• Title, Summary, Keyword: urokinase-type plasminogen activator

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Secretion of Active Urokinase-type Plasminogen Activator from the Yeast Yarrowia lipolytica

  • Ryu, Ho-Myoung;Kang, Woo-Kyu;Kang, Hyun-Ah;Kim, Jeong-Yoon
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.8 no.2
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    • pp.162-165
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    • 2003
  • In order to study the secretion of the human urokinase-type plasminogen activator, u-PA, from the yeast yarrowia lipolytica, three kinds of integrative expression vector were constructed. These vectors differed only in their secretion control legions, pre-, pre-dip-(dipeptide Stretch) or pre-dip-pro sequences of the alkaline extracellular protease, which were joined inflame to the human u-PA cDNA. The recombinant Y. lipolytica Strains, transformed with the expression vectors, secreted the hyperglycosylated u-PA. A fibrin plate assay of the culture supernatants showed that the hyperglycosylated u-PA proteins could catalyze fibrinolysis, and that the pre-dip sequence was the most efficient secretory signal for the secretion of the u-PA from Y. lipolyica. This result suggests that Y. lipolytica can be developed as a potential host for the production of recombinant human u-PA.

Kinetics of Converting Single Chain Urokinase Type Plasminogen Activator into Two Chain Plasminogen Activator in Cultivating HEK Cells with Low Serum Containing Medium (저혈청 배지에서 생산된 scu-PA의 tc-PA로의 전환에 관한 연구)

  • 김영남;김현구
    • KSBB Journal
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    • v.9 no.1
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    • pp.48-54
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    • 1994
  • A modified amidolytic assay and a fibrin plate method were used to accurately measure the concentration of single chain urokinase type plasminogen activator (scu-PA) and two-chain urokinase type plasminogen activator (tc-PA) in the spent media. $1.65{\times}10^6$(viable cells/ml) of maximum cell density and 1670(IU/ml) of scu-PA concentration were obtained in 1% serum containing medium. The overall conversion ratio from scu-PA to tc-PA was less than 10%. In the results of batch cultivation in a spinner vessel, $4.43{\times}10^6(total cells/ml)$ of maximum cell density and 1560(IU/ml) of scu-PA concentration was observed. The maximum scu-PA concentration and specific scu-PA Productivity were obtained in 1760(IU/ml) and $3.13{\times}10^{-4}(IU/cell)$, respectively, from perfusion cultivation. The conveysion ratios from batch, fed-batch and perfusion cultivations were less than 12%, which means that about 90% of scu-PA secreted from the cells can be maintained during the cultivations.

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In vitro Angiogenic Activity of Aloe vera Gel on Calf Pulmonary Artery Endothelial (CPAE) Cells

  • Lee, Myoung-Jin;Lee, Ok-Hee;Yoon, Soo-Hong;Lee, Seung-Ki;Chung, Myung-Hee;Park, Young-In;Sung, Chung-Ki;Choi, Jae-Sue;Kim, Kyu-Won
    • Archives of Pharmacal Research
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    • v.21 no.3
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    • pp.260-265
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    • 1998
  • Angiogenic activity of Aloe vera gel was investigated by in vitro assay. We obtained the most active fraction from dichloromethane extract of Aloe vera gel by partitioning between hexane and 90% aqueous methanol. The most active fraction (F3) increased the proliferation of calf pulmonary artery endothelial (CPAE) cells. In addition, F3 fraction induced CPAE cells to invade type I collagen gel and form capillary-like tube through in vitro angiogenesis assay, and increased the invasion of CPAE cells into matrigel through in vitro invasion assay. Furthermore, the effect on the MRNA expression of proteolytic enzymes which are key participants in the regulation of extracellular matrix degradation was investigated by northern blot analysis. F3 fraction enhanced mRNA expression of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase-2 (MMP-2), and membrane-type MMP (MT-MMP) in CPAE cells whereas the expression of plasminogen activator inhibitory (PAl-1) mRNA was not changed.

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High-Level Expression and Characterization of Single Chain Urokinase-type Plasminogen Activator(scu-PA) Produced in Recombinant Chinese Hamster Ovary(CHO) Cells

  • Kim, Jung-Seob;Min, Mi-Kyung;Jo, Eui-Cheol
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.6 no.2
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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.

