Academic literature on the topic 'Therapeutic proteins; Recombinant; Protein C'
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Journal articles on the topic "Therapeutic proteins; Recombinant; Protein C"
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Quinn, Louise M., Clive Drakeford, James S. O’Donnell, and Roger J. S. Preston. "Engineering activated protein C to maximize therapeutic efficacy." Biochemical Society Transactions 43, no. 4 (August 1, 2015): 691–95. http://dx.doi.org/10.1042/bst20140312.
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The anticoagulant-activated protein C (APC) acts not solely as a crucial regulator of thrombus formation following vascular injury, but also as a potent signalling enzyme with important functions in the control of both acute and chronic inflammatory disease. These properties have been exploited to therapeutic effect in diverse animal models of inflammatory disease, wherein recombinant APC administration has proven to effectively limit disease progression. Subsequent clinical trials led to the use of recombinant APC (Xigris) for the treatment of severe sepsis. Although originally deemed successful, Xigris was ultimately withdrawn due to lack of efficacy and an unacceptable bleeding risk. Despite this apparent failure, the problems that beset Xigris usage may be tractable using protein engineering approaches. In this review, we detail the protein engineering approaches that have been utilized to improve the therapeutic characteristics of recombinant APC, from early studies in which the distinct anti-coagulant and signalling activities of APC were separated to reduce bleeding risk, to current attempts to enhance APC cytoprotective signalling output for increased therapeutic efficacy at lower APC dosage. These novel engineered variants represent the next stage in the development of safer, more efficacious APC therapy in disease settings in which APC plays a protective role.
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Kerschen, Edward J., Brian C. Cooley, Francis J. Castellino, John H. Griffin, and Hartmut Weiler. "Protective Effect of Activated Protein C in Murine Endotoxemia: Mechanism of Action." Blood 106, no. 11 (November 16, 2005): 26. http://dx.doi.org/10.1182/blood.v106.11.26.26.
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Abstract Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory disease associated with multi-organ failure. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects. The contribution of these distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. The aim of the study is to delineate the mechanism underlying the protective effect of APC in mouse endotoxemia. We first establish an experimental mouse model to demonstrate that recombinant murine APC reduces 6 day mortality of mice subjected to LPS-induced endotoxemia. APC treatment did not alter the extent of inflammatory cytokine release. Recombinant human APC did not exhibit therapeutic efficacy in this model. In contrast, recombinant human and mouse APC reduced to a similar extent experimentally induced arterial thrombus formation. The therapeutic efficacy of wild type recombinant murine APC was abolished in genetically engineered mice with reduced expression of the endothelial protein C receptor (EPCR). Recombinant mutant murine APC with greatly reduced anticoagulant potency was as effective as wild type murine APC in reducing mortality of mice subjected to LPS-induced septicemia. Mice homozygous for the Leiden polymorphism in the factor V (fV) gene, which renders coagulation factor V partially resistant to the anticoagulant effect of APC secondary to blocked fV proteolysis at R504 (R506 in humans), were refractory to the therapeutic benefit conveyed by administration of recombinant wild type mouse APC. In summary, these findings provide evidence that the therapeutic efficacy of recombinant APC is predominantly based on the ability of APC to interact with the endothelial protein C receptor, and that the anticoagulant activity of APC is not sufficient for achieving protection against mortality in a mouse model of endotoxemia. On the other hand, cleavage of fV at R506 appears necessary for retaining therapeutic efficacy in carriers of the fV Leiden allele.
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Fei, Dongliang, Yaxi Guo, Qiong Fan, Ming Li, Li Sun, Mingxiao Ma, and Yijing Li. "Codon optimization, expression in Escherichia coli, and immunogenicity analysis of deformed wing virus (DWV) structural protein." PeerJ 8 (March 11, 2020): e8750. http://dx.doi.org/10.7717/peerj.8750.
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Background Deformed wing virus (DWV) is a serious threat to honey bees (Apis mellifera) and is considered a major cause of elevated losses of honey bee colonies. However, lack of information on the immunogenicity of DWV structural proteins has hindered the development of effective biocontrol drugs. Methods We optimized the VP1, VP2 and VP3 codons of DWV surface capsid protein genes on the basis of an Escherichia coli codon bias, and the optimized genes of roVP1, roVP2 and roVP3 were separately expressed in E. coli and purified. Next, the three recombinant proteins of roVP1, roVP2 and roVP3 were intramuscularly injected into BALB/c and the immunogenicity was evaluated by the levels of specific IgG and cytokines. Furthermore, anti-roVP-antisera (roVP1 or roVP2 or roVP3) from the immunized mice was incubated with DWV for injecting healthy white-eyed pupae for the viral challenge test, respectively. Results The optimized genes roVP1, roVP2 and roVP3 achieved the expression in E. coli using SDS-PAGE and Western blotting. Post-immunization, roVP2 and roVP3 exhibited higher immunogenicity than roVP1 and stimulated a stronger humoral immune response in the mice, which showed that the recombinant proteins of roVP3 and roVP2 induced a specific immune response in the mice. In the challenge test, data regarding quantitative real-time RT-PCR (qRT-PCR) from challenged pupae showed that the level of virus copies in the recombinant protein groups was significantly lower than that of the virus-only group at 96 h post-inoculation (P < 0.05). Among them, the degree of neutralization using antibodies raised to the recombinant proteins are between approximately 2-fold and 4-fold and the virus copies of the roVP3 group are the lowest in the three recombinant protein groups, which indicated that specific antibodies against recombinant proteins roVP1, roVP2 and roVP3 of DWV could neutralize DWV to reduce the virus titer in the pupae. Collectively, these results demonstrated that the surface capsid protein of DWV acted as candidates for the development of therapeutic antibodies against the virus.
