• Bulletin of the Korean Chemical Society
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• 제31권9호
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• pp.2607-2612
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• 2010

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.

• KSBB Journal
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• 제17권6호
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• pp.582-585
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• 2002

백합(Lilium longiflorum)으로부터 수집한 화분에 대하여 기내배양, Agrobacterium 및 vacuum infiltration과정을 이용한 형질전환 그리고 kanamycin배지에서의 선별배양을 통하여 PCR에 의하여 증폭된 1.7 kb의 human tissue plasminogen activator(tPA) cDNA의 발현을 분석하였다. 16시간 정도 배양한 신장화분관의 western blotting결과, human standard와 유사한 크기로의 발현을 확인하였다. 이로써 백합화분은 신속한 단백질발현의 분석을 위한 일회성 생산숙주로서의 가능성을 제시하고 있다.

• 한국수정란이식학회지
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• 제28권3호
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• pp.257-263
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• 2013

The endometrium undergoes a cyclic growth and tissue remodeling as changes of epithelial cells, and plasminogen activators (PAs) are related to endometrium tissue remodeling. This study was to evulate expression of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) in porcine uterine epithelial cells. In results, the uPA and tPA were expressed in uterine tissue, epithelium and secretory glands in porcine endometrial cell. In addition, the uPA and tPA were expressed in cultured epithelial cells, and it were mainly expressed in cytoplasm. In porcine uterine tissue and epithelial cells, uPA activity was higher than activity in tPA. In PAs mRNA expression levels, uPA mRNA level was significantly higher than tPA mRNA level (P<0.05). The fluorescence intensity of uPA protein was also higher than fluorescence intensity of tPA protein, and uPA protein expression was significantly higher than in tPA protein expression (P<0.05). Therefore, we suggest that a physiological function in porcine uterine epithelial cells should be more influenced by uPA than in tPA during pre-ovulatory phase.

• Biotechnology and Bioprocess Engineering:BBE
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• 제1권1호
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• pp.32-35
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• 1996

Amoving aeration-membrane (MAM) bioreactor was employed for the production of 2$\mu$g/mL of tissue type Plasminogen Activator (tPA)in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating condition, shear stress was as low as 0.65 dynes/$\textrm{cm}^2$ at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2$\mu$g tPA/mL while maintaining a high cell denisty of 1.0$\times$107 viable cell/mL.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.522-525
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• 1988

무혈청 배지에 생산촉진제로 30$\mu$g/m$\ell$의 Heparin 을 첨가해 정상인의 섬유 세포로부터 상업적으로 tPA를 생산할 수 있는 방법의 개발과, 효과적인 tPA 생산을 위해 대량 배양에 적합한 무혈청 배지의 조성을 확립했다. 이 방법으로 연속배양 공법하에서 매일 1.1gram의 tPA가 생산될 수 있으며, 이 생산성은 tPA 생산 단가를 크게 낮출 분만 아니라 무혈청 배지의 사용으로 tPA의 순수 정제 과정을 크게 단축시킬 수 있다. 또한 이 세포에서 생산되는 tPA 는 fibrin lysis 시험결과 섬유질 분해능력이 높음이 입증되었으며, ELISA결과와도 상충했다.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.75-82
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• 2005

This study were examined whether plasminogen activators (PAs) are produced by porcine fresh or frozen-thawed cumulus-oocytes complexes (COCs) and cumulus cell free-oocytes. In fresh or frozen-thawed COCs and oocytes for 0 hour cultured, no activity of PAs was detected. However, at 24 hours of culture urokinase-type plasminogen activator (uPA) was detected in COCs and denuded oocytes. In the frozen-thawed COCs and cumulus cell free-oocytes cultured for 24 hours, no PAs were observed. After COCs were cultured for 48 hours, tissue-type plasminogen activator (tPA) and tPA-PAI were observed in COCs only. In the frozen-thawed COCs and cumulus cell free-oocytes cultured for 48 hours, no PAs were observed. These results suggest that uPA, tPA and tPA-PAI are produced by porcine COCs, but only uPA by oocytes during maturation for 24 hours. Only tPA, and tPA-PAI are produced by COCs cultured for 48 hours, and no PAs are produced by denuded-oocytes cultured for 48 hours. In all of the frozen-thawed groups, no PAs are observed by COCs and denuded-oocytes.

• Animal cells and systems
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• 제10권2호
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• pp.79-83
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• 2006

Tissue plasminogen activator (tPA) is used to lyse clots and reperfuse brain in ischemic stroke. However, sideeffects of intracerebral hemorrhage (ICH) and edema limit their clinical application. In part, these phenomena has been linked with elevations in matrix metalloproteinase-9 (MMP-9) in neurovascular unit. However little is known about their regulatory signaling pathways in brain cells. Here, I examine the role of MAP kinase pathways in tPA-induced MMP-9 regulation in rat cortical astrocytes. tPA $(1-10\;{\mu}g/ml)$ induced dose-dependent elevations in MMP-9 and MMP-2 in conditioned media. Although tPA increased phosphorylation in two MAP kinases (ERK, JNK), only inhibition of the JNK pathway by the JNK inhibitor SP600126 significantly reduced MMP-9 upregulation. Neither ERK inhibition with U0126 nor p38 inhibition with SB203580 had any significant effects. Taken together, these results suggest that c-jun N-terminal kinase (JNK) plays an essential role for tPA-induced MMP-9 upregulation.

