinolytic activity potential were identified in terms of their morphology and biochemistry. In this study, three bacteria with thrombolytic activity potential were successfully isolated. Fibrin zymography indicated that the molecular weights of the proteins that play roles in the fibrinolytic activity ranged from approximately 30 to 153 kDa, which were suspected to correspond to serine protease, cysteine protease, and/or metalloprotease. All three isolates were identified as belonging to the genus Corynebacterium. This is the first report discussing the thrombolytic and fibrinolytic activities of Corynebacterium sp. derived from fermented food.

Exploring Agaricomycetes from the Paranaense rainforest (Misiones, Argentina) as an unconventional source of fibrinolytic enzymes

ABSTRACT Fungal fibrinolytic enzymes, secreted by some Agaricomycetes, are recognized as important thrombolytic agents due to their ability to rapidly dissolve thromboembolic clots. The present work evaluated fibrinolytic and proteolytic secretion abilities of 35 Agaricomycetes isolates from the Paranaense rainforest (Misiones, Argentina). We detected proteolytic activity in 40% of the strains while nine strains showed fibrinolytic activity. Schizophyllum commune LBM 026, Schizophyllum commune LBM 223, and Hornodermoporus martius LBM 224 exhibited the highest levels of fibrinolytic activity. Fibrin zymography from S. commune LBM 026 and LBM 223 showed an enzyme of 27.5 kDa, while H. martius LBM 224 presented an enzyme of 29 kDa. The evaluation of the enzymatic stability of culture supernatant of these strains revealed that the fibrinolytic activity was highly stable over a wide temperature and pH range. Long-term stability of fibrinolytic activity at physiological conditions evidenced that the strains had a half­life of at least 72 h. Fibrinolytic enzymes produced by S. commune LBM 026 and LBM 223 were inhibited in the presence of EDTA indicating that they are metalloproteases. This work reveals the potential of S. commune LBM 026, S. commune LBM 223, and H. martius LBM 224 as an unconventional source of thrombolytic agents.

Production and purification of nattokinase from Bacillus subtilis

ABSTRACT The present study is focused on the production and purification of nattokinase, a fibrinolytic protease, from B. subtilis TH9 (NatTH9) based on an aqueous two-phase system (ATPS) technique. The results showed that the optimal ATPS for NatTH9 recovery was 20% (w/v) polyethylene glycol 6000 and 15% (w/v) potassium phosphate at pH 8. The partitioning coefficient, the partitioning yield, and the activity of NatTH9 were 6.25, 76.7%, and 547.02 U/mg, respectively. The purified NatTH9 demonstrated the ability to degrade fibrin and dissolve the clot. Fibrin zymography showed three clear zones on the gel with molecular weights of approximately 37, 27, and 21 kDa. The optimal pH and temperature of purified NatTH9 were 8 and 39°C, respectively.

Characterization of a salt-resistant fibrinolytic protease of Bacillus licheniformis HJ4 isolated from Hwangseokae jeotgal, a traditional Korean fermented seafood.

Bacillus licheniformis HJ4 showing strong fibrinolytic activity was isolated from Hwangseokae jeotgal. aprEHJ4, a major fibrinolytic gene, was cloned by PCR, and an ORF consisting of 379 amino acids was located. The mature enzyme was expected to be 27kDa in size after processing, but a 24-kDa protein was observed by SDS-PAGE and fibrin zymography, indicating additional processing. RT-qPCR showed that expression level of aprEHJ4 in culture with 0% salt (control) was the highest followed by culture with 8% salt (89.7% of control) and 5% salt (74.2%) at 84h. The expression level in culture with 15% salt was 46.9%. The results matched with the fibrinolytic activity measurements of cultures and indicated that AprEHJ4 maintained significant activity in the presence of salt up to 15% (w/v). AprEHJ4 was overproduced in Escherichia coli, and mature 27kDa protein was purified after in vitro renaturation. The optimum pH and temperature of AprEHJ4 were pH 8 and 40 ℃, respectively.

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement of aprE3-5 from Bacillus subtilis CH3-5.

