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Journal articles on the topic "Apc protein"
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Brugge, Jeroen, Guido Tans, Jan Rosing, and Elisabetta Castoldi. "Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels." Thrombosis and Haemostasis 95, no. 02 (2006): 236–42. http://dx.doi.org/10.1160/th05-08-0582.
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SummaryElevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, area risk factor for venous thrombosis,partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated proteinC (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels asa modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and proteinS had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of proteinS confirmed that proteinS levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.
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Sen, Prosenjit, Sanghamitra Sahoo, Usha Pendurthi, and L. Vijaya Mohan Rao. "Zinc Binding to Protein C and Activated Protein C Modulates Their Interaction with Endothelial Cell Protein C Receptor." Blood 114, no. 22 (November 20, 2009): 331. http://dx.doi.org/10.1182/blood.v114.22.331.331.
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Abstract Abstract 331 Introduction/background: Zinc is a multi-functional element that is essential for life and the second most abundant metal ion, after iron in eukaryotic organisms. Zinc deficiency has been associated with bleeding disorders and with defective platelet aggregation, suggesting it may play an important role in hemostasis. Zinc ions have been shown to enhance activation of the intrinsic pathway of coagulation but to down-regulate the extrinsic pathway of coagulation. All vitamin K-dependent coagulation proteins have calcium binding sites and may therefore to some extent, interact with other divalent metal ions, including zinc, through these sites. Recent crystallography studies identified a pair of Zn2+ binding sites in FVIIa protease domain, and with the exception of Glu220, all the side chains involved in both the Zn1 and Zn2 coordination in FVIIa are unique to FVIIa and are not present in other vitamin K-dependent clotting factors (Bajaj et al., J Biol Chem 2006; 281:24873-88). Nonetheless, Zn2+ may bind to other vitamin K-dependent clotting factors at sites different from those identified in FVIIa. Objective: The aim of the present study is to investigate the effect of zinc ions on the protein C pathway, particularly on protein C/APC binding to EPCR, protein C activation and APC catalytic activity. Methods: Protein C and APC binding to EPCR on endothelial cells was examined by radioligand binding studies. Protein C activation and APC catalytic activity were evaluated in chromogenic assays. Equilibrium dialysis was used to measure zinc binding to protein C/APC. Conformational changes in protein C/APC were monitored by intrinsic fluorescence quenching. Results: Zn2+ does not replace the Ca2+ as a mandatory cofactor for protein C/APC binding to EPCR but Zn2+ at physiologically relevant concentrations (10 to 25 μM) markedly increased Ca2+-dependent protein C and APC binding to EPCR (∼2 to 5-fold). The kinetic analysis of protein C and APC binding to EPCR suggested that Zn2+ enhanced protein C/APC binding to EPCR by increasing the binding affinity of protein C/APC to its receptor (Kd for APC: – Zn2+, 117 ± 27 nM; + Zn2+, 9.3 ± 3.3 nM; Kd, for protein C: – Zn2+, 96 ± 26 nM; + Zn2+, 21.4 ± 6.6 nM). The enhancing effect of Zn2+ on APC binding to EPCR was also observed in the presence of physiological concentrations of Mg2+, which itself increased the APC binding to EPCR, two-fold. Zn2+-mediated increased protein C binding to EPCR resulted in increased APC generation. The effect of Zn2+ was not limited to enhancing protein C and APC binding to EPCR but also affected the catalytic activity of APC. Zn2+ inhibited the amidolytic activity of APC half-maximally at 50 to 100 μM. Zn2+ also inhibited the amidolytic activity of Gla domain deleted (GD)-APC in a similar fashion. The inhibitory effect of Zn2+ was partially reversed by physiological concentrations of calcium. Addition of Zn2+ to protein C or APC quenched the intrinsic fluorescence of both APC and GD-APC. Data from the equilibrium binding studies performed with 65Zn2+ revealed that Zn2+ binds to both GD-APC and APC, but that the amount of Zn2+ bound to APC was 3 to 4-fold higher than the amount bound to GD-APC. Kinetic analysis of equilibrium binding studies suggested that two Zn2+ atoms bind to APC outside the Gla domain with relatively high affinity (∼70 μM). At least one of the Zn2+ sites may overlap with the Ca2+ binding site as the Zn2+ binding to GD-APC was inhibited by approximately 50% by saturating concentrations of Ca2+. The substantially increased Zn2+ binding to the APC compared to GD-APC suggested that the N-terminus of the Gla domain of protein C contains multiple Zn2+ binding sites. Interestingly, Zn2+ bound to APC and GD-APC with a similar high affinity suggesting that the Gla domain, as well as the protease domain, may contain high affinity binding sites for Zn2+. A majority of the Zn2+ binding sites in the Gla domain appear to be distinct from the Ca2+ binding sites as less than 40% of the maximal Zn2+ binding could be blocked by Ca2+. The putative zinc binding sites in protein C/APC appeared to be unique as no consensus canonical zinc binding sequences homologous to other known zinc binding proteins were found in protein C. Conclusions: Our present data show that Zn2+ binds to protein C/APC and induces a conformational change in these proteins, which in turn leads to higher affinity binding to their cellular receptor EPCR. Overall our results suggest that zinc ions may play an important regulatory role in the protein C pathway. Disclosures: No relevant conflicts of interest to declare.
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Preston, Roger JS, Shona Harmon, Fionnuala B. Ni Ainle, Jennifer A. Johnson, Moya Cunningham, O. Smith, Barry White, and James S. O’Donnell. "Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement." Blood 112, no. 11 (November 16, 2008): 21. http://dx.doi.org/10.1182/blood.v112.11.21.21.
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Abstract Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (<2-fold) anticoagulant activity compared to wild type APC. The anticoagulant activity of APC-L38D, however, was severely impaired. APC-L38D was unable to achieve half-maximal inhibition of endogenous thrombin potential (ETP) at APC concentrations as high as 150nM, compared to wild type APC, which achieved half-maximal inhibition at 7.2nM APC. To clarify the role of Leu-38 in facilitating APC anticoagulant function, we further studied the ability of APC-L38D to be stimulated in protein S-deficient plasma reconstituted with plasma-purified protein S. Co-incubation of wild type APC with increasing protein S concentration (12.5–200nM) caused a corresponding reduction in ETP (IC50 = 24nM protein S). In contrast, APC-L38D was unresponsive to protein S. In the presence of APC-L38D, ETP was reduced only 22% at 1.5μM protein S (10-fold higher than plasma free protein S). In a phospholipid-dependent FVa proteolysis time course assay, both wild type APC and APC-L38D rapidly reduced FVa cofactor activity, indicating that the observed impaired plasma anticoagulant activity of APC-L38D is not mediated by impaired interaction with anionic phospholipids or FVa. In a modified version of this assay, wild type APC-mediated FVa proteolysis was rapidly enhanced by added protein S, with half-maximal inhibition observed at 5nM protein S. In contrast, APC-L38D exhibited no protein S-enhanced FVa proteolysis. Cumulatively, these data confirm that Leu-38 mediates APC anticoagulant function in plasma by facilitating critical protein S cofactor enhancement of FVa proteolysis. Previous studies have shown that APC Gla domain mutations can influence EPCR binding, a pre-requisite for PAR-1 mediated cytoprotective signalling. Consequently, we assessed APC binding to sEPCR using surface plasmon resonance. Binding affinities of APC-L38D and wild type APC were very similar (KD 112±25nM versus 117±36nM). Furthermore, the ability of APC-L38D to protect EAhy926 cells from staurosporine-induced apoptosis was also investigated using RT-PCR quantification of pro- (bax) and anti- (bcl-2) apoptotic gene expression. Pre-incubation with APC-L38D significantly reduced the bax/bcl-2 ratio to the same extent as wild type APC. The EPCR-dependence of these anti-apoptotic activities was confirmed using RCR-252, (an inhibitory anti-EPCR antibody) which ablated the cytoprotective effect of both APC species. In conclusion, we demonstrate that a single amino acid substitution (L38D) can significantly impair APC anticoagulant activity due to elimination of protein S cofactor enhancement. However, despite the location of Leu-38 in the Gla domain, APC-L38D retains its ability to bind EPCR, and trigger PAR-1 mediated cytoprotective signalling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to dissociate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.
