• BMB Reports
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• 제40권5호
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• pp.832-838
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• 2007

A novel inhibitory protein against blood coagulation factor Va (FVa) was purified from muscle protein of granulated ark (Tegillarca granosa, order Arcoida, marine bivalvia) by consecutive FPLC method using anion exchange and gel permeation chromatography. In the results of ESI-QTOF tandem mass analysis and database research, it was revealed that the purified T. granosa anticoagulant protein (TGAP) has 7.7 kDa of molecular mass and its partial sequence, HTHLQRAPHPNALGYHGK, has a high identity (64%) with serine/threonine kinase derived from Rhodopirellula baltica (order Planctomycetales, marine bacteria). TGAP could potently prolong thrombin time (TT), corresponding to inhibition of thrombin (FIIa) formation. Specific factor inhibitory assay showed that TGAP inhibits FVa among the major components of prothrombinase complex. In vitro assay for direct-binding affinity using surface plasmon resonance (SPR) spectrometer indicated that TGAP could be directly bound with FVa. In addition, the binding affinity of FVa to FII was decreased by addition of TGAP in dose-dependant manner ($IC_{50}$ value = 77.9 nM). These results illustrated that TGAP might interact with a heavy chain of FVa ($FVa_H$) bound to FII in prothrombin complex. The present study elucidated that non-cytotoxic T. granosa anticoagulant protein (TGAP) bound to FVa can prolong blood coagulation time by inhibiting conversion of FII to FIIa in blood coagulation cascade. In addition, TGAP did not significantly (P < 0.05) show fibrinolytic activity and cytotoxicity on venous endothelial cell line (ECV 304).

• Bulletin of the Korean Chemical Society
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• 제33권10호
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• pp.3248-3252
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• 2012

Vibrio extracellular metalloprotease (vEP), secreted from Vibrio vulnificus, shows various proteolytic function such as prothrombin activation and fibrinolytic activities. Premature form of vEP has an N-terminal (nPP) and a C-terminal (C-ter100) region. The nPP and C-ter100 regions are autocleaved for the matured metalloprotease activity. It has been proposed that two regions play a key role in regulating enzymatic activity of vEP. Especially, C-ter100 has a regulatory function on proteolytic activity of vEP. C-ter100 domain has been cloned into the E. coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using gel-filtration chromatography followed by affinity chromatography. To understand how C-ter100 modulates proteolytic activity of vEP, structural studies were performed by heteronuclar multi-dimensional NMR spectroscopy. Backbone $^1H$, $^{15}N$ and $^{13}C$ resonances were assigned by data from standard triple resonance and HCCH-TOCSY experiments. The secondary structures of vEP C-ter100 were determined by TALOS+ and CSI software based on hydrogen/deuterium exchange. NMR data show that C-ter100 of vEP forms a ${\beta}$-barrel structure consisting of eight ${\beta}$-strands.

• KSBB Journal
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• 제16권3호
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• pp.237-240
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• 2001

본 연구에서는 keratinocytes 세포를 이용하여 VEGF가 세포의 증식, MMP-2 및 plasmin 분비에 미치는 영향을 조사하였다. 그 결과 VEGF는 keratinocytes의 세포증식을 약 2.5배 상승시켜 내피세포 뿐반 아니라 상피세포에서도 강력한 mitogen임을 확인하였으며 submaximal effect를 보이는 농도는 10 ng/mL이었다. VEGF의 농도 증가에 따른 MMP-2의 분비에 미치는 효과 역시 10 ng/mL에서 최대 효과를 나타냈으며 VEGF 처리 후 1시간만에 확인 가능한 MMP-2 밴드가 나타났다. 한편, MMP-2의 분비 증가와 함께 plasmin의 분비 증가가 수반되는지를 확인한 결과 VEGF를 첨가했을 때 약 3배 정도 plasmin의 분비량이 증가함을 알 수 었었다. 이러한 결과는 VEGF의 성장촉진 효과와 관련자어 볼 때 MMP-2와 plasmin이 keratinocytes 세포의 성장과 이동에 부분적으로 관여할 것임을 시사하고 있다.

