• 한국미생물·생명공학회지
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• 제51권2호
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• pp.167-173
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• 2023

Proteolytic enzymes secreted by Aspergillus, as pathogenicity factors, affect blood coagulation and fibrinolysis, and therefore the target proteins of their action in the bloodstream are of significant interest. In the present study, the action of the isolated protease of A. terreus 2 on different human plasma proteins was shown. The protease of A. terreus 2 exhibited the highest proteolytic activity against hemoglobin, which was 2.5 times higher than the albuminolytic activity shown in both of the protein substrates used. In addition, the protease has significant ability to hydrolyze both fibrin and fibrinogen. However, the inability of the A. terreus 2 protease to coagulate rabbit blood plasma and coagulate human and bovine fibrinogen indicates the severity of the enzyme's action on human blood coagulation factors. It should be considered as a potential indicator of this isolated protease's participation in fungal pathogenesis. The protease shows no hemolytic activity. Furthermore, its activity is insignificantly inhibited by thrombin inhibitors, and is not inhibited by plasmin inhibitors.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2090-2099
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• 2015

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40oC), metal ions (Mn²⁺, Ca²⁺, K²⁺, and Mg²⁺), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

• 한국양봉학회지
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• 제33권4호
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• pp.303-306
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• 2018

Drones of honeybee (Apis mellifera) have been regarded as a useful value only when mating with queen bee. However, the drone pupae have been reported to be nutritionally valuable, and a potential beekeeping product. In this study, drone pupae extracted with 5% acetic acid were used to measure anti-thrombosis related fibrinolytic activity using Strup and Mullertz fibrin plate method. As a result, the drone pupae extract showed higher effect of fibrinolytic activity(clear zone diameter 20.83mm) compared to the human plasmin (clear zone diameter 12.93mm) used as a positive control. It was suggested that the extract of drone pupae can be developed as a functional material helping prevention or treatment of various vascular diseases.

• KSBB Journal
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• 제5권2호
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• pp.183-189
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• 1990

1,000l 규모의 인뇨로부터 순수한 urokinase를 정제하는 새로운 공정이 고안되었다. 2가 금속이온인 아연을 첨가하여 선택적으로 urokinase를 침전시켰으며, 이 방법은 뇨 중에 포함된 다른 단백질을 100~1,000배 농축할때도 사용할 수 있으며 10~20배의 경제효과를 얻었다. 침전물 속에 포함된 urokinate를 경제적으로 용출할기 위하여 0.1M EDTA/0.5M glycine, pH 9.0용액이 조성되었고, 용출액속에 포함된 높은 농도의 salt와 ion,뇨색소 등이 CM-Toyopeal column에 의해 신속하고 효과적으로 제거되었다. 최종적으로 사용된 benzamidine-Sepharose chromatog-raphy는 urokinase 정졔에 매우 효과적이었다. 이상의 공정을 통해, 1,000liter의 인뇨로부터 3$\times$$O^6IO$의 urokinase가 순수 정제 되었으며, 수욜은 약 50%, 정제도는 1,300배이었다. 또한 urokinase중에 12~14%의 pro-urokinase가 함유되어 있음이 확인되었다.

• 동의생리병리학회지
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• 제18권5호
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• pp.1494-1504
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• 2004

The present paper reports the effects of Hwaotang an atherosclerosis using a spontaneous experimental model, We have also investigated the pharmacological effect of Hwaotang on collagen- and ADP-induced blood platelet aggregation, thrombin-induced conversion of fibrinogen and fibrinolysis in in vitro experiments, and various effects on stimuli-induced superoxide generation in human neutrophils. Hwao-tang was shown to have inhibitory effect on collagen- and ADP-induced blood platelet aggregation, on thrombin-induced conversion of fibrinogen to fibrin and on the activity of plasminogen or plasmin. Hwao-tang also significantly inhibited fMLP-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid. Hwao-tang inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. In conclusion, the protection of extracts of Hwao-tang on the ischemic infarction induced artificially might be involved to their inhibition of thrombotic action. The results also indicate that Hwao-tang exerts the effects on superoxide generation related to the inhibition of neutrophil functions.

