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#27772757   2016/10/24 Save this To Up

Recombinant tissue plasminogen activator enhances microparticle release from mouse brain-derived endothelial cells through plasmin.

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, rt-PA exhibits vascular toxicity mainly due to endothelial damage. To investigate the mechanisms underlying rt-PA-induced endothelial alterations, we assessed the role of rt-PA in the generation of endothelial microparticles (EMPs), emerging biological markers and effectors of endothelial dysfunction. The mouse brain-derived endothelial cell line bEnd.3 was used. Cells were treated with rt-PA at 20, 40 or 80μg/ml for 15 or 24h, and EMPs were quantified in the culture media using Annexin-V staining coupled with flow cytometry. Rt-PA enhanced EMP release from bEnd.3 cells with a maximal increase at the 40μg/ml dose for 24h (+78% compared to controls). Using tranexamic acid and aprotinin we demonstrated that plasmin is responsible for rt-PA-induced EMP release. The p38 MAPK inhibitor SB203580 and the poly(ADP-ribose)polymerase (PARP) inhibitor PJ34 also reduced rt-PA-induced EMP production, suggesting that p38 MAPK and PARP are downstream intracellular effectors of rt-PA/plasmin. Rt-PA also altered through plasmin the morphology and the confluence of bEnd.3 cells. By contrast, these changes did not implicate p38 MAPK and PARP. This study demonstrates that rt-PA induces the production of microparticles by cerebral endothelial cells, through plasmin, p38 MAPK and PARP pathways. Determining the phenotype of these EMPs to clarify their role on the endothelium in ischemic conditions could thus be of particular interest.

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Mouse Brain Microvascular GFP Expressing Mouse Brai Mouse anti-Tissue type Pl Mouse Anti-Human Tissue P Rabbit Anti-Mouse Tissue Human Brain Microvascular GFP Expressing Human Brai Human Cord Blood CD34+ Ce Mouse Anti-Human Endothel Mouse Anti-Pig Endothelia Mouse Anti-Human Endothel Human tissue plasminogen

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#27089366   2016/04/19 Save this To Up

The Kunitz-Type Protein ShPI-1 Inhibits Serine Proteases and Voltage-Gated Potassium Channels.

The bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type protein ShPI-1 (UniProt: P31713) is the major protease inhibitor from the sea anemone Stichodactyla helianthus. This molecule is used in biotechnology and has biomedical potential related to its anti-parasitic effect. A pseudo wild-type variant, rShPI-1A, with additional residues at the N- and C-terminal, has a similar three-dimensional structure and comparable trypsin inhibition strength. Further insights into the structure-function relationship of rShPI-1A are required in order to obtain a better understanding of the mechanism of action of this sea anemone peptide. Using enzyme kinetics, we now investigated its activity against other serine proteases. Considering previous reports of bifunctional Kunitz-type proteins from anemones, we also studied the effect of rShPI-1A on voltage-gated potassium (Kv) channels. rShPI-1A binds Kv1.1, Kv1.2, and Kv1.6 channels with IC50 values in the nM range. Hence, ShPI-1 is the first member of the sea anemone type 2 potassium channel toxins family with tight-binding potency against several proteases and different Kv1 channels. In depth sequence analysis and structural comparison of ShPI-1 with similar protease inhibitors and Kv channel toxins showed apparent non-sequence conservation for known key residues. However, we detected two subtle patterns of coordinated amino acid substitutions flanking the conserved cysteine residues at the N- and C-terminal ends.

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#27035617   2016/04/07 Save this To Up

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro.

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

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#26463744   2015/11/26 Save this To Up

A novel coagulation inhibitor from Schistosoma japonicum.

Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µ m, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca(++). We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

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#26341789   2016/03/19 Save this To Up

Applications of pressure perturbation calorimetry to study factors contributing to the volume changes upon protein unfolding.

Pressure perturbation calorimetry (PPC) is a biophysical method that allows direct determination of the volume changes upon conformational transitions in macromolecules.

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#26183462   2016/01/28 Save this To Up

Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice.

Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N-terminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged β-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps.

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#26183121   2015/11/23 Save this To Up

Forum for debate: Safety of allogeneic blood transfusion alternatives in the surgical/critically ill patient.

In recent years, several safety alerts have questioned or restricted the use of some pharmacological alternatives to allogeneic blood transfusion in established indications. In contrast, there seems to be a promotion of other alternatives, based on blood products and/or antifibrinolytic drugs, which lack a solid scientific basis. The Multidisciplinary Autotransfusion Study Group and the Anemia Working Group España convened a multidisciplinary panel of 23 experts belonging to different healthcare areas in a forum for debate to: 1) analyze the different safety alerts referred to certain transfusion alternatives; 2) study the background leading to such alternatives, the evidence supporting them, and their consequences for everyday clinical practice, and 3) issue a weighted statement on the safety of each questioned transfusion alternative, according to its clinical use. The members of the forum maintained telematics contact for the exchange of information and the distribution of tasks, and a joint meeting was held where the conclusions on each of the items examined were presented and discussed. A first version of the document was drafted, and subjected to 4 rounds of review and updating until consensus was reached (unanimously in most cases). We present the final version of the document, approved by all panel members, and hope it will be useful for our colleagues.

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#26166362   2015/08/08 Save this To Up

Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

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#25707103   2015/02/24 Save this To Up

[Production and properties of recombinant glutenin-cleaving proteinases from Eurygaster integriceps Put].

cDNAs coding for a mature form of glutenin-cleaving trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest-Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin, treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes:

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#25460544   2015/01/17 Save this To Up

Effect of protease inhibitors on pulmonary bioavailability of therapeutic proteins and peptides in the rat.

The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2 kD), salmon calcitonin (3.4 kD), human insulin (5.8 kD), human leptin (16.0 kD), and human chorionic gonadotropin (HCG) (36.5 kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1 mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

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