• Preventive Nutrition and Food Science
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• 제17권1호
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• pp.36-45
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• 2012

To produce novel cheese-like fermented soybean, the solid-state fermentation of roasted soybean flour (RSF) was performed using 1.0% inoculum Bacillus subtilis HA and Lactobacillus plantarum, with the initial 60% substrate moisture for 10 hr at $42^{\circ}$, resulting in pH 6.5, 0.82% acidity, 3.5% mucilage, 14.3 unit/g protease activity, 7.6 unit/g fibrinolytic activity, 216 mg% tyrosine content and $1.7{\times}10^{10}$ CFU/g of viable cell counts. After the second lactic acid fermentation with 10~30% skim milk powder, the fermented RSF resulted in an increase in acidity with 1.64~1.99%, tyrosine content with 246~308 mg% and protease activity in the range of 5.2~17.5 unit/g and 0.966 water activity. Viable cell counts as probiotics indicated $1.6{\times}10^8$ CFU/g of B. subtilis and $7.3{\times}10^{10}$ CFU/g of L. plantarum. The firmness of the first fermented RSF with 2,491 $g{\cdot}{\o}mm^{-1}$ greatly decreased to 1,533 $g{\cdot}{\o}mm^{-1}$ in the second fermented RSF, although firmness was slightly increased by adding a higher content of skim milk. The consistency of the second fermented RSF also decreased greatly from 55,640 to 3,264~ 3,998 in the presence of 10~30% skim milk. The effective hydrolysis of soy protein and skim milk protein in the fermented RSF was confirmed. Thus, the second fermented RSF with a sour taste and flavor showed similar textural properties to commercial soft cheese.

• BMB Reports
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• 제37권3호
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• pp.298-303
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• 2004

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

• 대한한방내과학회지
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• 제17권1호
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• pp.34-50
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• 1996

The present experiment was desinged to investigate the effects of Tongdosan water extracts on the Cardiovascular System in the Experimental Animals. Thus, the changes of blood pressure and heart rate were measured after oral admini- stration. Measurment of Mortality rate was observed for measuring the effect of Tongdosan water extract. Tongdosan water extract against pulmonary thrombo- embolism induced by collagen the mixture(0.1ml/10g, 2mg/kg B.W) plus serotonin(5mg/kg B.W) in mouse. The effect of Tongdosan water extract was examined by observing the change of collagen-induced platelet aggregation, coagulation activity, ex vivo and in vitro fibrinolytic activity of euglobulin fraction in rats. The results were summarized as followings. 1. Tongdosan dropped the blood pressure in spontaneous hypertensive rat. 2. The drug increased the auricular blood flow in rabbit. 3. The drug relaxed the artery contraction by pretreated norepinephrine in rat. 4. The drug inhibited the death rate of mouse which was led to thromboembo- lism by serotonin and collagen. 5. The drug inhibited the platelet aggregation in rat. 6. The drug prolonged the prothrombin time and activated partial thromboplastin time on the test of plasma coagulation factor activity in rat, but was not valuable. 7. The drug reduced the fibrinogen lyses time of rat ex vivo assay and lyses area was increased. 8. Tongdosan reduced fibrinogen lyses time of rat in vitro assay. According to the above mentioned results, Tongdosan increased the blood flow and dropped the blood pressure by the dilation of blood vessel. And the drug presented the antithrombin acivity, inhibited the platelet aggregation.

• 동의생리병리학회지
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• 제16권2호
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• pp.245-256
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• 2002

This studt was investigated to prove the effect of SPBST on the hypertension, the thrombosis and the brain damage. The results were as follows; 1. SPBST affected the htpertension as adepressant, but insignificant. 2. SPBST decreased significantly dopamine, aldosterone but ineffective on the epinephrine, norepinephrine and renin activity. 3. SPBST increased the NO product but insignificant. 4. SPBST had a death suppression effect by 50% in pulmonary thrombosis inducement experiment and activated slightly on the fibrinolytic activity. 5. SPBST suppressed significantly platelet diminution and prolonged insignificantly PT and APTT. 6. On the measure of the blood flow rate induced by the thrombus, in vivo SPBST accelerated the blood flow rate, in vitro insignificant. 7. SPBST had no toxicity on the PC12 cell and B103 cell induced by amyloid β protein (-35) and a protective effect, in proportion to the density. 8. SPBST decreased significantly coma duration time in a Infatal dose of KCN and showed 50% of survival rate in a fatal dose. 9. SPBST decreased significantly ischemic area and edema incited by the MCA blood flow block. These results indicate that SPBST can be used in hypertension, the thrombosis, the brain damage, the ischemic cerebral infarction and the acute stage of the brain damage. Further study will be needed about the functional mechanism and etc.