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Marx, Antje, Hans Weiler, Volker Liebe, Siegfried Lang, Jens Kaden, Wolfgang Zieger, Martin Borggrefe, Guenter Huhle, Karl Haase, and Martina Brueckmann. "Stabilization of monocyte chemoattractant protein-1-mRNA by activated protein C." Thrombosis and Haemostasis 89, no. 01 (2003): 149–60. http://dx.doi.org/10.1055/s-0037-1613554.
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SummaryThe activated protein C (APC) pathway has been suggested to be a common link between coagulation and inflammation. APC may function to restore hemostasis via modulation of cytokine expression. We investigated the effect of APC on the endothelial expression of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is controlled by the activation of central proinflammatory transcription factors, such as nuclear factor-kappa B (NF-κ B).We found that human APC (2.5-10 μ g/ml) upregulated the amount of MCP-1-mRNA in human umbilical vein endothelial cells (HUVEC) and caused a time- and dose-dependent increase in MCP-1 protein production (p <0.001 for APC 2.5 μg/ ml at 4 up to 24 h). In this cell culture model MCP-1 induced an improvement of cell migration and wound repair after injury to endothelial monolayers. After stimulation of MCP-1-mRNA-transcription with TNF-α (0.1-1 ng/ml), HUVEC’s were washed and an inhibitor of gene transcription, Actinomycin D (1 μg/ml), was added in the presence or absence of APC. HUVEC’s receiving APC contained more MCP-1-mRNA than controls after one hour and up to eight hours suggesting an inhibitory effect of APC on MCP-1-mRNA degradation (with APC: 753 ± 56 atto mol of MCP-1-mRNA per ml of cell lysate vs. 263 ± 60 atto mol/ml without APC at t =4 h; p <0.001). Electrophoretic mobility shift assays revealed that APC attenuated NF- κB DNA-binding capacity implying that NF- B may not be involved in the upregulatory effect of APC on MCP-1 production.The ability of APC to upregulate the production of MCP-1, most likely by increasing the stability of MCP-1-mRNA rather than by transcriptional activation via NF- B, identifies a novel immunomodulatory pathway, by which APC may control the local inflammatory reaction, thereby initiating wound repair and modulating the extent of endothelial injury.
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Maurissen, Lisbeth F. A., M. Christella L. G. D. Thomassen, Gerry A. F. Nicolaes, Björn Dahlbäck, Guido Tans, Jan Rosing, and Tilman M. Hackeng. "Re-evaluation of the role of the protein S-C4b binding protein complex in activated protein C-catalyzed factor Va-inactivation." Blood 111, no. 6 (March 15, 2008): 3034–41. http://dx.doi.org/10.1182/blood-2007-06-089987.
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AbstractProtein S expresses cofactor activity for activated protein C (APC) by enhancing the APC-catalyzed proteolysis at R306 in factor Va. It is generally accepted that only free protein S is active and that complex formation with C4b-binding protein (C4BP) inhibits the APC-cofactor activity of protein S. However, the present study shows that protein S-C4BP expresses APC-cofactor activity and stimulates APC-catalyzed proteolysis at R306 more than 10-fold, but instead inhibits proteolysis at R506 by APC 3- to 4-fold. Free protein S stimulates APC-catalyzed cleavage at R306 approximately 20-fold and has no effect on cleavage at R506. The resulting net effect of protein S-C4BP complex formation on APC-catalyzed factor Va inactivation is a 6- to 8-fold reduction in factor Va inactivation when compared with free protein S, which is not explained by inhibition of APC-cofactor activity of protein S at R306, but by generation of a specific inhibitor for APCcatalyzed proteolysis at R506 of factor Va. These results are of interest for carriers of the factor VLeiden mutation (R506Q), as protein S-C4BP effectively enhances APC-catalyzed factor Va (R306) inactivation in plasma containing factor VLeiden.
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Nishioka, Junji, Masaru Ido, Tatsuya Hayashi, and Koji Suzuki. "The Gla26 Residue of Protein C Is Required for the Binding of Protein C to Thrombomodulin and Endothelial Cell Protein C Receptor, but not to Protein S and Factor Va." Thrombosis and Haemostasis 75, no. 02 (1996): 275–82. http://dx.doi.org/10.1055/s-0038-1650260.
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SummaryA functionally defective protein C (PC)-Mie, detected in the plasma of a patient with hereditary thrombophilia, has Lys substituted for γ-carboxyglutamic acid (Gla)26 residue. The activation rate of PC-Mie by Protac or thrombin in the absence of Ca2+ and that by thrombin with native thrombomodulin (TM), recombinant soluble truncated TM or on cultured endothelial cells in the presence of Ca2+ were all apparently lower than that of normal PC. The anticoagulant activity of Protac-activated PC (APC)-Mie on the plasma clotting time and the rate of inactivation of factor Va by APC-Mie in the presence of phospholipids were lower than those seen with normal APC. APC-Mie and normal APC bound equally to protein S and to biotinyl-factor Va. However, neither PC-Mie nor APC-Mie bound to phospholipids and to cultured human endothelial cells. It was similar to that observed with Gla-domainless PC/APC, but different from that seen with normal PC/APC. These results suggest that Gla26-dependent conformation is required for the binding of PC/APC to phospholipids, TM and the surface of endothelial cell PC/APC receptor, but not to protein S and factor Va.
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Pérez-Casal, Margarita, Colin Downey, Kenji Fukudome, Gernot Marx, and Cheng Hock Toh. "Activated protein C induces the release of microparticle-associated endothelial protein C receptor." Blood 105, no. 4 (February 15, 2005): 1515–22. http://dx.doi.org/10.1182/blood-2004-05-1896.
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Abstract Activated protein C (APC) treatment is now used for patients with severe sepsis. We investigated its effect in vitro on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release that is not inhibited by metalloproteinase inhibitors. Exposure of human umbilical vein endothelial cells or monocytes to APC (6.25-100 nM) results in the release of EPCR-containing microparticles, as demonstrated by confocal microscopy and characterized through flow cytometry, enzyme-linked immunosorbent assay quantitation of isolated microparticles, and Western blotting. The phenomenon is time- and concentration-dependent and requires the APC active site, EPCR, and protease activated receptor 1 (PAR1) on endothelial cells. Neither protein C nor boiled or d-Phe-Pro-Arg-chloromethylketone–blocked APC can induce microparticle formation and antibody blockade of EPCR or PAR1 cleavage and activation abrogates this APC action. Coincubation with hirudin does not alter the APC effect. The released microparticle bound is full-length EPCR (49 kDa) and APC retains factor V–inactivating activity. Although tumor necrosis factor-α (10 ng/mL) can also induce microparticle-associated EPCR release to a similar extent as APC (100 nM), it is only APC-induced microparticles that contain bound APC. This novel observation could provide new insights into the consequences of APC therapy in the septic patient.
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Preston, Roger JS, Jennifer A. Johnson, Fionnuala Ni Ainle, Shona Harmon, Owen P. Smith, Barry White, and James S. O’Donnell. "Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement." Blood 112, no. 11 (November 16, 2008): 20. http://dx.doi.org/10.1182/blood.v112.11.20.20.
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Abstract Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.
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de Fouw, N. J., Y. F. de Jong, F. Haverkate, and R. M. Bertina. "Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S." Thrombosis and Haemostasis 60, no. 02 (1988): 328–33. http://dx.doi.org/10.1055/s-0038-1647055.
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summaryThe effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125T-fibrinogen and thrombin. All proteins were of human origin.In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.
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Deane, Rashid, Barbra LaRue, Abhay P. Sagare, Francis J. Castellino, Zhihui Zhong, and Berislav V. Zlokovic. "Endothelial Protein C Receptor-Assisted Transport of Activated Protein C across the Mouse Blood—Brain Barrier." Journal of Cerebral Blood Flow & Metabolism 29, no. 1 (October 8, 2008): 25–33. http://dx.doi.org/10.1038/jcbfm.2008.117.
