Tissue-type plasminogen activator (t-PA) is a thrombolytic agent important in fibirn clot lysis. T-PA causes fibirn-specific plasminogen activation. Six binary vectors harboring t-PA and its derivative genes were cloned and expressed in transgenic alfalfa plants. The insertion of the t-PA and its derivative genes in genomic DNA of alfalfa plants was confirmed by PCR. The presence of the t-PA and its derivative transcripts in total RNAs of the transgenic alfalfa leaves was verified by RT-PCR. ELISA experiments demonstrated that the highest level of recombinant t-PA expression was $75.1{\mu}g$/ total soluble protein (mg) in alfalfa plants. The amount of recombinant t-PA and its derivative proteins in transgenic plants was estimated to range from 9.7 to $39.5{\mu}g$/ total soluble proteins (mg). Western blot analysis of the transformed alfalfa leaves revealed bands of approximately 68-kDa recombinant t-PA and its derivative proteins. The fibrinolysis of recombinant t-PA and its derivative proteins was confirmed by a fibrin plate assay (range from 3.2 to 8.1 cm). The results presented provide information for the development of an additional production of recombinant human proteins having pharmaceutical applications using transgenic plants.
We present a case of a 57-year-old male patient who presented with squamous cell carcinoma on his mouth floor with cervical and mandibular metastases. Wide glossectomy with intergonial mandibular ostectomy, and sequential reconstruction using fibular osteomyocutaneous free flap were planned. When the anastomosis between the peroneal artery of the fibular free flap and the right lingual artery was performed, no venous flow was observed at the vena comitans. Then re-anastomosis followed by topical application of papaverine and lidocaine was attempted. However, the blood supply was not recovered. Warm saline irrigation over 30 minutes was also useless. Microvascular thromboses of donor vessels were clinically suspected, so a solution of 100,000 units of urokinase was infused once through a 26-gauge angiocatheter inserted into the recipient artery just at the arterial anastomotic site, until the solution gushed out through the flap vena comitans. Immediately after the application of urokinase, arterial flow and venous return were restored. There were no complications during the follow-up period of 11 months. We believe that vibrating injuries from the reciprocating saw during osteotomies and flap insetting might be the cause of microvascular thromboses. The use of urokinase may provide a viable option for the treatment of suspicious intraoperative arterial thrombosis.
Background: Tissue-type plasminogen activator is a physiologic activator, which has high affinity for fibrin and is activated by fibrin. Because of these properties, t-PA has the potential to induce effective thrombolysis without producing a systemic lytic state. In practice, however, therapeutically efficacious doses of t-PA has been associated with the development of a systemic lytic state. As experience with t-PA has accumulated, it has suggested that the fibrin selectivity is influenced by the dose and duration of t-PA infusion, and many studies have performed in an attempt to optimize the duration of t-PA regimen. Methods: This study was designed to assess the thrombolytic efficacy of t-PA and the differences of two dosing regimens of t-PA (infusion of 1 mg/kg t-PA over 15 or 180 minutes) in a canine model of pulmonary embolism, induced by injection of radioactive autologous blood clots. By continuously counting over both lung fields with a external gamma counter, we correlated rate and extent of pulmonary thrombolysis with corresponding pulmonary hemodynamics in addition to the gas analyses of arterial and mixed venous blood. Results: 1) While total clot lysis was similar ($36.2{\pm}3.3%$ and $39.6{\pm}2.3%$ respectively, p>0.05) when t-PA was infused over 15 or 180 minutes, the rate of lysis during infusion was markedly increased with the shorter infusion ($81.4{\pm}16.8%/hr$ vs $37.3{\pm}2.4%/hr$, p<0.05). 2) The duration of thrombolysis was $63.3{\pm}22.2$ minutes although t-PA was administered over 15 minutes, and it was only $148.5{\pm}14.0$ minutes in case of the infusion over 180 minutes (p<0.05). 3) The increased rate of thrombolysis with the shorter infusion was accompanied by a faster amelioration of cardiopulmonary impairment from pulmonary embolism (p<0.05). Conclusion: It is concluded that the shorter (15 minutes) infusion of t-PA is superior to the longer (180 minutes) infusion when the dose is equal, in consideration of the faster improvement in cardiopulmonary impairment from pulmonary embolism.
