• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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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.

• 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.

• 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.

• Archives of Pharmacal Research
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• 제21권3호
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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.

• Nutrition Research and Practice
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• 제14권5호
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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.

• Tuberculosis and Respiratory Diseases
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• 제44권3호
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• pp.516-524
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• 1997

목 적 : 악성 종양에 있어서 단백질 분해 효소에 의한 세포외기질의 분해는 종양 침습 및 전이에 있어서 중요한 역할을 한다. Urokinase-type plasminogen activator(u-PA)는 이러한 단백질 분해효소 중 하나이며, u-PA의 길항제인 plasminogen activator inhibitor(PAI-1, PAI-2)도 u-PA에 의한 종양조직 자체의 분해를 방어하여 종양의 성장, 침습 및 혈관신생을 촉진하는 역할을 한다. u-PA와 PAI-1은 폐암 등의 종양 조직내의 양이 증가하며, 침습도 및 예후 둥과 유의하게 상관되는 것으로 보고되고 있다. 저자 등은 폐암환자의 혈장 u-PA와 PAI-1의 농도를 측정하여 조직형 및 병기와의 관련성을 조사해 보고자 한다. 방 법 : 폐암 환자 37예, 여러 가지 양성 폐질환 환자 21예 및 유사연령의 정상 대조군 24예에서 혈장 u-PA 및 PAI-1 항원의 농도를 ELISA법으로 측정하였다. 결 과 : 혈장 u-PA 항원 농도는 정상 대조군에서 $1.0{\pm}0.3ng/mL$, 양성 폐질환군에서 $1.0{\pm}0.3ng/mL$, 폐암환자군에서 $0.9{\pm}0.3ng/mL$로 유의한 차이가 없었다. 혈장 PAI-1 항원 농도는 정상 대조군에서 $14.2{\pm}6.7ng/mL$, 양성 폐질환군에서 $14.9{\pm}6.3ng/mL$, 폐암 환자군에서 $22.1{\pm}9.8ng/mL$로 폐암 환자에서 정상 대조군 및 양성 폐질환군에 비하여 유의하게 높았다. 혈장 u-PA 항원 농도는 편평상피암에서 $0.7{\pm}0.4ng/mL$, 선암에서 $0.8{\pm}0.3ng/mL$, 대세포암에서 0.9ng/mL, 소세포암에서 $1.1{\pm}0.7ng/mL$로 유의한 차이가 없었다. 혈장 PAI-1 항원 농도는 편평상피암에서 $22.3{\pm}7.2ng/mL$, 선암에서 $22.6{\pm}9.9ng/mL$, 대세포암에서 42ng/mL, 소세포암에서 $16.0{\pm}14.2ng/mL$로 유의한 차이가 없었다. u-PA 항원 농도는 stage I에서 0.74ng/mL, stage II에서 $1.2{\pm}0.6ng/mL$, stage IIIA에서 $0.7{\pm}0.4ng/mL$, stage IIIB에서 $0.7{\pm}0.4ng/mL$, stage IV에서 $0.7{\pm}0.3ng/mL$였고, 혈장 PAI-1 항원 농도는 stage I에서 21.8ng/mL, stage II에서 $22.7{\pm}8.7ng/mL$, stage IIIA에서 $18.4{\pm}4.9ng/mL$, stage IIIB에서 $25.3{\pm}9.0ng/mL$, stage IV에서 $21.5{\pm}10.8ng/mL$로 유의한 차이가 없었다. 폐암 환자에서 T기를 T1-3 와 T4로 나누어서 비교를 한 결과, 혈장 u-PA 항원 농도는 T1-3에서 $0.8{\pm}0.4ng/mL$였고 T4에서 $0.7{\pm}0.4ng/mL$로 차이가 없었으나, 혈장 PAI-1 항원 농도는 T1-3에서 $17.9{\pm}5.6ng/mL$였고 T4에서 $26.1{\pm}9.1ng/mL$로 T4에서 유의하게 높았다. 혈장 u-PA 항원 농도는 M0에서 $0.8{\pm}0.4ng/mL$, M1에서 $0.7{\pm}0.3ng/mL$였고, 혈장 PAI-1 항원 농도는 M0에서 $23.6{\pm}8.3ng/mL$, M1에서 $21.5{\pm}10.8ng/mL$로 유의한 차이가 없었다. 결 론 : 이상의 결과에서 폐암 환자에서 혈장 PAI-1은 정상대조군 및 양성 폐질환에 비하여 특이적으로 증가하였고, 또한 폐암의 국소적 침습 정도와의 관련성을 보여 주었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.60-60
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• 2003