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Activated protein C (APC), a serine-protease with anticoagulant, anti-inflammatory, and cytoprotective activities, is neuroprotective and holds potential to treat different neurologic disorders. It is unknown whether APC crosses the blood—brain barrier (BBB) to reach its therapeutic targets in the brain. By using a brain vascular perfusion technique, we show that 125I-labeled plasma-derived mouse APC enters the brain from cerebrovascular circulation by a concentration-dependent mechanism. The permeability surface area product of 125I-APC (0.1 nmol/L) in different forebrain regions ranged from 3.11 to 4.13 μL/min/g brain. This was approximately 80- to 110-fold greater than for 14C-inulin, a simultaneously infused reference tracer. The Km value for APC BBB cortical transport was 1.6±0.2 nmol/L. Recombinant APC variants with reduced anticoagulant activity, 5A-APC and 3K3A-APC, but not protein C, exhibited high affinity for the APC BBB transport system. Blockade of APC-binding site on endothelial protein C receptor (EPCR), but not blockade of its protease-activated receptor-1 (PAR1) catalytic site, inhibited by > 85% APC entry into the brain. APC brain uptake was reduced by 64% in severely deficient EPCR mice, but not in PAR1 null mice. These data suggest that APC and its variants with reduced anticoagulant activity cross the BBB via EPCR-mediated saturable transport.
Dissertations / Theses on the topic "Apc protein"
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Tickenbrock, Lara. "Das Tumorsuppressor-Protein APC strukturelle und biochemische Aspekte /." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=966017064.
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Hankey, William C. IV. "Chromatin-associated functions of the APC tumor suppressor protein." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480198247672881.
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Cuddihy, Jane. "The non-Wnt functions of APC : unravelling the link between APC and apoptosis." Thesis, University of Dundee, 2016. https://discovery.dundee.ac.uk/en/studentTheses/bfb0d6ce-149b-4152-a591-943d61e2c714.
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Colorectal cancer (CRC) is the second most common cause of cancer-related death in the UK and Western world. More than 90% of sporadic CRCs harbour mutations in the multi-functional tumour suppressor gene Adenomatous polyposis coli (Apc). The most commonly studied function of APC is its role as a scaffold for the β-catenin destruction complex involved in Wnt signalling. However, APC binds many other proteins. For example, it directly binds to and stabilises microtubules and actin. These non-Wnt related functions of APC are poorly understood. My PhD examines non-Wnt functions of APC. To this end, I created degron-tagged APC in DT40 cells that allowed for the rapid, conditional degradation of endogenous APC. The aim was to identify the immediate effects on cellular processes. Then, to identify the contribution of different APC domains by measuring the ability to rescue any defects when reintroducing fragments of APC. However, creation of these degron-tagged Apc knock-in cell lines resulted in hypomorphic phenotypes and auxin-associated off-target effects. Nonetheless, I compared the response of APChigh, APClow, and APCminimal cells to DNA damaging agents and Taxol® but found no significant differences. Subsequently, I focused on the relationship between APC and apoptosis. Previous observations suggested that deficiency in Apc rendered cells less sensitive to low doses of Taxol®. However, Apc deficient cells were more readily killed when Taxol® was combined with the Bcl-2 inhibitor, ABT-737. One possible explanation is the increase in Bcl-2 protein upon Apc depletion. However, I found that ABT-737, Taxol® and Apc depletion each cause activation of the unfolded protein response. This suggests that these treatments elicit a stress response that can stimulate apoptosis. Moreover, the same treatments also cause changes in mitochondria. Importantly, all of these effects do not require an increase in the β-catenin protein. Together, my data reveal novel links between APC and apoptosis that could be exploited clinically.
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Vijaya, Chandra Shree Harsha [Verfasser], and Jürgen [Akademischer Betreuer] Behrens. "Functional analysis of truncated APC protein in human colorectal cancers = Funktionelle Analyse von verkürztem APC Protein in humanem kolorektalen Krebs / Shree Harsha Vijaya Chandra. Betreuer: Jürgen Behrens." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2011. http://d-nb.info/1015474802/34.
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Yu, Yao. "Endothelial cell signalling of APC and thrombin : the involvement of protein kinase C." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/18048.
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Activated protein C (APC) is a natural anticoagulant. In addition to this well-described function, APC also mediates endothelial cell (EC) cytoprotection, including anti-apoptotic, anti-inflammatory and endothelial barrier integrity stabilising effects. APC confers these effects through proteolytic activation of protease activated receptor 1 (PAR1), but only when it is bound to its receptor, endothelial protein C receptor (EPCR). Interestingly, thrombin exerts opposing cellular responses (including induction of endothelial hyperpermeability) through activation of PAR1. How activation of the same receptor can mediate such different signals remains unclear. I hypothesised that, downstream of PAR1 cleavage, activation of different protein kinase C (PKC) isoforms may account for the divergent signal transduction. The aim of my thesis was to explore the contribution of specific PKC isozyme(s) that are involved in APC and thrombin mediated signalling. To do this, I expressed, purified and characterised a panel of APC variants lacking either anticoagulant activity, EPCR binding, PAR1 binding or proteolytic activity. I demonstrated that both APC and thrombin activate Erk1/2 pathway in the endothelial cells in a dose and time-dependent manner. APC also dose-dependently stabilised endothelial monolayer integrity, whereas thrombin disrupted this. APC’s cytoprotective properties, based on this established cell assay model, were found to be PAR1 and EPCR dependent. Using peptides that inhibit specific PKC isozymes, I found that the atypical PKCζ isozyme, was involved in Erk1/2 phosphorylation triggered by thrombin and both classical PKC isozymes (PKCβ1) and PKCζ were involved in the hyperpermeability induced by thrombin. In contrast, no PKC isozyme was found to play roles in the APC-mediated Erk1/2 activation as well as EC permeability reduction. These findings might indicate that the difference in PKC involvement may contribute to the diverse intracellular signalling pathways of APC and thrombin, and hence their opposite cellular responses.
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Shen, Ying. "Regulation of EphA4 expression through the APC-mediated ubiquitin-proteasome pathway /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BICH%202007%20SHEN.
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Mills, Kate May. "A novel role for Adenomatous Polyposis Coli protein in the transport of mitochondria." Thesis, The University of Sydney, 2015. http://hdl.handle.net/2123/14184.
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Adenomatous Polyposis Coli (APC) is a multifunctional tumour suppressor protein, contributing to pathways in normal cell growth and differentiation. APC gene mutation is one of the earliest events in the progression of colorectal cancer (CRC), and typically gives rise to a truncated protein lacking C-terminal sequences, initiating deregulation of key cellular pathways. This thesis describes a new role for APC in mitochondrial transport. Silencing of wild-type APC by siRNA induced a redistribution of mitochondria from the cell periphery to the perinuclear region. Subsequently, novel interactions for APC were identified at the mitochondria with kinesin-motor complex proteins Miro/Milton. These interactions were mapped to the C-terminus of APC, correlating with defective mitochondrial transport and loss of Miro/Milton binding in CRC cells, which were restored by reconstitution of wild-type APC. Analysis by live cell imaging showed that loss of APC slowed the frequency of mitochondrial anterograde transport towards the cell periphery. It is proposed that APC drives mitochondria to the membrane to supply energy required for directed cell migration, a process disrupted in CRC. This opens up a new route through which CRC-associated APC mutations may contribute to carcinogenesis.
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Dieckhoff, Patrick. "Protein modification and degradation in the cell cycle of the yeast Saccharomyces cerevisiae." Doctoral thesis, [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972638644.
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Quyn, Aaron J. "The role of the APC protein in mitotic spindle orientation and tissue organisation in gut epithelium." Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505629.
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Zimmermann, Julia Janina [Verfasser]. "Untersuchung des Einflusses verschiedender Liganden auf die Inaktivierungskinetik von aktiviertem Protein C (APC) / Julia Janina Zimmermann." Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/1080591761/34.
Full textBooks on the topic "Apc protein"
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Näthke, Inke S. APC proteins. New York, N.Y: Springer Science+Business Media, 2009.
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S, Näthke Inke, and McCartney Brooke M, eds. APC proteins. New York, N.Y: Springer Science+Business Media, 2009.
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S, Näthke Inke, and McCartney Brooke M, eds. APC proteins. New York, N.Y: Springer Science+Business Media, 2009.