• Journal of Ginseng Research
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• 제37권4호
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• pp.401-412
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• 2013

Korean Red Ginseng (KRG) is an oriental herbal preparation obtained from Panax ginseng Meyer (Araliaceae). To expand our understanding of the action of KRG on central nervous system (CNS) function, we examined the effects of KRG on tissue plasminogen activator (tPA)/plasminogen activator inhibitor-1 (PAI-1) expression in rat primary astrocytes. KRG extract was treated in cultured rat primary astrocytes and neuron in a concentration range of 0.1 to 1.0 mg/mL and the expression of functional tPA/PAI-1 was examined by casein zymography, Western blot and reverse transcription-polymerase chain reaction. KRG extracts increased PAI-1 expression in rat primary astrocytes in a concentration dependent manner (0.1 to 1.0 mg/mL) without affecting the expression of tPA itself. Treatment of 1.0 mg/mL KRG increased PAI-1 protein expression in rat primary astrocytes to $319.3{\pm}65.9%$ as compared with control. The increased PAI-1 expression mediated the overall decrease in tPA activity in rat primary astrocytes. Due to the lack of PAI-1 expression in neuron, KRG did not affect tPA activity in neuron. KRG treatment induced a concentration dependent activation of PI3K, p38, ERK1/2, and JNK in rat primary astrocytes and treatment of PI3K or MAPK inhibitors such as LY294002, U0126, SB203580, and SP600125 (10 ${\mu}M$ each), significantly inhibited 1.0 mg/mL KRG-induced expression of PAI-1 and down-regulation of tPA activity in rat primary astrocytes. Furthermore, compound K but not other ginsenosides such as Rb1 and Rg1 induced PAI-1 expression. KRG-induced up-regulation of PAI-1 in astrocytes may play important role in the regulation of overall tPA activity in brain, which might underlie some of the beneficial effects of KRG on CNS such as neuroprotection in ischemia and brain damaging condition as well as prevention or recovery from addiction.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.463-468
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• 2011

This study investigated the changes of plasminogen activators (PAs) activity, expression and localization of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) during the estrous cycle in pigs. Estrous cycle was sorted into three group by pre-ovulation (Pre-Ov), post-ovulation (Post-Ov) and early to mid-luteal stages (Early to mid-L). Analysis for immunohistochemistry was confirmed by location of tPA and uPA. Porcine uterus tissue was cut into $1{\times}1$ cm squares, and were incubated in DMEM/F-12 medium for 1 h at $38^{\circ}C$, 5% $CO_2$ for measurement of PA activity. Western blotting was implemented for measurement of PA quantity. In results, the blood vessels and secretory glands were increased in Post-Ov stage than Pre-Ov and Early to mid-L stages. The tPA and uPA was located mainly within lumen of blood vessels and secretory glands. The PA activity in Post-Ov ($0.99{\pm}0.03$) stage were significantly (p<0.01) higher than Pre-Ov stage ($0.51{\pm}0.03$) and Early to mid-L stage ($0.21{\pm}0.04$). Expression of PAs were significantly (p<0.05) higher in Early to mid-L stage than other stages. These results indicate that PAs activity and expression may change in uterus tissue during the estrous cycle in pigs.

• Journal of Korean Neurosurgical Society
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• 제66권2호
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• pp.144-154
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• 2023

Objective : Stroke caused from large vessel occlusion (LVO) has emerged as the most common stroke subtype worldwide. Intravenous tissue plasminogen activator administration (IV-tPA) and additional intraarterial thrombectomy (IA-Tx) is regarded as standard treatment. In this study, the authors try to find the early recanalization rate of IV-tPA in LVO stroke patients. Methods : Total 300 patients undertook IA-Tx with confirmed anterior circulation LVO, were analyzed retrospectively. Brain computed tomography angiography (CTA) was the initial imaging study and acute stroke magnetic resonance angiography (MRA) followed after finished IV-tPA. Early recanalization rate was evaluated by acute stroke MRA within 2 hours after the IV-tPA. In 167 patients undertook IV-tPA only and 133 non-recanalized patients by IV-tPA, additional IA-Tx tried (IV-tPA + IA-Tx group). And 131 patients, non-recanalized by IV-tPA (IV-tPA group) additional IA-Tx recommend and tried according to the patient condition and compliance. Results : Early recanalization rate of LVO after IV-tPA was 12.0% (36/300). In recanalized patients, favorable outcome (modified Rankin Scale, 0-2) was 69.4% (25/36) while it was 32.1% (42/131, p<0.001) in non-recanalized patients. Among 133 patients, non-recanalized after intravenous recombinant tissue plasminogen activator and undertook additional IA-Tx, the clinical outcome was better than not undertaken additional IA-Tx (favorable outcome was 42.9% vs. 32.1%, p=0.046). Analysis according to the perfusion/diffusion (P/D)-mismatching or not, in patient with IV-tPA with IA-Tx (133 patients), favorable outcome was higher in P/D-mismatching patient (52/104; 50.0%) than P/D-matching patients (5/29; 17.2%; p=0.001). Which treatment tired, P/D-mismatching was favored in clinical outcome (iv-tPA only, p=0.008 and IV-tPA with IA-Tx, p=0.001). Conclusion : The P/D-mismatching influences on the recanalization and clinical outcomes of IV-tPA and IA-Tx. The authors would like to propose that we had better prepare IA-Tx when LVO is diagnosed on initial diagnostic imaging. Furthermore, if the patient shows P/D-mismatching on MRA after IV-tPA, additional IA-Tx improves treatment results and lessen the futile recanalization.