• 한국동물학회지
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• 제32권1호
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• pp.48-54
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• 1989

인체의 자궁암과 간암조직들이 나타내는 몇 종류의 단백질 분해효소들과 Anti-trypsin의 활성도를 정상조직의 것들과 비교하여서, 암의 종양성 형질과 단백질 분해활성의 변화사이에 어떤 상관관계가 있는지를 조사하였다. Casein과 Insulin의 분해 활성도는, 자궁암에서 2-3배 정도 증가하는 반면, 간암에서는 1/2에서 1/5정도로 감소하였다. 이와는 대조적으로, Anti-trypsin의 활성도는 자궁암에서 약 1/10정도로 감소하였고 간암에서는 2배 가량 증가하였다. 한편, Plamin-like enzyme과 Plasminogen activator의 활성도는 자궁암과 간암조직 모두에서 정상 조직에서보다 10-20% 정도 높게 나타났다. 이러한 결과는, 정상조직 내의 단백질 분해활성도가 단백질 분해효소들과 이들의 활성을 저해하는 단백질들의 균형에 의하여 조절됨을 시사하며, 암조직들에서는 각 암조직들의 종양특이성에 따라 단백질 분해효소와 저해단백질들 사이의 균형이 깨어짐에 따라 단백질 분해활성도가 다르게 나타남을 보여준다.

• BMB Reports
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• 제47권12호
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• pp.691-696
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• 2014

${\alpha}$-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. ${\alpha}$-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, ${\alpha}$-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of ${\alpha}$-enolase. In this study, we report that this peptide competes with cellular ${\alpha}$-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer.

• BMB Reports
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• 제33권2호
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• pp.112-119
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• 2000

Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitor, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1, 2 and 3 (DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki = $3.6{\times}10^{-9}\;M$), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki = $6.2{\times}10^{-8}\;M$). Pre-treatment of the 33 mg/kg of DI-mixture (active fractions from $C_{18}$ open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권2호
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• pp.117-127
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• 2001

The high-level expression of a human single chain urokinase-type plasminogen activator (scu-PA) was achieved by employing a methotrexate (MTX)-dependent gene amplification system in Chinese hamster ovary (CHO) cells. By cotransfecting and coamplifying a scu-PA expression plasmid and dihydrofolate reductase (DHFR) minigene, several scu-PA expressing CHO cell lines were selected and gene-amplified. These recombinant cell lines, NGpUKs, secreted a completely processed scu-PA of 54 kD and up to 60mg/L was accumulated in the culture medium when they were adapted to an optimal MTX concentration. Over 95% of the scu-PA expressed was secreted in the culture medium and identified having the proper function of a plasminogen activator when activated by plasmin. Based on a genomic Southern analysis, a representative subclone, MGpUK-5, exhibited MTX-dependent scu-PA gene amplification, plus the initial single-copy gene of scu-PA eventually turned into about 150 copies of the amplified gene of scu-PA after gradual adaptation to 2.0$\mu$M of MTX. Meanwhile, the transcripts kof the scu-PA gene increased, although -early saturation of transcription was identified at 0.1$\mu$M of MTX. The scu-PA production by the MGpUK-5 subclone also increased relative to the gene amplification and increased transcripts, however, the relationship was not linearly proportional. Accordingly, since the MGpUK cell lines expressed elevated levels of enzymatically active scu-PA, these cell lines could be applied to the largescale production of scu-PA.

• Journal of Periodontal and Implant Science
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• 제38권4호
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• pp.629-638
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• 2008

Purpose: The purposes of this study were to compare and quantify the expression of Stromelysin-1 and MT-MMP-1 in the gingival tissues of patients with type 2 diabetes mellitus(DM) and healthy adults with chronic periodontitis. Materials and Methods: Gingival tissue samples were obtained during periodontal surgery or tooth extraction. According to the patient's systemic condition & clinical criteria of gingiva, each gingival sample was devided into three groups. Group 1 (n=8) is clinically healthy gingiva without bleeding and no evidence of bone resorption or periodontal pockets, obtained from systemically healthy 8 patients. Group 2 (n=8) is inflammed gingiva from patients with chronic periodontitis. Group 3 (n=8) is inflammed gingiva from patients with chronic periodontitis associated with type 2 DM. Tissue samples were prepared and analyzed by Western blotting. The quantification of Stromelysin-1 and MT-MMP-1 were performed using a densitometer and statistically analyzed by one-way ANOVA followed by Tukey test. Results: In the analysis of expression levels, Stromelysin-1 and MT-MMP-1 expressions were similar in group 1 and 2. Stromelysin-1 and MT-MMP-1 expressions was more increased in group 3 than group 1, 2. The difference between group 3 and group 1, 2 was statistically significant. Also, in the interrelationship of Stromelysin-1 and MT-MMP-1 expressions, expressions of Stromelysin-1 and MT-MMP-1 showed increasing tendency in chronic periodontitis associated with type 2 DM and it seems that the MT-MMP-1 expressions were increasing in proportion to Stromelysin-1 expressions. Conclusion: It is suggested that Stromelysin-1 and MT-MMP-1 may be partly involved in the progression of periodontal inflammation associated with type 2 DM, as related to a metabolism of other factors, such as AGE, plasmin and other inflammatory mediators. Therefore, the expression levels of Stromelysin-1 and MT-MMP-1 can be inflammatory markers of periodontal inflammed tissue with type 2 DM.