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S, Näthke Inke, and McCartney Brooke M, eds. APC proteins. New York, N.Y: Springer Science+Business Media, 2009.
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Inuzuka, Hiroyuki. SCF and APC E3 ubiquitin ligases in tumorigenesis. Cham: Springer, 2014.
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Näthke, Inke S., and Brooke M. McCartney, eds. APC Proteins. New York, NY: Springer New York, 2009. http://dx.doi.org/10.1007/978-1-4419-1145-2.
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Scott, Deborah. Determination of the effect of activated protein C (APC) upon the anticoagulant activity of various forms of heparin as determined in-vitro in normal human plasma. [S.l: The Author], 2003.
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B, Holland I., ed. ABC proteins: From bacteria to man. Amsterdam: Academic Press, 2003.
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1958-, Kuchler Karl, Rubartelli Anna 1956-, and Holland Barry, eds. Unusual secretory pathways: From bacteria to man. New York: Chapman & hall, 1997.
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Ahcène, Boumendjel, Boutonnat Jean, and Robert Jacques M. D, eds. ABC transporters and multidrug resistance. Hoboken, N.J: John Wiley & Sons, 2009.
Find full textBook chapters on the topic "Apc protein"
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Tripodi, A. "Activated protein C (APC) resistance." In Laboratory Techniques in Thrombosis - a Manual, 163–69. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4722-4_17.
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Prasad, Ramesh, Abhishek Roy, and Prosenjit Sen. "Functional Aspects of Activated Protein C (APC) in Regulating Homeostasis and Disease." In Proteases in Human Diseases, 395–408. Singapore: Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-3162-5_18.
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Hoffmann, J. N., M. W. Laschke, B. Vollmar, J. Fertmann, D. Inthorn, F. W. Schildberg, J. Römisch, and M. D. Menger. "Aktiviertes Protein C (APC) bei der Sepsis: Zelluläre und mikrohämodynamische Mechanismen der Protektion." In Zurück in die Zukunft, 564–65. Berlin, Heidelberg: Springer Berlin Heidelberg, 2003. http://dx.doi.org/10.1007/978-3-642-55611-1_378.
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Hoffmann, Johannes N., B. Vollmar, M. W. Laschke, D. Inthorn, F. W. Schildberg, and M. D. Menger. "Aktiviertes Protein C (APC) bei der Sepsis: Zelluläre und mikrohämodynamische Mechanismen der Protektion." In Deutsche Gesellschaft für Chirurgie, 327–29. Berlin, Heidelberg: Springer Berlin Heidelberg, 2003. http://dx.doi.org/10.1007/978-3-642-19024-7_90.
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Brocardo, Mariana, and Beric R. Henderson. "Detection of Cytoplasmic and Nuclear Localization of Adenomatous Polyposis Coli (APC) Protein in Cells." In Methods in Molecular Biology, 77–89. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-249-6_6.
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Uttenreuther-Fischer, M. M., B. Vetter, C. Hellmann, U. Otting, S. Ziemer, G. Gaedicke, and A. E. Kulozik. "Thromboembolien im Kindesalter: Einflüsse nichtgenetischer Faktoren und die Rolle der Resistenz gegen aktiviertes Protein C (APC-R) und des Protein-C-Mangels." In 28. Hämophilie-Symposion Hamburg 1997, 117–22. Berlin, Heidelberg: Springer Berlin Heidelberg, 1999. http://dx.doi.org/10.1007/978-3-642-59915-6_16.
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Gradehandt, Gernot, Johannes Hampl, Nadja Kleber, Christina Lobron, Silke Milbradt, Burkhard Schmidt, and Erwin Rüde. "Requirements of Exogenous Protein Antigens for Presentation to CD4+ T lymphocytes By MHC Class II-Positive APC." In Advances in Experimental Medicine and Biology, 23–27. Boston, MA: Springer US, 1993. http://dx.doi.org/10.1007/978-1-4615-2930-9_4.
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Catimel, Bruno, John D. Wade, M. Faux, Anthony Burgess, Laszlo Otvos, and Edouard Nice. "The Use of a Synthetic Biotinylated Peptide Probe for the Isolation of Adenomatous Polyposis Coli (APC) Tumor Suppressor Protein Complexes." In Peptides: The Wave of the Future, 998–99. Dordrecht: Springer Netherlands, 2001. http://dx.doi.org/10.1007/978-94-010-0464-0_466.
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Bachmaier, Sabine, and Michael Boshart. "Kinetoplastid AGC Kinases." In Protein Phosphorylation in Parasites, 99–122. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013. http://dx.doi.org/10.1002/9783527675401.ch05.
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Roston, Rebecca L., Anna K. Hurlock, and Christoph Benning. "Plastidic ABC Proteins." In Signaling and Communication in Plants, 103–36. Cham: Springer International Publishing, 2014. http://dx.doi.org/10.1007/978-3-319-06511-3_7.
Full textConference papers on the topic "Apc protein"
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de Four, N. J., R. M. Bertina, and F. Havgrkate. "STIMULATION OF FIBRINOLYSIS BY ACTIVATED PROTEIN C (APC)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642961.
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In 1960 Mammen and Seegers reported the discovery of a new protein (autoprothrombin II-A, APC) with both anticoagulant and profibrinolytic activity. They found that APC accelerated clot lysis in vitro and proposed that this was due to a reduction of plasmin - inhibitory activity. Many years later Comp et al (J Clin Inv 68: 1221) reported that the infusion of APC into dogs resulted in an increase in circulating plasminogen activator activity. This observation stimulated more extensive studies of the profibrinolytic effects of APC.In our laboratories we have studied the effect of human APC on clot lysis both in whole blood (human) and in a system of purified human proteins. In these systems 125I-labelled fibrinogen was incorporated in a clot formed after the addition of Jombin (complete clot formation within 5 min) and the subsequent lysis of this clot was followed by measuring the release of I-labelled fibrin degradation products (FDP) into the supernatant. Human t-PA was added to the system to achieve complete lysis of the clot within a few hours.When APC was added to citrated whole blood before clot formation, it was found to accelerate clot lysis in a dose dependent way. This effeg| was specific for APC and dependent on an intact active site, on the presence of protein S (the protein cofactor of APC) and Ca . The presence of APC did not influence the composition of the FDP formed, as analysed by means of SDS-polyacry-1 amide gel electroforesis, and its effect was found to be independent of the presence or absence of a.-antiplasmin.Subsequently we developped a clot lysis system using the purified human proteins of the fibrinolytic system: fibrinogen, FXIII, t-PA, PAI-1 (from human endothelial cells), glu-plasminogen and a -antiplasmin. In this system clot lysis was dependent on the concentrations of plasminogen, -antiplasmin, t-PA and PAI-1, but independent on the thrombin concentration and the presence or absence of phospholipids (purified from human brain). In the absence of PAI-1, no effect of APC on clot lysis was observed. However, in the presence of PAI-1, APC accelerated clot lysis. This effect was independent of the presence or absence of phospholipids and/or protein S and could be explained by the observation that APC can form a complex with PAI-1 (~ 95 kd) and under certain conditions even can convert active PAI-1 (~ 46 kd) into an inactive degradation product (~ 42 kd). However, complex formation is relatively slow anti high PAI-1 concentrations are needed to observe the reaction. The addition of protein S or phospholipids in the presence of Ca did not stimulate complex formation. Therefore, it seems highly unlikely that neutralization of PAI-1 by APC is responsible for the profibrinolytic effect of APC in the whole blood clot lysis.A completely different explanation for the profibrinolytic effect of APC was suggested by the observation that the addition of blood-platelets to the system of purified fibrinolytic components introduced a dependence of the clot lysis rate on the thrombin concentration (decrease in clot lysis at increasing thrombin concentration). This finding opened the possibility that APC stimulated fibrinolysis by reducing the effective thrombin concentration. Subsequent experiments using the whole blood clot lysis system revealed that in the presence of anti-FX antibodies clot lysis was no longer accelerated by APC, while the actual rate of clot lysis depended on the concentration of thrombin added.We like to propose, that in a blood clot lysis system APC most likely accelerates fibrinolysis by reducing the effective thrombin concentration; if at all, neutralization of PAI-1 may play only a minor role.