소의 난포란과 난구세포의 체외배양시 plasminogen activators(PAs)의 생산을 보고하였다 (Choi 등, 1998). 따라서 본 연구는 돼지 난포란 및 난구세포의 체외성숙시 PAs의 생산을 SDS-PAGE와 Zymogram을 이용하여 조사하였다. PCOCs는 도축암퇘지의 난소로부터 채취하여, 난구세포가 충실한 것만 선별하였으며, 실험구에 사용될 난구세포는 pipetting에 의해 분리하여 이용하였다. 돼지 신선정액은 D-PBS로 1,500 rpm, 5분간 2회 원심분리하여 정장물질을 제거하고, 3회째는 5mM caffein이 함유된 BO(Brackett과 Oliphant, 1985) 배양액으로 세정하였다. 처리한 돼지 정액은 1$\times$$10^{8}$ cells/$m\ell$로 조정하여 20${\mu}\ell$씩 분주하고 0, 1, 2, 3 또는 4시간 동안 39$^{\circ}C$ 5% $CO_2$, 95% 공기인 배양기에서 수정능획득을 유도하였다. 배양이 완료된 정액은 20${\mu}\ell$의 sample buffer(5% SDS, 20% glycerol, 0.0025% bromophenol blue 그리고 0.125M Tris HC1 buffer)에 넣어 -7$0^{\circ}C$ 동결기에 보관하였다. 전기영동은 4% stacking gel과 10% separating gel로 분리하였으며, 20 mA에서 90분간 실시하였다. Zymogram은 Choi 등(1988)의 방법에 따라 실시하여 PAs의 생산을 확인하였으며, 이상의 실험은 3반복을 실시하였다. 시험구 전체에서 urokinase type plasminogen activator(uPA)가 확인되었으며, 체외수정능 획득시간에는 차이가 없었다. 두 종류의 고분자량의 uPA의존성 영역이 나타났으며, 분자량은 65kD과 62 kD이었다. 이러한 결과로 볼 때 Hart 등(1986)이 uPA의 경우 다양한 영역의 분자량 변이를 확인할 수 있었다고 한 것과 동일하였으며, 돼지 정자가 체외수정능 획득시 uPA를 생산하는 것을 확인할 수 있었다.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.203-209
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• 2009

The present study was performed to identify changes of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in porcine oviduct epithelial cells (POECs) during the estrous cycle. POECs obtained from ovary in pre-ovulatory (Pre-Ov), early to mid-luteal stage (Early-mid L) and post-ovulatory stage (Post-Ov). For the examine of PA activity, $1{\times}10^5$ fresh cells of POECs were cultured in DMEM/Ham F-12 containing 10% FBS and 0.2% amphotericin under humidified atmosphere of 5% $CO_2$ in air and $38^{\circ}C$. The urokinase-type PA (uPA) was observed at 7 days of POECs culture. PA activity was measured with culture prolonged of 0, 3, 6, 12 and 24 h after culture of 7 days. The PA activity were high significantly (p<0.05) at 12 h of culture, but PA activity were decreased with culture periods increased. The PA activity in POECs of Post-Ov stage were higher significantly (p<0.05) than that of Early-mid L and Pre-Ov stage. When PAI-1 and PAI-2 were added during the POECs culture, the PA were observed significant low activity (p<0.05). The PA activity and protein expression were decreased by PA inhibitor. This results suggest that PAI-1 and PAI-2 have a suppressive action on change of PA activity during the estrous cycle of pigs. Specifically, this study using PA inhibitor was effect the PA activity and PAI expression in oviduct epithelial cells in pigs.

• Reproductive and Developmental Biology
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• 제38권2호
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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.

• Reproductive and Developmental Biology
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• 제30권2호
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• pp.135-141
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• 2006

The present study was conducted to investigate the effects of cumulus cells and porcine follicular fluid (pFF) on plasminogen activator (PA) activity and oocytes maturation in vitro in the pig. The cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were incubated in NCSU-23 medium with or without 10% pFF for 0, 24, or 48 hr. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P<0.05) higher in medium with pFF than without pFF (69.8 vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 hr than 24 hr. When COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. No PA activities were detected in DOs regardless of pFF supplementation. When porcine oocytes were cultured in the presence of pFF for 24 and 48 hrs, the activities of tPA-PAI, tPA, and uPA were observed in both COCs and DOs. In medium of absence of pFF, PA activities were observed in oocytes with cumulus cells only. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 hr of culture, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48 hr of culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.