• 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.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제33권5호
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• pp.445-454
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• 2007

Background: Phosphatidic acid(PA), an important second messenger, is involved in inflammation. Notably, cell-cell interactions via adhesion molecules playa central role in inflammation. This thesis show that PA induces expression of intercellular adhesion molecule-1(ICAM-1) on macrophages and describe the signaling pathways. Materials and methods: Macrophages were cultured in the presence of 10% FBS and assayed cell to cell adhesion using HUVEC. For the gene and protein analysis, RT-PCR, Western blot and flow cytometry were performed. In addition, overexpressed cell lines for dominant negative PKC-${\delta}$ mutant established and tested their effect on the promoter activity and expression of ICAM-1 protein by PA. Results: PA-activated macrophages significantly increased adhering to human umbilical vein endothelial cell and this adhesion was mediated by ICAM-1. Pretreatment with rottlerin(PKC-${\delta}$ inhibitor) or expression of a dominant negative PKC-${\delta}$ mutant, but not Go6976(classical PKC-${\alpha}$ inhibitor) and myristoylated PKC-${\xi}$ inhibitor, attenuated PA-induced ICAM-1 expression. The p38 mitogen-activated protein kinase(MAPK) inhibitor blocked PA-induced ICAM-1 expression in contrast, ERK upstream inhibitor didn't block ICAM-1. Conclusion: These data suggest that PA-induced ICAM-1 expression and cell-cell adhesion in macrophages requires PKC-${\delta}$ activation and that PKC-${\delta}$ activation is triggers to sequential activation of p38 MAPK.

• Biomolecules & Therapeutics
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• 제21권2호
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• pp.107-113
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• 2013

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제20권1호
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• pp.19-29
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• 1993

Plasminogen activator (PA)-plasmin system in follicular fluid is involved in the process leading to follicular rupture at ovulation. It is well known that PA is closely associated with cellular differentiation and tissue remodeling on evidences from the study of normal and malignant tissues. This study was designed to ascertain a potential role of PA in the ovarian folliculogenesis. Immature Sprague-Dawley rats were injected with pregnant mare serum gonadotropin, followed by injection of serine protease inhibitor (SPI; mixture of 1 mol/L benzamidine and 1 mol/L amino-caproic acid) into the unilateral ovarian bursa. In the control study, mechanical effect of bursal injection and contralateral ovarian effect SPI were ruled out. Total antral follicular areas relative to total ovarian cross-sectional areas was siginificantly lower in SPI-injected ovary than in saline-injected ovary. SPI injection decreased the relative antral follicular area by 33 % respectively. Electron microscopic finding of granulosa cell in the atretic follicle showed the presence of pyknotic nucleus, blurring of neucleolemma, degeneration of mitochondria and dilation of endoplasmic reticulum. After induction of ovulation with hCG, the number of oocytes released was significantly decreased in SPI-injected oviduct than in saline-injected oviduct. From above results, author discussed that PA may play a role not only in ovulation but also in some processes of folliculogenesis.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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• pp.134-134
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• 1998

The modulation of glycosidase activity by inhibitors is of great interest. Such compounds have been shown to be important tools in mechanistic studies on glycohydrolase as well as having promising therapeutic application. An ${\alpha}$-glucosidase inhibitor was isolated from culture filterates of Penicillium sp. The inhibitor was active against ${\alpha}$-glucosidase isolated from yeast and porcine small intestine. However, it showed no inhibition to Aspergillus ${\alpha}$-galactosidase, Escherichia coli ${\beta}$-galactosidase, and jack bean ${\alpha}$-mannosidase. The inhibitor was highly soluble in ether, methanol and chloroform. The inhibitor was purified using silica gel, Sephadex LH-20 column chromatography and reverse-phase HPLC. The inhibitory compound designated PA-7(IC$\sub$50/=35$\mu\textrm{g}$) was obtained as white powder. The structure of PA-7 was determined with spectroscopic data of EI-MS, FAB-MS, $^1$H, and $\^$13/C NMR. The inhibitor has a diketopiperazine moiety.

• Biomolecules & Therapeutics
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• 제15권3호
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• pp.150-155
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• 2007

In the present study, we have tested the effect of dioleoyl phosphatidic acid (PA) on intracellular $Ca_{2+}$ concentration ($[Ca^{2+}]_{i}$) in two human colon cancer cell lines (HCT116 and HT29). PA and lysophosphatidic acid (LPA), a bioactive lysolipid, increased $[Ca^{2+}]_{i}$ in both HCT116 and HT29 cell lines. Increases of $[Ca^{2+}]_{i}$ by PA and LPA were more robust in HCT116 cells than in HT29 cells. A specific inhibitor of phospholipase C (U73122), however, was not inhibitory to the cell responses. Pertussis toxin, a specific inhibitor of $G_{i/o}$ type G proteins, however, had an inhibitory effect on the responses except for an LPA-induced one in HT29 cells. Ruthenium red, an inhibitor of the ryanodine receptor, was not inhibitory on the responses, however, 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor, completely inhibited both lipid-induced $Ca^{2+}$ increases in both cell types. Furthermore, by using Ki16425 and VPC32183, two structurally dissimilar specific antagonists for $LPA_{1}/LPA_{3}$ receptors, an involvement of endogenous LPA receptors in the $Ca^{2+}$ responses was observed. Ki16425 completely inhibited the responses but the susceptibility to VPC32183 was different to PA and LPA in the two cell types. Expression levels of five LPA receptors in the HCT116 and HT29 cells were also assessed. Our data support the notion that PA could increase $[Ca^{2+}]_{i}$ in human colon cancer cells, probably via endogenous LPA receptors, G proteins and $IP_{3}$ receptors, thereby suggesting a role of PA as an intercellular lipid mediator.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1299-1305
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• 2011

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncated-mutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncated-mutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

• BMB Reports
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• 제44권1호
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• pp.34-39
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• 2011

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase$^{(R)}$. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, $570\;IU/{\mu}g$, was found to be similar to those found in full length t-PA (Alteplase$^{(R)}$), $580\;IU/{\mu}g$. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.

• 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.