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Heeb, M. J., F. Espana, M. Geiger, D. Collen, D. C. Stump, and J. H. Griffin. "IMMUNOLOGICAL SIMILARITIES BETWEEN PLASMA AND URINARY PROTEIN C INHIBITORS (PCIs) AND URINARY UROKINASE INHIBITOR (UKI)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643816.
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Plasma PCIs have similar MW (∼ 57K), amino acid composition, and heparin dependence (Suzuki et al 1983, JBC 258:163) as urinary UKI (Stump et al 1986, JBC 261:12759). Urinary PCI of ∼ 50K MW has a similar heparin dependence and urokinase (UK) competes with activated protein C (APC) for this PCI (Geiger et al 1986, Circ. 74:11-234). For comparison, three forms of PCI, one from urine and two from plasma, were purified, and each exhibited heparin-dependent UK and APC inhibitory activity and formed heparin-dependent complexes with APC. The APC-PCI complexes were visible on immunoblots (nondenaturing gels) developed using: A) monoclonal anti-UKI + 125I-antimouse IgG; B) polyclonal anti-plasma PCI + 125I-plasma PCI; and C) monoclonal anti-protein C + 125I-protein C. The three forms of purified PCI were detected by methods A and B. Two new bands of APC-inhibitor complexes were seen upon incubation of plasma with APC in the presence of heparin, and the same pattern was visualized by methods A, B, and C. In the absence of heparin, only one APC-inhibitor band was visualized by methods A and B, but two bands were visualized by method C. Plasma immunodepleted of UKI by monoclonal anti-UKI-Sepharose showed no detectable antigen or complexes with APC as visualized by methods A and B. However, the UKI-depleted plasma contained components which formed a reduced amount of complexes with APC as visualized with protein C antibodies, i.e. method C. Heparin stimulates tenfold the PCI activity of normal plasma. Based on amidolytic assays of APC using S-2366, the UKI-depleted plasma was very deficient (< 15%) in heparin-dependent PCI activity, whereas the weak heparin-independent PCI activity was slightly reduced. This indicates that the majority of heparin-dependent PCI activity of plasma is immunologically.related to UKI. These studies suggest that the two slightly different forms of plasma PCI, the urinary UKI, and the urinary PCI are very similar if not identical proteins and that plasma may contain a minor heparin-independent PCI which is not immunologically related to these proteins.
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van der Meer, F. J. M., N. H. van Tilburg, I. K. van der Linden, E. Briét, and R. M. Bertina. "A SECOND INHIBITOR OF ACTIVATED PROTEIN C (APC)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643817.
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The inactivation of APC in plasma, as measured with the chromogenic substrate S2366, follows, in the absence of heparin, pseudo-first order kinetics. The ti of about 20 minutes is independent of the APC concentration (31-500 nM) and increases linearly with the dilution of the plasma. These observations suggest that the concentration of the APC inhibitor in plasma is much higher than 500 nM, which is much higher than the concentration of 88 nM reported by Suzuki for the protein C inhibitor (PCI).In the presence of heparin (5 IU/ml) the inactivation of APC becomes biphasic. Fast inactivation with an apparent ti of 8 minutes is followed by slower inactivation with a ti of 20 minutes.Removal of PCI from the plasma with α-PCI antibodies (kindly provided by Dr. Suzuki) has no effect on APC inactivation in the absence of heparin. However, in this plasma APC inactivation could not be stimulated by addition of heparin (absence of fast phase of APC-inactivation). These data suggest the presence of two APC inhibitors in plasma: the heparin dependent PCI (PCI-I), earlier reported by Suzuki, and a sofar unknown heparin-independent inhibitor (PCI-II).Further experiments showed that this PC I-11 has a molecular weight of 60 kD and is different from the APC-binding protein reported by Kisiel. Using Heparin-Sepharose affinity chromatography we could separate PCI -II from both PCI-I and the APC binding protein. In this PC I-11 preparation APC inactivation was accompanied by the formation of an APC-PCI -11 complex of about 105 kD as demonstrated by immunoblotting with a-PC antibodies after SDS-PAGE. The identity of PCI-II is unknown; however, it is different from antithrombin III, heparin cofactor II, α1-antitrypsin and β2-antiplasmin.The demonstration of the presence of two APC inhibitors in plasma will require a re-evaluation of the current functional assays for the APC inhibitor.
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Joost, S., A. Koedam, Joost C. M. Meijers, Jan J. Sixma, and Bonno N. Bouma. "VON WILLEBRAND FACTOR PROTECTS FACTOR VIII FROM INACTIVATION BY ACTIVATED PROTEIN C AND PROTEIN S." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643618.
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Activated protein C (APC) inactivated the cofactors factor V (FV) and factor VIII (FVIII). In the case of FV, this reaction and the respective roles of Ca2+ , phospholipids and protein S have been well documented. We investigated the role of protein S and von Willebrand factor (VWF) on the inactivation of FVIII.Purified human factor VIII (3 units/ml) was incubated with protein C (0.2 μg/ml) in the presence of 8 μg/ml phospholipid, 5 mM CaCl, and 1 unit/ml hirudin. Factor VIII coagulant activity decreased with a pseudo first-order rate constant of 0.09 min . The reaction rate increased linearly with the concentration of prot^ig S in the incubation mixture. 12I-FVIII was incubated under the same conditions. SDS-polyacrylamide gel electrophoresis showed cleavage products of Mr 43 and 22 kDa. High Mr bands (FVIII-heavy chain) ranging fromMr 108 to208 kDa disappeared while the Mr 80 kDa FVIII-lightchain remained unchanged. The degradationpattern was not changed by addition of protein S.The FVIII-VWF complex was reconstitutedby mixing the two components (±2 units VWF/units FVIII) and lowering the calcium concentration to 2 mM. The inactivation of the FVIII-VWF complex by APC proceeded at a 15- to 20-fold slower rate as compared to the isolated FVIII, indicating a protection of FVIII by VWF. Protein S exhibited no cofactor activity on the inactivation of FVIII-VWF by APC. The protective effect of VWF was lost completely after activation of the FVIII-VWF complexwith thrombin (0.05 units/ml).When FVIII (0.1 units/ml) was added toplasma of a patient with severe von Willebrand's disease, 96% of its activitywas lost in 20 min after the addition of APC. All of the FVIII activity was retained when haemophilic plasma was used. Mixing experiments showed that one unit ofVWF unit FVIII is needed to fully protec FVIII against APC. These results may explain the observed lability of FVIII in von Willebrand's disease patients.
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Hessing, Martin, Joost C. M. Meijers, Jan A. van Mourik, and Bonno N. Bouma. "MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S AND C4b-BINDING PROTEIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644291.
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Protein S (PS) circulates in plasma both free and in reversible association with the complement component C4b-binding protein (C4bp). Only free PS is functional as a cofactor for activated protein C (APC). Cleavage of PS by thrombin at a site near the r-carboxyglutamic acid domain is associated with a loss of cofactor activity. This may be a control mechanism for the anticoagulant activity of APC. These observations led us to investigate the role of C4bp and thrombin in the regulation of PS. Complex formation between purified PS and C4bp was studied in plasma and in a system with purified components. 125I-labeled PS was first incubated with either C4bp or citrated plasma and then subjected to polyacrylamide gelelectrophoresis in the absence of SDS. The formation of the C4bp-PS complex in plasma and in the purified system was demonstrated by autoradiography. Crossed immuno-electrophoresis using an antiserum against PS was performed in the presence of 8 mM EDTA. Human citrated plasma showed two precipitin peaks. Free PS migrated rapidly in the first dimension, whereas the C4bp-PS complex was just anodal to the application slot. The addition of C4bp to either plasma or purified PS resulted in the disappearance of the free PS peak and an increase of the slower migrating peak. The effect of purified C4bp on the PS-cofactor function of APC was studied in citrated plasma. The prolongation of the APTT induced by the addition of APC could be inhibited by the addition of increasing amounts of C4bp. Monoclonal antibodies to PS and C4bp were prepared and characterized. The monoclonal antibodies to either PS or C4bp did not block the complex formation between and PS, as was demonstrated by dot blotting of C4bp with 125I-PS and agarose gelelectrophoresis followed by Western blotting. Three out of 7 monoclonal antibodies to PS did not detect PS after thrombin cleavage on an immunoblot after non-reduced SDS polyacrylamide gelelectrophoresis. These 3 antibodies gave a significant shortening of the prolonged APTT induced by the addition of APC to normal plasma, indicating that these monoclonals inhibited the cofactor function of PS. The other 4 monoclonals to PS that did detect PS after thrombin cleavage on an immunoblot, gave only a minor inhibition of the PS cofactor function.