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A Successfully Treated Case of Recurrent Focal Segmental Glomerulosclerosis (FSGS) with Plasmapheresis and High dose Methylprednisolone Pulse Therapy

  • Her, Sun Mi;Lee, Keum Hwa;Shin, Jae Il
    • Childhood Kidney Diseases
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    • v.21 no.2
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    • pp.165-168
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    • 2017
  • Focal segmental glomerulosclerosis (FSGS) in children, which is a kind of nephrotic syndrome showing steroid resistance, usually progresses to a substantial number of end stage renal disease (ESRD). Although the pathogenesis of primary FSGS is unclear, several recent studies have reported that FSGS is associated with circulating immune factors such as soluble urokinase-type plasminogen activator receptor (suPAR) or anti-CD40 autoantibody. We report a successfully treated case of a 19-year-old female patient who experienced a recurrence of primary FSGS. After the diagnosis of FSGS, the patient progressed to ESRD and received a kidney transplantation (KT). Three days later, recurrence was suspected through proteinuria and hypoalbuminemia. She has been performed plasmapheresis and high dose methylprednisolone pulse therapy and shown remission status without increasing proteinuria for four years after KT. In conclusion, strong immunosuppressive therapy may be helpful for a good prognosis of recurrent FSGS, suppressing several immunologic circulating factors related disease pathogenesis.

Expression of Plasminogen Activators in Uterine Epithelial Cells of Pre-ovulatory Phase in Pigs (돼지의 배란 전 자궁내막 상피세포 내 Plasminogen Activators의 발현)

  • HwangBo, Yong;Lee, Sang-Hee;Cha, Hye-Jin;Song, Eun-Ji;Lee, Seung-Tae;Lee, Eun-Song;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Journal of Embryo Transfer
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    • v.28 no.3
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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.

Rhus verniciflua Stokes extract suppresses migration and invasion in human gastric adenocarcinoma AGS cells

  • Lee, Hyun Sook;Jung, Jae In;Kim, Kyeong-Hee;Park, Sang Jae;Kim, Eun Ji
    • Nutrition Research and Practice
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    • v.14 no.5
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    • pp.463-477
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    • 2020
  • BACKGROUND/OBJECTIVES: Many studies have suggested that Rhus verniciflua Stokes (RVS) and its extract are anticancer agents. However, RVS had limited use because it contains urushiol, an allergenic toxin. By improving an existing allergen-removal extraction method, we developed a new allergen-free Rhus verniciflua Stokes extract (RVSE) with higher flavonoid content. In this study, we examined whether RVSE inhibits the ability of AGS gastric cancer cells to migrate and invade. MATERIALS/METHODS: The flavonoids content of RVSE was analyzed by HPLC. The effects of RVSE on migration and invasion in AGS cells were analyzed by each assay kit. Matrix metalloproteinase (MMP)-9, plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) protein expression was analyzed by protein antibody array. The Phosphorylation of signal transducer and activator of transcription (STAT) 3 were assayed by Western blot analysis. RESULTS: RVSE treatment with 0-100 ㎍/mL dose-dependently reduced the ability of AGS cells to migrate and invade. Notably, treatment with RVSE strongly inhibited the expression of MMP-9 and uPA and the phosphorylation of STAT3. In contrast, RVSE treatment dramatically increased the expression of PAI-1. These results indicate that the inhibition of MMP-9 and uPA expression and STAT3 phosphorylation and the stimulation of PAI-1 expression contributed to the decreased migration and invasion of AGS cells treated with RVSE. CONCLUSIONS: These results suggest that RVSE may be used as a natural herbal agent to reduce gastric cancer metastasis.

Production of Plasminogen Activators during In Vitro Maturation of Fresh or Frozen- Thawed Oocytes in the Pig

  • Chen J. B.;Sa S. J.;Cao Y.;Choi S. H.;Cheong H. T.;Yang B. K.;Park C. K.
    • Reproductive and Developmental Biology
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    • v.29 no.2
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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.

The Significance of Plasma Urokinase-type Plasminogen Activator and Type 1 Plasminogen Activator Inhibitor in Lung Cancer (폐암에서 혈장 Urokinase-Type Plasminogen Activator 및 Type 1 Plasminogen Activator Inhibitor의 의의)