• 대한본초학회지
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• 제25권4호
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• pp.1-6
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• 2010

Objective : The purpose of this study is to compare anti-thrombotic activities of 4 Rhubarb species. Methods : Rhubarb has been used as medical and edible resources worldwide for the therapy of cancer, constipation and inflammation, etc. To compare the anti-thrombotic activities among the species within rhubarb, we investigated and compared the inhibitory properties of water extracts from R. palmatum(RPE), R. officinale(ROE), R. nobile(RNE), and R. franzenbachii (RFE) on the fibrinolysis, blood coagulation and platelet aggregation. APTT and PT, blood coagulation time, were measured by apparatus of auto blood coagulation analysis that called ACL-7000. The measurement of fibrinolysis was measured and compares with the width of clear zone from melted fibrin plate. The platelet aggregation was measured and compared with inhibition rate of aggregation. Results : We found out the best species from rhubarb showing the significant fibrinolytic, anti-coagulant and anti-platelet aggregation activity. Our results show that Rheum nobile (RNE) has the most effective anti-thrombotic activity among 4 tested rhubarb. Conclusions : This study, therefore, RNE will be able to be used the development of drug which is thrombosis treatment.

• 한국균학회지
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• 제30권2호
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• pp.142-146
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• 2002

눈꽃동충하초(Paecilomyces japonica)를 이용한 새로운 약용민속주를 개발하기 위하여 먼저 알콜발효조건을 검토하였다. 1.0%의 눈꽃동충하초를 함유한 덧밥에 코지를 10%첨가하고 Saccharomyces cerevisiae를 이용하여 제조한 주모를 첨가한 후 $25^{\circ}C$에서 15일간 발효 시켰을 때 에탄올이 가장 많이 생성되었다. 눈꽃동충하초를 0.1%, 0.5%, 1.0%, 2.0%씩 각각 첨가하여 제조한 동충하초 민속주들의 관능검사와 생리기능성을 조사한 결과 눈꽃동충하초를 0.1% 첨가하여 제조한 D-1 눈꽃동충하초 민속주가 기호도가 제일 높았고 혈전용해활성과 아질산염 제거 작용이 각각 11.2U와 19.8%로 대조구인 동충하초를 첨가하지 않고 제조한 민속주보다 우수하였다.

• 대한한방내과학회지
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• 제18권1호
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• pp.97-113
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• 1997

The present experiment was designed to investigate the effects of Sambutang water extracts on the serum cholesterol levels and the cardiovascular system in the experimental animals. Thus, the changes of blood pressure and heart rate were measured after oral administration. Measurment of Mortality rate was observed for measuring the effect of Sambutang water extract. Sambutang water extract against pulmonary thromboembolism induced by collagen the mixture(0.1 ml/10 g, 2 mg/kg) plus serotonin(5 mg/kg) in mouse. The effect of Sambutang water extract was examined by observing the change of collagen-induced platelet aggregation, coagulation activity, ex vivo and in vitro fibrinolytic activity of euglobulin fraction in rats. The results were summarized as follows. 1. Sambutang decreased the serum cholesterol levels in rats. 2. Sambutang dropped the blood pressure in spontaneous hypertensive rat. 3. The drug increased the auricular blood flow in rabbit. 4. The drug relaxed the artery contraction by pretreated norepinephrine in rat. 5. The drug inhibited the death rate of mouse which was led to thromboembolism by serotonin and collagen. 6. The drug inhibited the platelet aggregation in rat. 7. The drug prolonged the prothrombin time and activated partial thromboplastin time on the test of plasma coagulation factor activity in rat, but was not valuable. 8. The drug increased the antithrombin activity in rat and the fibrinogen lysis time was reduced and lysis area was increased. 9. Sambutang reduced fibrinogen lysis time of rat in vitro assay. According to the above mentioned results. Sambutang increased the blood flow and dropped the blood pressure by the dilation of blood vessel. And the drug presented the antithrombin activity, inhibited the platelet aggregation.