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Dahiback, Bjorn, Ake Lundwall, Andreas Hillarp, Johan Malm, and Johan Stenflo. "STRUCTURE AND FUNCTION OF VITAMIN K-DEPENDENT PROTEIN S, a cofactor to activated protein C which also interacts with the complement protein C4b-binding protein." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642960.
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Protein S is a single chain (Mr 75.000) plasma protein. It is a cofactor to activated protein C (APC) in the regulation of coagulation factors Va and Villa. It has high affinity for negatively charged phospolipids and it forms a 1:1 complex with APC on phospholipid surfaces, platelets and on endothelial cells. Patients with heterozygous protein S deficiency have a high incidence of thrombosis. Protein S is cleaved by thrombin, which leads to a loss of calcium binding sites and of APC cofactor activity. Protein S has two to three high affinity (KD 20uM) calcium binding sites - unrelated to the Gla-region - that are unaffected by the thrombin cleavage. In human plasma protein S (25 mg/liter) circulates in two forms; free (approx. 40%) and in a 1:1 noncovalent complex (KD 1× 10-7M) with the complement protein C4b-binding protein (C4BP). C4BP (Mr 570.000) is composed of seven identical 70 kDa subunits that are linked by disulfide bonds. When visualized by electron microscopy, C4BP has a spiderlike structure with the single protein S binding site located close to the central core and one C4b-binding site on each of the seven tentacles. When bound to C4BP, protein S looses its APC cofactor activity, whereas the function-of C4BP is not directly affected by the protein S binding. Chymotrypsin cleaves each of the seven C4BP subunits close to the central core which results in the liberation of multiple 48 kDa “tentacte” fragments and the formation of a 160 kDa central core fragment. We have successfully isolated a 160 kDa central core fragment with essentially intact protein S binding ability.The primary structure of both bovine and human protein S has been determined and found to contain 635 and 634 amino acids, respectively, with 82 % homology to each other. Four different regions were distinguished; the N-terminal Gla-domain (position 1-45) was followed by a region which has two thrombin-sensitive bonds positioned within a disulfide loop. Position 76 to 244 was occupied by four repeats homologous to the epidermal growth factor (EGF) precursor. In the first EGF-domain a modified aspartic acid was identified at position 95, B-hydroxaspartic acid (Hya), and in corresponding positions in the three following EGF-domains (positions 136,178 and 217) we found B-hydroxyasparagine (Hyn). Hyn has not previously been identified in proteins. The C-terminal half of protein S (from position 245) shows no homology to the serine proteases but instead to human Sexual Hormon Binding Globulin (SHBG)(see separate abstract). To study the structure-function relationship we made eighteen monoclonal antibodies to human protein S. The effects of the monoclonals on the C4BP-protein S interaction and on the APC cofactor activity were analysed. Eight of the antibodies were calciumdependent, four of these were against the Gla-domain, two against the thrombin sensitive portion and two against the region bearing the high affinity calcium binding sites. Three of the monoclonals were dependent on the presence of chelating agents, EDTA or EGTA, and were probably directed against the high affinity calcium binding region. Three other monoclonals inhibited the protein S-C4BP interaction. At present, efforts are made to localize the epitopes to gain information about functionally important regions of protein S.
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Schwarz, H. P., and W. Muntean. "LOW TOTAL PROTEIN S ANTIGEN BUT HIGH PROTEIN S ACTIVITY DUE TO DECREASED C4b-BINDING PROTEIN (C4b-BP) LEVELS IN NEWBORNS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643610.
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Vitamin K-dependent coagulation proteins are known to be decreased in the neonatal period. So far no data have been published on protein S (PS), the vitamin K-dependent cofactor for the antithrombotic enzyme, activated protein C (APC) in this period. We determined, therefore, PS antigen, PS activity and C4b-BP,a regulatory protein of the classical complement pathway to which PS is complexed, in 36 neonates. Total PS antigen in newborns was below the range associated with thromboembolism in patients congenitally deficient in this protein (22±9.6%, mean±SD). None of these infants had clinical or laboratory evidence of thromboembolism or DIC. In contrast to the PS antigen level PS activity measured by the ability of APC to prolong the clotting time of a modified APTT assay using PS-immunodep1eted plasma was significantly higher (77.6±14%, mean±SD, p< 0,001), suggesting a shift in PS to the free form. In fact two dimensional immunoe1ectrophoresis studies revealed the absence of protein S-C4b-BP complexes and only one precipitation indicating free PS was seen in 15 out of the 36 infants. In these 15 neonates C4b-BP was below the limit of detection by sensitive quantitative immunob1otting techniques using monoclonal or polyclonal antibodies. In the remaining 21 infants PS-C4b-BP complexes were detected, but in contrast to adult normal plasma approximately 80% of PS was found in the free form. Mixing experiments with normal human plasma and newborn’s plasma indicate that PS in neonate deficient of C4b-BP can bind normally to C4bp. Absence of C4b-BP did not correlate to gestational age. If an equilibrium distribution of PS between bound and free form regulates the cofactor activity of PS for the anticoagulant and profibrino 1ytic properties of APC in normal adults, our study demonstrates that the absence of PS-C4b-BP complexes in newborns and the presence of free PS only may contribute to the increased bleeding risk of premature infants.
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Suzuki, Koji, Yoshihiro Deyashiki, Junji Nishioka, Kazunori Toma, and Shuji Yamamoto. "THE INHIBITOR OF ACTIVATED PROTEIN C: STRUCTURE AND FUNCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642963.
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In the final step of protein C pathway, activated protein C (APC) is neutralized with a plasma inhibitor, termed protein C inhibitor (PCI). PCI was first described by Marlar and Griffin (1980) and then isolated from human plasma as a homogeneous form and characterized by the authors (1983). PCI is a single chain glycoprotein with M 57,000 and a plasma concentration of 5 ug/ml. Analysis of a cDNA nucleotide sequence has clarified that a precursor of human PCI consists of a mature protein of 387 amino acid residues (M 43,759) and a signal peptide of 19 amino acid residues. Only one cysteine residue is present in the entire protein as in α1antitrypsin (α1AT) and α1antichymotrypsin (α1ACT). Three Asn-X-Ser/Thr sequences and two Ser/Thr-X-X-Pro sequences are present as potential attachment sites of carbohydrate chains. Based on the amino acid sequence of the carboxyl-terminal peptide released from the inhibitor by APC digestion, the reactive site peptide bond of PCI was found to be Arg(354)-Ser(355). It is similar to the reactive sites of the other serine protease inhibitors which are located to their carboxyl-terminal Arg(393)-Ser (394), Met(358)-Ser(359) and Leu(358)-Ser(359) in antithrombin III, α1AT and α1ACT, respectively. The alignment of the amino acid sequence of PCI with heparin cofactor II, α1plasmin inhibitor, ovalbumin, angiotensinogen and the above noted plasma inhibitors showed that PCI is a member of serine protease inhibitor superfamily. PCI inhibits APC noncompetitively in a 1:1 stoichiometry and forms a covalent acyl-bond with a Ser residue in the active center of APC. The half life of APC in plasma approximately 30 min, which is rather slow compared with the other protease inhibitors. However, optimal concentrations of heparin, dextran sulfate and its derivatives potentiate the rate of inhibition 30-60 fold. PCI has Ki of 10-8m for APC, and can inhibit thrombin, Factor Xa, urokinase and tissue plasminogen activator as well in the presence of heparin or dextran sulfate, though the Ki for these enzymes is slightly higher. During the complex formation with APC, PCI is cleaved by the complexed APC to form a modified form with M 54,000. PCI is synthesized in several hepatoma cell lines and decreased in plasma of patients with liver cirrhosis. It is also decreased in patients with DIC or those during cardiopulmonary bypass in parallel with the decrease in protein C, suggesting that PCI participates in regulation of the protein C pathway in intravascular coagulation. Recently, we have obtained the recombinant PCI from COS-1 cells which were transfected with expression vector pSV2 containing the cDNA of PCI. The recombinant PCI had the same Mr and specific activity as the protein purified from plasma. It also had an affinity for heparin and dextran sulfate. Moreover, we have predicted a three dimentional structure of the proteolytically modified PCI with computer graphics based on its amino acid sequence homology with the modified α1AT whose structure had been elucidated with X-ray crystallography. All potential carbohydrate attachment sites were estimated to exist on the surface of the protein. Succesively we have constructed the interaction model between the intact PCI predicted from the modified form and the active center of APC which was simulated from that of trypsin. From the model, it was observed that the amino-group of Arg (354, PI site) of PCI could strongly interact with the carboxy1-group of Asp (88, SI site) of the heavy chain of APC at the base of the active center pocket of the enzyme.