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

Isolation of 2 Bacillus Strains with Strong Fibrinolytic Activities from Kimchi

Zhuang Yao , Yu Meng , Huong Giang Le , Se Jin Lee , Hye Seong Jeon , Ji Yeon Yoo , Diana Nur Afifah and Jeong Hwan Kim . Microbiol. Biotechnol. Lett. 2020;48:439-46. https://doi.org/10.48022/mbl.2003.03008

피브린 Zymographic Gel에서 Bacillus amyloliquefaciens G-13으로부터 혈전용해효소의 생성에 영향을 미치는 물리화학적 요인들의 비교

Our previous research demonstrated the characteristics and fibrinolytic activities of NaCl- and capsaicin-resistant bacterium Bacillus amyloliquefaciens G-13, isolated from mustard leaf kimchi [1]. In this study, we extended the work to the comparison of various physicochemical factors (e.g., temperature, pH, NaCl, capsaicin, metal ions, and inhibitors) influencing the production of fibrinolytic enzyme by strain G-13. Initially, a time course of the fibrinolytic activity of strain G-13 was measured by the fibrin plate assay, and after 84 hours of incubation, the maximum fibrinolytic activity was about 3.42 times that of plasmin used as the standard. Through a fibrin zymographic assay, four bands (23, 34, 45, and 67 kDa) of fibrinolytic enzyme from strain G-13 were compared in fibrin zymographic gels. The strain could grow and produce the fibrinolytic enzyme in the presence of NaCl (1-10%) or capsaicin (0-300 ㎍/mL), respectively. The optimum pH and temperature for the enzymatic activity were 8 and 35℃. In the study of the effects of metal ions on fibrinolytic activity, K⁺, CaSUP2+/SUP, and MgSUP2+/SUP ions increased the activity and maintained it overall, but FeSUP3+/SUP, ZnSUP2+/SUP, BaSUP2+/SUP, and HgSUP2+/SUP ions rapidly decreased the activity of the fibrinolytic enzyme. The enzyme was completely inhibited by phenylmethanesulfonyl fluroide (PMSF), suggesting that it was a serine protease. These results provide important clues for understanding the characteristics of fibrinolytic bacteria by confirming the effects of B. amyloliquefaciens G-13 on various physicochemical factors influencing the production of fibrinolytic enzymes through fibrin zymography.

Fibrinolytic enzyme from Arthrospira platensis cultivated in medium culture supplemented with corn steep liquor

Artrhospira (Spirulina) platensis produced fibrinolytic enzyme under mixotrophic conditions using corn steep liquor (CSL). The enzyme was extracted, purified by combination of two chromatographic techniques and biochemically characterized. Maximum fibrinolytic production (268.14 U mg−1) was obtained using liquid medium culture composed by 0.2% CLS after 10th day of cultivation. Fibrinolytic activity was higher when extracted by homogenization methods and was purified 32.72–fold with specific activity of 7988 U mg−1. Fibrin zymography showed an active band, indicated acts as a plasmin-like protein with molecular weight of 72 kDa. Fibrinolytic enzyme have optimum pH of 6.0, stable in the range of 6.0 to 10.0 during 24 h and optimum temperature at 40 °C with a stability below 50 °C. Fibrinolytic enzyme is a serine metalloprotease by to be enhanced by Fe2+ and inhibited by PMSF. The enzyme has higher enzymatic activity than most other fibrinolytic enzymes and is stable at temperature and pH human physiological. Overall, the fibrinolytic enzyme from A. platensis has attractive biochemical properties to potential applications in the treatment of thrombosis.

Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods.

Previously, the crude extracts of recombinant Nattokinase (NK) variants i.e. NatTK and NatOC and one wild type Douchi Fibrinolytic Enzyme (DFE) from Indonesian traditional fermented foods has been shown to demonstrate fibrinolytic activity. Both NKs contain substitutions of D41N, V192A and 252-RLQHTLEALSTM-263 but NatOC has additional V4F. In the present study, the effects of amino acid substitutions in NK variants and G169A in DFE on their enzyme characteristics were evaluated. Pure proteins were obtained using two sequential steps chromatography using ion exchange and a gel filtration columns. Their activities were determined with fibrin plate, fibrin zymography, fibrinogen hydrolysis, and chromogenic assays. The fibrinogen degradation profile of the wild type NK (NatWT) was different to the NK variants but similar to DFEs. Optimum activity of all the NKs and DFEs was achieved at 50°C while the optimum pH for NatWT/DFEs and NK variants were 8 and 7, respectively. DFEG169A exhibited higher fibrinogen degradation rate and fibrin specific activity than DFE. PMSF inhibited all the NKs and DFEs while SDS and EDTA caused lower activity. The NK variants were more resistant towards Na+ and Ca2+ but more sensitive to K+. The amino acid substitutions in NK variants alter their fibrinogen degradation profile, optimum working pH, working pH range, and resistance to some ions. Substitutions in NK variants likely promote structural changes, particularly with the binding mode of the calcium ion cofactor. The results provide a beneficial basis for future development of fibrino(gen)olytic proteins with improved properties for cardiovascular diseases therapy.