Im, Jun Hyung;Yeo, In Jun;Hwang, Chul Ju;Lee, Kyung Sun;Hong, Jin Tae
Biomolecules & Therapeutics
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v.28
no.2
/
pp.152-162
/
2020
Cerebral ischemia exhibits a multiplicity of pathophysiological mechanisms. During ischemic stroke, the reactive oxygen species (ROS) concentration rises to a peak during reperfusion, possibly underlying neuronal death. Recombinant human erythropoietin (EPO) supplementation is one method of treating neurodegenerative disease by reducing the generation of ROS. We investigated the therapeutic effect of PEGylated EPO (P-EPO) on ischemic stroke. Mice were administered P-EPO (5,000 U/kg) via intravenous injection, and middle cerebral artery occlusion (MCAO) followed by reperfusion was performed to induce in vivo ischemic stroke. P-EPO ameliorated MCAO-induced neurological deficit and reduced behavioral disorder and the infarct area. Moreover, lipid peroxidation, expression of inflammatory proteins (cyclooxygenase-2 and inducible nitric oxide synthase), and cytokine levels in blood were reduced by the P-EPO treatment. In addition, higher activation of nuclear factor kappa B (NF-κB) was found in the brain after MCAO, but NF-κB activation was reduced in the P-EPO-injected group. Treatment with the NF-κB inhibitor PS-1145 (5 mg/kg) abolished the P-EPO-induced reduction of infarct volume, neuronal death, neuroinflammation, and oxidative stress. Moreover, P-EPO was more effective than EPO (5,000 U/kg) and similar to a tissue plasminogen activator (10 mg/kg). An in vitro study revealed that P-EPO (25, 50, and 100 U/mL) treatment protected against rotenone (100 nM)-induced neuronal loss, neuroinflammation, oxidative stress, and NF-κB activity. These results indicate that the administration of P-EPO exerted neuroprotective effects on cerebral ischemia damage through anti-oxidant and anti-inflammatory properties by inhibiting NF-κB activation.
The earliest stages of mammalian embryogenesis are governed by the activity of maternally inherited transcripts and proteins. Cytoplasmic polyadenylation of selected maternal mRNA has been reported to be a major control mechanism of delayed translation during preimplantation embryogenesis in mice. The presence of cis-elements required for cytoplasmic polyadenylation (e.g., CPE) can serve as a useful tag in the screening of maternal genes partaking in key functions in the transcriptionally dormant egg and early embryo. However, due to its relative simplicity, UA-rich sequences satisfying the canonical rule of known CPE consensus sequences are often found in the 3'-UTR of maternal transcripts that do not actually undergo cytoplasmic polyadenylation. In this study, we developed a method to confirm the validity of candidate CPE sequences in a given gene by a multiplex comparison of 3'-UTR sequences between mammalian homologs. We found that genes undergoing cytoplasmic polyadenylation tend to create a conserved block around the CPE, while CPE-like sequences in the 3'-UTR of genes lacking cytoplasmic polyadenylation do not exhibit such conservation between species. Through this cross-species comparison, we also identified an alternative CPE in the 3'-UTR of tissue-type plasminogen activator (tPA), which is more likely to serve as a functional element. We suggest that verification of CPEs based on sequence conservation can provide a convenient tool for mass screening of factors governing the earliest processes of mammalian embryogenesis.
Kim, Hye Jin;Shim, Kyou Hee;Yeon, Seung Ju;Shin, Hwa Sung
Journal of Microbiology and Biotechnology
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v.28
no.2
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pp.275-283
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2018
Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature ($37^{\circ}C-70^{\circ}C$) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the ${\alpha}$-, ${\beta}$-, and ${\gamma}$-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.
Kim, Baek-Chul;Kim, Hong-Rye;Kim, Myung-Yoon;Park, Chang-Sik;Jin, Dong-Il
Reproductive and Developmental Biology
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v.33
no.2
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pp.113-117
/
2009
Animals produced by somatic cell nuclear transfer (SCNT) using genetically modified cells are almost always transgenic, implying that this method is more efficient than the traditional pronuclear microinjection method. Most somatic cells for SCNT in animals are fetus-derived primary cells and successful gene integration in somatic cells will depend on transfection condition. The objective of this study is to evaluate the efficiency of electroporation (Microporator) and liposome reagents (F-6, F-HD, W-EX, W-Q, W-M) for tissue-type plasminogen activator (tPA) gene transfection and to estimate the overall efficiency of transfection of Korean native pig fetal fibroblast cells (KNPFF). Electroporation showed significantly higher transfection efficiency than liposome reagents with regard to the transfection of in vitro cultures in the early stages of development (41.7% with Microporator vs. 18.3% with F-6, 20.0% with F-HD 18.5% with W-EX, 5.0% with W-M and 6.3% W-Q,). Colonies identified as tPA-positives were treated once more with G418 for 10 to 14 days and growing colonies were selected again. When the cells of newly selected colonies were subjected to single-cell PCR, reselection of colonies following second round of G418 selection increased the rate of transgene integration per each colony. These results suggest that transfection with electroporation is the most efficient and the second rounds of G418 selection may be an effective method for transfection of porcine fetal fibroblast cells.