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Nicham, F., and J. L. Martinoli. "AMIDOLYTIC DETERMINATION OF ANTI-ACTIVATED PROTEIN C ACTIVITY IN PLASMA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644316.
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Anti-activated protein C (anti-APC) potency of plasma was studied using purified bovine activated protein C (Bovine APC) and the chromogenic peptide substrate CBS 65.25. The choice of bovine instead of human APC was justified by a better sensitivity (Km = 0.14 and 0.42 mM respectively). Inhibition was shown to be dramatically enhanced by the presence of Heparin and calcium. No significant difference occurred for pH values up to 8.2 for both inhibition and hydrolysis reactions.In the final test, O.l ml of 1:5 diluted plasma (Tris buffer saline, pH 8.4, containing 5 U/ml of Heparin) were incubated at 37°C with 0.2 ml of Bovine APC (0.125 U/ml). After 10 minutes of inhibition, 0.2 ml of CBS 65.25 (1.5 mM/1) were added to the mixture and the change in absorbance was recorded at 405 nm for 2 minutes. In these conditions linearity of the dose-response curve was ensured from O up to 130 % of activity (normal plasma pool being assigned to 100 %) ; day to day precision was 1.9 %. When a normal plasma was overloaded with different purified inhibitors such as antithrombin III, cl-esterase inactivator, alpha 2 macroglobulin, the measured anti-APC activities were not affected at all. It could be concluded that this test measures protein C inhibitor described by Suzuki.Levels in 23 normal individuals averaged 97.7 %, giving a normal range of 77 - 118 %. Levels were below normal in 6 of 10 patients after surgery (54.1 +/- 4.8 %), in 18 of 19 patients with liver disease (49.5 +/- 9.6 %) and in 4 of 18 coumarin treated patients (54.9 +/- 6.5 %). In 9 of 10 patients previou sly characterized as type I protein C deficient, a statistically significant increase in anti-APC activity was observed (mean 110.7 +/- 7.7 %).The use of a chromogenic peptide substrate has led to a sensitive and fast assay for anti-APC activity in plasma. That could be of interest in clinical investigations and knowledge of regulatory mechanisms in thrombotic disorders.
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Laurell, M., T. Carlsson, and J. Stenflo. "IMMUNOAFFINITY PURIFICATION OF PLASMA INHIBITOR FOR ACTIVATED PROTEIN C." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643814.
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Activated protein C (APC) is an important regulator of blood coagulation in vivo. In plasma this serine protease is slowly inhibited by a specific inhibitor, activated protein C inhibitor (PCI), (Suzuki et al. (1983) J.Biol.Chem. 258 , 163-168). We have now made monoclonal antibodies against PCI by immunizing with the APC-PCI complex. Positive clones were identified by solid phase immunoassay with 125I labelled partially purified inhibitor. After subcloning and expansion in mice, one of the monoclonal antibodies was immobilized on Sepharose 4B and used in the purification of the inhibitor. A two step purification procedure was deviced starting with passage of fresh human plasma over the column. Following extensive washing the inhibitor was eluted with 50 mM triethylamine- HCI,0.5 M NaCl, pH 11.0, from the column together with a small amount of high molecular weight material. After gel filtration on a column packed with AcA 44 the inhibitor appeared homogenous on SDS - PAGE. Approximatly 0.5 mg inhibitor was obtained from 200 ml of fresh plasma. The apparent Mr of the inhibitor was 57000 kDa on SDS -PAGE. The purified protein formed a complex (Mr =110000 kDa) with human APC. At the same time a band (Mr = 54000 kDa ) appeared that represented the modified inhibitor. When analyzed on agarose gel electrophoresis (75mM barbital buffer, 2mM EDTA at pH 8.7 ) the PCI migrated to the β2- region, whereas the modified inhibitor had a slightly more anodal mobility. The APC-PCI komplex migrated to the α2- region.Two immunoradiometric assays were constructed with the monoclonal antibodies. One measured the complexes between APC and PCI while the other one measured the total amount of PCI present. These assays were used to study complex formation in buffer and plasma.
Reports on the topic "Apc protein"
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Ray, Ranjan. The Regulation of the Angiogenic Factor FGF-Binding Protein (FGF-BP) by the APC/beta-Catenin Signaling Pathway in the Progression of Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, May 2002. http://dx.doi.org/10.21236/ada406733.
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Stylianou, Dora, and Anna T. Riegel. The Regulation of the Angiogenic Factor FGF Binding Protein (FGF-BP) by the APC/Beta-Catenin Signaling Pathway in the Progression of Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, May 2003. http://dx.doi.org/10.21236/ada423021.
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Stylianou, Dora C. The Regulation of the Angiogenic Factor FGF Binding Protein (FGF-BP) by the APC/Beta-Catenin Signaling Pathway in the Progression of Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, May 2004. http://dx.doi.org/10.21236/ada425854.
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Bercovier, Herve, Raul Barletta, and Shlomo Sela. Characterization and Immunogenicity of Mycobacterium paratuberculosis Secreted and Cellular Proteins. United States Department of Agriculture, January 1996. http://dx.doi.org/10.32747/1996.7573078.bard.
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Our long-term goal is to develop an efficient acellular vaccine against paratuberculosis based on protein antigen(s). A prerequisite to achieve this goal is to analyze and characterize Mycobacterium paratuberculosis (Mpt) secreted and cellular proteins eliciting a protective immune response. In the context of this general objective, we proposed to identify, clone, produce, and characterize: the Mpt 85B antigen and other Mpt immunoreactive secreted proteins, the Mpt L7/L12 ribosomal protein and other immunoreactive cellular proteins, Mpt protein determinants involved in invasion of epithelial cells, and Mpt protein antigens specifically expressed in macrophages. Paratuberculosis is still a very serious problem in Israel and in the USA. In the USA, a recent survey evaluated that 21.6% of the dairy herd were infected with Mpt resulting in 200-250 million dollars in annual losses. Very little is known on the virulence factors and on protective antigens of Mpt. At present, the only means of controlling this disease are culling or vaccination. The current vaccines do not allow a clear differentiation between infected and vaccinated animals. Our long-term goal is to develop an efficient acellular paratuberculosis vaccine based on Mpt protein antigen(s) compatible with diagnostic tests. To achieve this goal it is necessary to analyze and characterize secreted and cellular proteins candidate for such a vaccine. Representative Mpt libraries (shuttle plasmid and phage) were constructed and used to study Mpt genes and gene products described below and will be made available to other research groups. In addition, two approaches were performed which did not yield the expected results. Mav or Mpt DNA genes that confer upon Msg or E. coli the ability to invade and/or survive within HEp-2 cells were not identified. Likewise, we were unable to characterize the 34-39 kDa induced secreted proteins induced by stress factors due to technical difficulties inherent to the complexity of the media needed to support substantial M. pt growth. We identified, isolated, sequenced five Mpt proteins and expressed four of them as recombinant proteins that allowed the study of their immunological properties in sensitized mice. The AphC protein, found to be up regulated by low iron environment, and the SOD protein are both involved in protecting mycobacteria against damage and killing by reactive oxygen (Sod) and nitrogen (AhpC) intermediates, the main bactericidal mechanisms of phagocytic cells. SOD and L7/L12 ribosomal proteins are structural proteins constitutively expressed. 85B and CFP20 are both secreted proteins. SOD, L7/L12, 85B and CFP20 were shown to induce a Th1 response in immunized mice whereas AphC was shown by others to have a similar activity. These proteins did not interfere with the DTH reaction of naturally infected cows. Cellular immunity provides protection in mycobacterial infections, therefore molecules inducing cellular immunity and preferentially a Th1 pathway will be the best candidate for the development of an acellular vaccine. The proteins characterized in this grant that induce a cell-mediated immunity and seem compatible with diagnostic tests, are good candidates for the construction of a future acellular vaccine.