Stimulation of Phagocytic Activity in Cultured Human Corneal Fibroblasts by Plasminogen.

Plasminogen has been detected in the corneal stroma after tissue injury and interacts with corneal fibroblasts during wound healing. We examined the effect of plasminogen on phagocytic activity of corneal fibroblasts. Cultured human corneal fibroblasts were exposed to plasminogen and then incubated with fluorescent microparticles before measurement of phagocytic activity by confocal microscopy or flow cytometry. The binding of corneal fibroblasts to immobilized plasminogen was quantitated with a real-time biomolecular interaction assay. The production of urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), and IL-1β by corneal fibroblasts was measured by fibrin zymography, by immunoblot analysis or gelatin zymography, or with an enzyme-linked immunosorbent assay, respectively. Plasminogen increased phagocytic activity of corneal fibroblasts in a concentration- and time-dependent manner, with the maximal effect apparent at 30 μg/mL and 24 hours. Corneal fibroblasts bound to immobilized plasminogen in a manner dependent on time and cell number, and the stimulatory effect of plasminogen on phagocytic activity was blocked in the presence of epsilon-aminocaproic acid, an inhibitor of plasminogen binding to cell surface receptors. Plasminogen-induced phagocytic activity was not associated with changes in the production of uPA, MMPs, or IL-1β by corneal fibroblasts. Plasminogen induced phagocytic activity in corneal fibroblasts in a manner dependent on its binding to the cell surface. This effect was not associated with increased production of proteases or IL-1β. Thus, plasminogen may promote the clearance of foreign particles or damaged tissue components by corneal fibroblasts early after tissue injury.

Inhibition by Epigallocatechin Gallate of IL-1-Induced Urokinase-Type Plasminogen Activator Expression and Collagen Degradation by Corneal Fibroblasts.

The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration and promotes collagen degradation by corneal fibroblasts cultured in a three-dimensional (3D) collagen gel. Epigallocatechin-3-gallate (EGCG), the principal polyphenol in extracts of green tea, has various beneficial health effects, some of which appear to be mediated through direct or indirect inhibition of protease activity. We therefore examined the effect of EGCG on IL-1β-induced collagen degradation by corneal fibroblasts embedded in a collagen gel. Human corneal fibroblasts were cultured in a type I collagen gel. Collagen degradation was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. The expression of urokinase-type plasminogen activator (uPA) was examined by real-time and RT-PCR analysis and by fibrin zymography, and that of the collagenase matrix metalloproteinase 1 (MMP1) was detected by immunoblot analysis. EGCG inhibited IL-1β-induced, plasminogen-dependent collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also attenuated the IL-1β-induced expression of uPA at both mRNA and protein levels. EGCG inhibited the IL-1β-induced conversion of exogenous plasminogen to plasmin as well as the plasminogen-dependent activation of pro-MMP1 in the 3D cultures without a substantial effect on pro-MMP1 abundance. EGCG inhibits IL-1β-induced collagen degradation by corneal fibroblasts, with this effect likely being mediated by suppression of the upregulation of uPA, the uPA-mediated conversion of plasminogen to plasmin, and the plasmin-mediated activation of pro-MMP1. EGCG thus warrants further investigation as a potential treatment for corneal ulcer.

Characterization of a Fibrinolytic Enzyme Secreted by Bacillus velezensis BS2 Isolated from Sea Squirt Jeotgal.

Bacillus sp. BS2 showing strong fibrinolytic activity was isolated from sea squirt (munggae) jeotgal, a traditional Korean fermented seafood. BS2 was identified as B. velezensis by molecular biological methods.B. velezensis BS2 grows well at 15% NaCl and at 10oC. When B. velezensis BS2 was cultivated in TSB broth for 96 h at 37°C, the culture showed the highest fibrinolytic activity (131.15 mU/µl) at 96 h. Three bands of 27, 35 and 60 kDa were observed from culture supernatant by SDS-PAGE, and fibrin zymography showed that the major fibrinolytic protein was the 27 kDa band. The gene (aprEBS2) encoding the major fibrinolytic protein was cloned, and overexpressed in heterologous hosts, B. subtilis WB600 and E. coli BL21 (DE3). B. subtilis transformant showed 1.5-fold higher fibrinolytic activity than B. velezensis BS2. Overproduced AprEBS2 in E. coli was purified by affinity chromatography. The optimum pH and temperature were pH 8.0 and 37°C, respectively. Km and Vmax were 0.15 mM and 39.68 µM/l/min, respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA was used as the substrate. AprEBS2 has strong α-fibrinogenase and moderate β-fibrinogenase activity. Considering its high fibrinolytic activity, significant salt tolerance, and ability to grow at 10°C, B. velezensis BS2 can be used as a starter for jeotgal.