  • Park, Kwang-Joo;Kim, Hyung-Jung;Ahn, Chul-Min;Lee, Doo-Yun;Chang, Joon;Kim, Sung-Kyu;Lee, Won-Young
    • Tuberculosis and Respiratory Diseases
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    • v.44 no.3
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    • pp.516-524
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    • 1997
  • Background : Cancer invasion and metastasis require the dissolution of the extracellular matrix in which several proteolytic enzymes are involved. One of these enzymes is the urokinase-type plasminogen activator(u-PA), and plasminogen activator inhibitors(PAI-1, PAI-2) also have a possible role in cancer invasion and metastasis by protection of cancer itself from proteolysis by u-PA. It has been reported that the levels of u-PA and plasminogen activator inhibitors in various cancer tissues are significantly higher than those in normal tissues and have significant correlations with tumor size and lymph node involvement. Here, we measured the concentration of plasma u-PA and PAI-1 antigens in the patients with lung cancer and compared the concentration of them with histologic types and staging parameters. Methods : We measured the concentration of plasma u-PA and PAI-1 antigens using commercial ELISA kit in 37 lung cancer patients, 21 benign lung disease patients and 24 age-matched healthy controls, and we compared the concentration of them with histologic types and staging parameters in lung cancer patients. Results : The concentration of u-PA was $1.0{\pm}0.3ng/mL$ in controls, $1.0{\pm}0.3ng/mL$ in benign lung disease patients and $0.9{\pm}0.3ng/mL$ in lung cancer patients. The concentration of PAI-1 was $14.2{\pm}6.7ng/mL$ in controls, $14.9{\pm}6.3ng/mL$ in benign lung disease patients, and $22.1{\pm}9.8ng/mL$ in lung cancer patients. The concentration of PAI-1 in lung cancer patients was higher than those of benign lung disease patients and controls. The concentration of u-PA was $0.7{\pm}0.4ng/mL$ in squamous cell carcinoma, $0.8{\pm}0.3ng/mL$ in adenocarcinoma, 0.9ng/mL in large cell carcinoma, and $1.1{\pm}0.7ng/mL$ in small cell carcinoma. The concentration of PAI-1 was $22.3{\pm}7.2ng/mL$ in squamous cell carcinoma, $22.6{\pm}9.9ng/mL$ in adenocarcinoma, 42 ng/mL in large cell carcinoma, and $16.0{\pm}14.2ng/mL$ in small cell carcinoma. The concentration of u-PA was 0.74ng/mL in stage I, $1.2{\pm}0.6ng/mL$ in stage II, $0.7{\pm}0.4ng/mL$ in stage IIIA, $0.7{\pm}0.4ng/mL$ in stage IIIB, and $0.7{\pm}0.3ng/mL$ in stage IV. The concentration of PAI-1 was 21.8ng/mL in stage I, $22.7{\pm}8.7ng/mL$ in stage II, $18.4{\pm}4.9ng/mL$ in stage IIIA, $25.3{\pm}9.0ng/mL$ in stage IIIB, and $21.5{\pm}10.8ng/mL$ in stage IV. When we divided T stage into T1-3 and T4, the concentration of u-PA was $0.8{\pm}0.4ng/mL$ in T1-3 and $0.7{\pm}0.4ng/mL$ in T4, and the concentration of PAI-1 was $17.9{\pm}5.6ng/mL$ in T1-3 and $26.1{\pm}9.1ng/mL$ in T4. The concentration of PAI-1 in T4 was significantly higher than that in T1-3. The concentration of u-PA was $0.8{\pm}0.4ng/mL$ in M0 and $0.7{\pm}0.3ng/mL$ in M1, and the concentration of PAI-1 was $23.6{\pm}8.3ng/mL$ in M0 and $21.5{\pm}10.8ng/mL$ in M1. Conclusions : The plasma levels of PAI-1 in lung cancer were higher than benign lung disease and controls, and the plasma levels of PAI-1 in T4 were significantly higher than T1-3. These findings suggest involvement of PAI-1 with local invasion of lung cancer, but it should be confirmed by the data on comparison with pathological staging and tissue level in lung cancer.

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A Role of Plasminogen Activators in Animal Reproductive Cells and Organs

  • HwangBo, Yong;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.38 no.2
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    • pp.63-70
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    • 2014
  • Plasminogen activators (PAs) are serine proteases that convert plasminogen to plasmin. Two type of PAs are urokinase-type PA (uPA) and tissue-type PA (tPA). Plasminogen is present in most extracellular fluids. PAs play in various reproductive processes including implantation, ovulation and fertilization. In the spermatozoa, PAs and PAIs play a role in sperm motility and fertilization. PAs in the sertoli cell are stimulated spermatozoa maturation and sperm activation through the phospholipase A2. The oocyte maturation is the process for fertilization and implantation. PAs in cumulus-oocyte complexes (COCs) are related to oocyte maturation by protein kinase A and C. In the ovulatory process, PAs activity are changed and it are related to reducing the tensile strength of ovarian follicle wall. The uterine environment is important for reproduction and the uterus undergo tissue remodeling. In the uterus and oviduct of mammals, expression and activity of PAs are changed during estrous cycle. Thus, expression and activity of PAs are concerned to many reproductive functions. Therefore, PAs seem to important factor of regulator in reproductive events.