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Lekostaj, Jacqueline K. The Role of ABC Proteins in Drug Resistant Breast Cancer Cells. Fort Belvoir, VA: Defense Technical Information Center, April 2008. http://dx.doi.org/10.21236/ada485613.
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Pleeter, Perri, and Jacqueline K. Lekostaj. The Role of ABC Proteins in Drug Resistant Breast Cancer Cells. Fort Belvoir, VA: Defense Technical Information Center, March 2009. http://dx.doi.org/10.21236/ada504701.
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Lekostaj, Jacqueline K. The Role of ABC Proteins in Drug-Resistant Breast Cancer Cells. Fort Belvoir, VA: Defense Technical Information Center, April 2007. http://dx.doi.org/10.21236/ada470298.
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Reichel, Ina, Michael Zisman, and Massimo Placidi. Aperture studies for the AP2 anti-proton Line at Fermilab. Office of Scientific and Technical Information (OSTI), December 2003. http://dx.doi.org/10.2172/821765.
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O'Neill, Sharman, Abraham Halevy, and Amihud Borochov. Molecular Genetic Analysis of Pollination-Induced Senescence in Phalaenopsis Orchids. United States Department of Agriculture, 1991. http://dx.doi.org/10.32747/1991.7612837.bard.
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The project investigated the molecular genetic and biochemical basis of pollination-induced senescence of Phalaenopsis flowers. This experimental system offered unique advantages in that senescence is strictly regulated by pollination, providing the basis to experimentally initiate and synchronize senescence in populations of flowers. The postpollination syndrome in the Phalaenopsis orchid system was dissected by investigating the temporal and spatial regulation of ACC synthase gene expression. In the stigma, pollen-borne auxin induces the expression of the auxin-regulated ACC synthase (PS-ACS2) gene, resulting in ACC synthesis within 1 h following pollination. Newly formed ACC is oxidized by basal constitutive ACC oxidase to ethylene, which then induces the expression of the ethylene-regulated ACC synthase(PS-ACS1) and oxidase (ACO1) genes for further autocatalytic production of ethylene. It is speculated that during the 6-h period following pollination, emasculation leads to the production or release of a sensitivity factor that sensitizes the cells of the stigma to ethylene. ACC and ethylene molecules are translocated from the stigma to the labellum and perianth where ethylene induces the expression of PS-ACS1 and ACO1 resulting in an increased production of ACC and ethylene. Organ-localized ethylene is responsible for inrolling and senescence of the labellum and perianth. The regulation of ethylene sensitivity and signal transduction events in pollinated flowers was also investigated. The increase in ethylene sensitivity appeared in both the flower column and the perianth, and was detected as early as 4 h after pollination. The increase in ethylene sensitivity following pollination was not dependent on endogenous ethylene production. Application of linoleic and linoleic acids to Phalaenopsis and Dendrobium flowers enhanced their senescence and promoted ethylene production. Several major lipoxygenase pathway products including JA-ME, traumatic acid, trans-2-hexenal and cis-3-hexenol, also enhanced flower senescence. However, lipoxygenase appears to not be directly involved in the endogenous regulation of pollination-induced Phalaenopsis and Dendrobium flower senescence. The data suggest that short-chain saturated fatty acids may be the ethylene "sensitivity factors" produced following pollination, and that their mode of action involves a decrease in the order of specific regions i the membrane lipid bilayer, consequently altering ethylene action. Examination of potential signal transduction intermediates indicate a direct involvement of GTP-binding proteins, calcium ions and protein phosphorylation in the cellular signal transduction response to ethylene following pollination. Modulations of cytosolic calcium levels allowed us to modify the flowers responsiveness to ethylene.
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Dubcovsky, Jorge, Tzion Fahima, and Ann Blechl. Molecular characterization and deployment of the high-temperature adult plant stripe rust resistance gene Yr36 from wheat. United States Department of Agriculture, November 2013. http://dx.doi.org/10.32747/2013.7699860.bard.
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Stripe rust, caused by Puccinia striiformis f. sp. tritici is one of the most destructive fungal diseases of wheat. Virulent races that appeared within the last decade caused drastic cuts in yields. The incorporation of genetic resistance against this pathogen is the most cost-effective and environmentally friendly solution to this problem. However, race specific seedling resistance genes provide only a temporary solution because fungal populations rapidly evolve to overcome this type of resistance. In contrast, high temperature adult plant (HTAP) resistance genes provide a broad spectrum resistance that is partial and more durable. The cloning of the first wheat HTAP stripe rust resistance gene Yr36 (Science 2009, 323:1357), funded by our previous (2007-2010) BARD grant, provided us for the first time with an entry point for understanding the mechanism of broad spectrum resistance. Two paralogous copies of this gene are tightly linked at the Yr36 locus (WKS1 and WKS2). The main objectives of the current study were to characterize the Yr36 (WKS) resistance mechanism and to identify and characterize alternative WKSgenes in wheat and wild relatives. We report here that the protein coded by Yr36, designated WKS1, that has a novel architecture with a functional kinase and a lipid binding START domain, is localized to chloroplast. Our results suggest that the presence of the START domain may affect the kinase activity. We have found that the WKS1 was over-expressed on leaf necrosis in wheat transgenic plants. When the isolated WKS1.1 splice variant transcript was transformed into susceptible wheat it conferred resistance to stripe rust, but the truncated variant WKS1.2 did not confer resistance. WKS1.1 and WKS1.2 showed different lipid binding profiling. WKS1.1 enters the chloroplast membrane, while WKS1.2 is only attached outside of the chloroplast membrane. The ascorbate peroxidase (APX) activity of the recombinant protein of TmtAPXwas found to be reduced by WKS1.1 protein in vitro. The WKS1.1 mature protein in the chloroplast is able to phosphorylate TmtAPXprotein in vivo. WKS1.1 induced cell death by suppressing APX activity and reducing the ability of the cell to detoxify reactive oxygen. The decrease of APX activity reduces the ability of the plant to detoxify the reactive H2O2 and is the possible mechanism underlying the accelerated cell death observed in the transgenic plants overexpressing WKS1.1 and in the regions surrounding a stripe rust infection in the wheat plants carrying the natural WKS1.1 gene. WKS2 is a nonfunctional paralog of WKS1 in wild emmer wheat, probably due to a retrotransposon insertion close to the alternative splicing site. In some other wild relatives of wheat, such as Aegilops comosa, there is only one copy of this gene, highly similar to WKS2, which is lucking the retrotransposon insertion. WKS2 gene present in wheat and WKS2-Ae from A. showed a different pattern of alternative splice variants, regardless of the presence of the retrotransposon insertion. Susceptible Bobwhite transformed with WKS2-Ae (without retrotansposon insertion in intron10), which derived from Aegilops comosaconferred resistance to stripe rust in wheat. The expression of WKS2-Ae in transgenic plants is up-regulated by temperature and pathogen infection. Combination of WKS1 and WKS2-Ae shows improved stripe rust resistance in WKS1×WKS2-Ae F1 hybrid plants. The obtained results show that WKS1 protein is accelerating programmed cell death observed in the regions surrounding a stripe rust infection in the wheat plants carrying the natural or transgenic WKS1 gene. Furthermore, characterization of the epistatic interactions of Yr36 and Yr18 demonstrated that these two genes have additive effects and can therefore be combined to increase partial resistance to this devastating pathogen of wheat. These achievements may have a broad impact on wheat breeding efforts attempting to protect wheat yields against one of the most devastating wheat pathogen.