FIRST REPORT ON FIBRINOLYTIC AND THROMBOLYTIC ACTIVITY OF EUTYPHOEUS GAMMIEI AN EARTHWORM SPECIES COLLECTED FROM TRIPURA, NORTHEAST INDIA

Objective: The present investigation for the first time evaluated the in vitro fibrinolytic and thrombolytic activities of crude extracts from Eutyphoeus gammiei, native, large size earthworm of Tripura, Northeast, India. The present study was designed to evaluate the therapeutic use of the organism E. gammiei as a source of fibrinolytic and thrombolytic agent(s).Methods: The fibrinolytic activity was studied using by fibrin plate and zymography assays. Thrombolytic assay was carried out according to Prasad et al. (2006) using whole blood.Results: The results obtained clearly indicated E. gammiei as a potential source of fibrinolytic and thrombolytic agents. Both in fibrin plate assay and thrombolytic assay with whole blood, E. gammiei crude homogenate showed similar and close results in respect to that of streptokinase. Fibrin zymography also showed antifibrinolytic activity with producing clear bands. Dose and time dependency also is evident from the results.Conclusion: The results of the present study conclude that the studied earthworm species E. gammiei possessed profound fibrinolytic and thrombolytic activity on human blood and E. gammiei might prove to be useful alternative source for the development of new drugs for treatments involving blood coagulation and fibrinolysis.

DOWN REGULATION OF u-PA BY A NUTRIENT MIXTURE IN HEMANGIOMA (EOMA) CELLS BY INDUCING CASPASE-DEPENDENT APOPTOSIS

To examine, whether the antitumor effects of a specific nutrient mixture are due to induction of apoptosis by inhibition of u-PA. A nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract which has showed anticancer activity against a number of cancer cell lines was used as an experimental composition. EOMA cells were grown in appropriate media with antibiotics in 24-well tissue culture plates. At near confluence, the cells were treated with nutrition mixture at 10, 100, 1000µg/ml in triplicate. Analysis of u-PA activity was carried out by fibrin zymography. Morphological changes and caspase activation associated with apoptosis induction was checked by H&E staining and Live Green caspase assay, respectively. Apoptosis inducing anticancer drug camptothecin (10µM) was used as positive control. The nutrition mixture exhibited dose response toxicity with maximum toxicity 55% (p < 0.001) at 1000µg/ml. EOMA cells expressed u-PA, which was inhibited by nutrition mixture in a dose-dependent manner. The caspase analysis revealed a dose dependent increase in apoptosis of EOMA hemangioma cells, with an increasing apoptosis observed at 100μg/ml, and maximum at 1000μg/ml. Cells treated with nutrition mixture showed significantly more apoptotic changes than the control or camptothecin-treated cells. These results suggest that NM may induce apoptosis of hemangioma cells in vitro thus warranting further investigation.

Gene Cloning, Expression, and Properties of a Fibrinolytic Enzyme Secreted by Bacillus pumilus BS15 Isolated from Gul (Oyster) Jeotgal

A Bacillus strain, BS15, showing strong fibrinolytic activity, antibacterial activity, and salt tolerance was isolated from gul (oyster) jeotgal, a Korean fermented sea food. BS15 was identified as B. pumilus. B. pumilus BS15 was able to grow in LB broth with 18% (w/v) NaCl. When culture supernatant was analyzed by SDS-PAGE, 22, 27, 35, and 60 kDa proteins were observed. The 27 kDa protein was determined to be major fibrinolytic enzyme by fibrin zymography. The gene (aprEBS15) was cloned in pHY300PLK, a Bacillus-E. coli shuttle vector. A B. subtilis transformant (TF) harboring pHYBS15 showed higher fibrinolytic activity than B. pumilus BS15, and produced the same 27 kDa protein. aprEBS15 was overexpressed in E. coli BL21 (DE3), and recombinant enzyme (AprEBS15) was purified. The optimum pH and temperature of AprEBS15 were pH 8.0 and 40°C, respectively. Km and Vmax values were 0.26 mM and 21.88 µmol/L/min, respectively. B. pumilus BS15 can be used as a starter for jeotgals and other fermented foods with high salinities.