• Journal of Microbiology and Biotechnology
• /
• v.17 no.4
• /
• pp.594-599
• /
• 2007

It is well known that the Escherichia coli inner membrane-bound protease DegS is a periplasmic stress sensor for unfolded outer membrane proteins (OMPs). Previous studies have also shown that the outer membrane protease OmpT activates plasminogen in vitro and this may be exploited by bacteria in the course of pathogenesis. However, there has been no research on the plasminogen activation ability of the important periplasmic protein DegS. Accordingly, in this study, the whole-length and truncated degS genes were separately overexpressed in Escherichia coli, the recombinant proteins purified by affinity chromatography, and their plasminogen activator role tested in vitro. The results suggested that the whole-length DegS was able to activate plasminogen on a plasma plate. The truncated form of DegS (residues 80-345), designated ${\Delta}DegS$, also acted as a plasminogen activator, as confirmed by different assays. The serine protease property of ${\Delta}DegS$ was verified based on the complete inhibition of its enzyme activity by PMSF (phenylmethanesulfonyl fluoride). Therefore, the present results indicate that DegS is a plasminogen activator in vitro.

• Bulletin of the Korean Chemical Society
• /
• v.31 no.9
• /
• pp.2607-2612
• /
• 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
• /
• v.17 no.6
• /
• pp.582-585
• /
• 2002

In an effort to use plant biotechnology for the production of biopharmaceuticals, pollens collected from lily (Lilium longiflorum) were grown in vitro and transformed with a PCR-amplified 1.7 kb cDNA encoding human tissue plasminogen activator (tPA) using Agrobacterium via a vacuum infiltration process. Western blotting showed that transgenic lily pollen tubes selected on kanamycin for 16 hrs expressed a tPA protein with a size similar to the human standard, suggesting their possible use as a disposable host for rapid foreign protein production.

• Journal of Korean Academy of Nursing
• /
• v.28 no.1
• /
• pp.184-192
• /
• 1998

This study was done to examine the correlations between weight, BMI and risk factors of coronary heart disease in men and women in their forties and fifties. The subjects were 412 adults. who had regular health examinations between January and December of 1996 at S-Hospital in Seoul. The data were analyzed using ANOVA, Scheffe test, and Pearson correlation coefficient. The results are as follows : 1. The men between 50 and 59 years of age had higher levels for BMI, weight, systolic blood pressure, diastolic blood pressure, total cholesterol. LDL-cholesterol, triglyceride, fasting blood sugar, plasminogen activator-1, and hemoglobin A,C than the group of women in their forties. Yet. HDL-cholesterol was lower than in the former group. 2. In the group of men in their forties, weight was significantly correlated to diastolic blood pressure(r=.22), LDL-cholesterol(r=.20), plasminogen activator-inhibitor-1(r=.35) HDL-cholesterol(r=-.19). Their BMI was significantly correlted to systolic blood pressure(r=.27), diastolic blood pressure (r=.33), total cholesterol(r=.23), LDL-cholesterol (r=.26), plasminogen activator-1(r=.36) and HDL-cholesterol(r=-.25). 3. As for the group of women in their forties weight was significantly correlated to systolic blood pressure(r=.20), diastolic blood pressure(r=.22), triglyceride(r=.32) , plasminogen activator inhibitor-1 (r=.30) and HDL-cholesterol(r= -.37). Their BMI was significantly correlated to diastolic blood pressure (r=.25) triglyceride(r=.47), plasminogen activator-1 (r=.35), fibrinogen(r=.27) and HDL-cholesterol(r=-.47). 4. In the group of men in their fifties. weight was significantly correlated to total cholesterol (r=32), LDL-cholesterol(r=.29). plasminogen activator inhibitor-1(r=.26). Their BMI was significantly correlated to systolic blood pressure(r=.24), diastolic blood pressure (r=.22), total cholesterol (r=.34), LDL-cholesterol (r=.32), and plasminogen activator-1(r=.25). 5. In the group of women in their fifties, weight was significantly correlated to diastolic blood pressure(r=.33), total cholesterol(r=.21), LDL-cholesterol(r=.20), plasminogen activator inhibitor-1 (r=.43) and HDL-cholesterol(r=-.21). Their BMI was significantly correlated to systolic blood pressure(r=.25), diastolic blood pressure(r=.40), total cholesterol(r=.24), LDL-cholesterol(r=.24), triglyceride(r=22), and HDL-cholesterol (r=-.30). The above findings indicate that the BMI was more predictive than weight as a risk factor for coronary artery disease for men and women in their forties and fifties.

• Archives of Pharmacal Research
• /
• v.20 no.6
• /
• pp.566-571
• /
• 1997

To gain further insight into how estrogens modulate cell function, the effects of estrogen on cell proliferation were studied inhuman breast cancer cells. We examined the effects of estrogen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Ten nM estradiol markedly stimulated the proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor $1.15{\pm}0.03 pmole/mg protein)$(over that of control. In T47D cells that contained low levels of estrogen receptor $0.23{\pm}0.05 pmole/mg protein)$, Ten nM estrogen slightly stimulated the proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by estrogen. These results showed their sensitivity to growth stimulation by estrogen correlated well with their estrogen receptor content. Also we examined the effect of estrogen on cellular progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. Ten nM estradiol showed maximal stimulation of progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. It is not clear whether these stimulations of progesterone receptor and plasminogen activator activity by estrogen are related to the estrogen stimulation of cell proliferation of MCF-7 cells. Studies with estrogen in human breast cancer cells in culture indicate that sensitivity to growth stimulation by estrogen correlates well with estrogen receptor contents.

• Journal of Korean Neurosurgical Society
• /
• v.37 no.4
• /
• pp.287-292
• /
• 2005

Objective: Recent clinical studies have demonstrated that intracisternal administration of recombinant tissue plasminogen activator(rt-PA) can facilitate the normal clearing of blood from the subarachnoid space. Urokinase, a first generation fibrinolytic agent, has been used to liquify such clots with some success. Therefore, recombinant tissue plasminogen activator, a second generation fibrinolytic drug that may be safer and more effective, is studied to evaluate its dosage to lyse clots in vitro and reactivity in the brain parenchyme. Methods: Intracerebral hematomas were created by stereotactically injecting 2ml of clotted autogenous blood into the brain parenchyme of total 28 anesthetized adult cats (weighting 3.8 to 4.1 kg). The control animals (group A) received 1 ml of normal saline injected into the clots and the experimental animals received each 0.1 mg of rt-PA (group B), 0.5mg of rt-PA (group C) and 1 mg of rt-PA (group D) at 6 hours after the clot injection. Results: 1. The amount of remained clots after lysing the hematomas were as follows: $1.80{\pm}0.17ml$ in group A, $1.65{\pm}0.23ml$ in group B, $0.61{\pm}0.37ml$ in group C and $0.52{\pm}0.34$ in group D. The result indicated that hematomas in rt-PA treated groups (C & D) were lysed better than the control group. 2. At least 0.5mg of rt-PA should be required for the lysis of 2ml of hematomas. 3. Light microscopic examination revealed no histological evidence of hemorrhage in tissue sections from each brain. Conclusion: Recombinant tissue plasminogen activator may be safely and effectively employed for the lysis of intracerebral hematomas in animal model.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.8 no.2
• /
• pp.162-165
• /
• 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.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.1 no.1
• /
• pp.32-35
• /
• 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.

• Journal of Ginseng Research
• /
• v.37 no.4
• /
• pp.401-412
• /
• 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.

• Nutrition Research and Practice
• /
• v.14 no.5
• /
• pp.463-477
• /
• 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.