Ki, Sung Hwan;Yang, Ji Hye;Ku, Sae Kwang;Kim, Sang Chan;Kim, Young Woo;Cho, Il Je
Journal of Ginseng Research
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v.37
no.1
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pp.45-53
/
2013
Korean red ginseng, the processed root of Panax ginseng Meyer, has been frequently used for various therapeutic purposes in oriental medicine. The present study investigated the possible effect of Korean red ginseng extract (RGE) for the treatment of liver fibrosis in mice injected with carbon tetrachloride ($CCl_4$) for 4 wk. Liver injuries were assessed by blood biochemistry and histopathology in mice treated with $CCl_4$ alone or $CCl_4$+ RGE (30, 100, and 300 mg/kg). Concomitant treatment with RGE and $CCl_4$ (three times/wk for 4 wk) effectively inhibited liver fibrosis as evidenced by decreases in plasma alanine and aspartate aminotransferases, as well as by the percentages of degenerative regions, numbers of degenerative hepatocytes, and collagen accumulation in hepatic parenchyma. Treatment with $CCl_4$ for 4 wk increased mRNA levels of transforming growth factor ${\beta}1$ and plasminogen activator inhibitor 1 in fibrogenic liver, whereas RGE (30, 100, and 300 mg/kg) significantly blocked the induction of fibrogenic genes by $CCl_4$. Similarly, RGE also prevented transforming growth factor ${\beta}1$-mediated induction of fibrogenic genes in human hepatic stellate cell lines. More importantly, RGE markedly reduced the number of ${\alpha}$-smooth muscle actin-positive cells in liver tissue. This study implies that RGE efficaciously protects against the liver fibrosis induced by chronic $CCl_4$ treatment, and may therefore have potential to treat liver disease.
Intravenous recombinant tissue plasminogen activator had been the only approved treatment for acute ischemic stroke since its approval in 1995. However, the restrictive time window, numerous contraindications, and its low recanalization rate were all limitations of this modality. Under those circumstances, endovascular stroke therapy went through a great evolution during the past two decades of intravenous thrombolysis. The results of the 2013 randomized trials for endovascular stroke therapy were neutral, although they were limited by insufficient imaging screening at enrollment, early-generation devices with less efficacy, and treatment delays. Huge progress was made in 2015, as there were five randomized clinical trials which all demonstrated the safety and efficacy of endovascular stroke treatment. Despite differences in detail patient enrollment criteria, all 5 trials employed key factors for good functional recovery; (1) screening with non-invasive imaging to identify the proximal occlusion and exclude a large infarct core, (2) using highly effective modern thrombectomy devices mainly with stent retriever, and (3) establishment of a fast workflow to achieve effective reperfusion. The results of those trials indicate that modern thrombectomy devices can allow for faster and more effective reperfusion, which can lead to improved clinical outcomes compared to intravenous thrombolysis alone. These advances in mechanical thrombectomy are promising in the global fight against ischemic stroke-related disability and mortality. Two current mainstreams among such mechanical thrombectomy techniques, "stent retriever thrombectomy" and "direct clot aspiration", are the topic of this review. Stent retriever thrombectomy using Solitaire and Trevo retriever will be firstly discussed. And, the commonalities and the differences between two major clot aspiration thrombectomy techniques; a direct aspiration first pass technique (ADAPT) and forced arterial suction thrombectomy (FAST), will be additionally explained. Finally, details regarding the combination of direct clot aspiration and stent retriever thrombectomy, the switching strategy and the Solumbra technique, will be described.
An, Gyu-Hwan;Sim, Sook-Young;Jwa, Cheol-Su;Kim, Gang-Hyeon;Lee, Jong-Yun;Kang, Jae-Kyu
Journal of Korean Neurosurgical Society
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v.50
no.1
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pp.1-5
/
2011
Objective : There is no proven regimen to reduce the severity of stroke in patients with acute cerebral infarction presenting beyond the thrombolytic time window. Ozagrel sodium, a selective thromboxane A2 synthetase inhibitor, has been known to suppress the development of infarction. The antiplatelet effect is improved when aspirin is used together with a thromboxane synthetase inhibitor. Methods : Patients with non-cardiogenic acute ischemic stroke who were not eligible for thrombolysis were randomly assigned to two groups; one group received ozagrel sodium plus 100 mg of aspirin (group 1, n=43) and the other 100 mg of aspirin alone (group 2, n=43). Demographic data, cardiovascular risk factors, initial stroke severity [National Institute of Health Stroke Scale (NIHSS) and motor strength scale] and stroke subtypes were analyzed in each group. Clinical outcomes were analyzed by NIHSS and motor strength scale at 14 days after the onset of stroke. Results : There were no significant differences in the mean age, gender proportion, the prevalence of cardiovascular risk factors, stroke subtypes, and baseline neurological severity between the two groups. However, the clinical outcome for group 1 was much better at 14 days after the onset of stroke compared to group 2 (NIHSS score, p=0.007, Motor strength scale score, p<0.001). There was one case of hemorrhagic transformation in group 1, but there was no statistically significant difference in bleeding tendency between two groups. Conclusion : In this preliminary study, thromboxane A2 synthetase inhibitor plus a low dose of aspirin seems to be safe and has a favorable outcome compared to aspirin alone in patients with acute ischemic stroke who presented beyond the thrombolytic time window.
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