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Monakhova, E. V., G. V. Demidova, R. V. Pisanov, O. V. Duvanova, and B. N. Mishan’kin. "Recombinant Escherichia coli Strain with Enhanced Production of Vibrio cholerae Neuraminidase." Problems of Particularly Dangerous Infections, no. 2 (July 3, 2019): 87–92. http://dx.doi.org/10.21055/0370-1069-2019-2-87-92.
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Objectiveof this work was cloning of the Vibrio cholerae nanH gene as part of a plasmid vector, providing expression of foreign genes under the control of T5 promoter, and construction of a E. coli strain – producer of V. cholerae recombinant neuraminidase.Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplifi ed, the cloning was carried out by means of conventional methods, performance of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Neuraminidase activity was identifi ed by fl uorescence in ultraviolet light after incubation with specifi c substrate (4-methylumbelliferyl-N-acetylneuraminic acid).Results and discussion. Recombinant plasmid pNanH, containing the cloned gene nanH V. cholerae, has been constructed. The gene is inserted into BamHI-PstI sites of the polylinker of pQE30. Expression of the cloned gene in the producer strain E. coli JM103pNanH occurs under the control of T5 promoter after isopropyl-1-thio-β-D-galactopyranoside (IPTG) induction. The strain shows neuraminidase activity. The recombinant NanH protein is accumulated in the producer’s cells in two forms. The fi rst form, with molecular mass (MM) of 89.5 kDa, is an unprocessed protein with the hexahistidine block (6His-tag) at its N-terminus, it is located in inclusion bodies. Its percentage is 5.6–6.6 % of the total cell proteins. The second one, with MM of 83 kDa, is found in the periplasmic space and corresponds to the mature NanH, its percentage being 3.4–3.8 %. The total percentage of both forms is 9–10 % of total cell proteins, which allows to consider the strain E. coli JM103pNanH to be a super-producer of the required protein. The strain may be used for purifi cation of NanH preparations for construction of specifi c diagnostic, therapeutic and pharmaceutical preparations as well as for investigation of the protein as a virulence/persistence factor of the pathogen.
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Watson, Alastair, Maximillian J. S. Phipps, Howard W. Clark, Chris-Kriton Skylaris, and Jens Madsen. "Surfactant Proteins A and D: Trimerized Innate Immunity Proteins with an Affinity for Viral Fusion Proteins." Journal of Innate Immunity 11, no. 1 (October 5, 2018): 13–28. http://dx.doi.org/10.1159/000492974.
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Innate recognition of viruses is an essential part of the immune response to viral pathogens. This is integral to the maintenance of healthy lungs, which are free from infection and efficient at gaseous exchange. An important component of innate immunity for identifying viruses is the family of C-type collagen-containing lectins, also known as collectins. These secreted, soluble proteins are pattern recognition receptors (PRRs) which recognise pathogen-associated molecular patterns (PAMPs), including viral glycoproteins. These innate immune proteins are composed of trimerized units which oligomerise into higher-order structures and facilitate the clearance of viral pathogens through multiple mechanisms. Similarly, many viral surface proteins form trimeric configurations, despite not showing primary protein sequence similarities across the virus classes and families to which they belong. In this review, we discuss the role of the lung collectins, i.e., surfactant proteins A and D (SP-A and SP-D) in viral recognition. We focus particularly on the structural similarity and complementarity of these trimeric collectins with the trimeric viral fusion proteins with which, we hypothesise, they have elegantly co-evolved. Recombinant versions of these innate immune proteins may have therapeutic potential in a range of infectious and inflammatory lung diseases including anti-viral therapeutics.
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Collier, Aaron M., Yuliya Nemtsova, Narendra Kuber, Whitney Banach-Petrosky, Anurag Modak, David E. Sleat, Vikas Nanda, and Peter Lobel. "Lysosomal protein thermal stability does not correlate with cellular half-life: global observations and a case study of tripeptidyl-peptidase 1." Biochemical Journal 477, no. 3 (February 14, 2020): 727–45. http://dx.doi.org/10.1042/bcj20190874.
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Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.
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Park, Jinseu, Jiyoon Ryu, Kyeong-Ae Kim, Hak Joo Lee, Jae Hoon Bahn, Kyuhyung Han, Eui Yul Choi, Kil Soo Lee, Hyeok Yil Kwon, and Soo Young Choi. "Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells." Journal of General Virology 83, no. 5 (May 1, 2002): 1173–81. http://dx.doi.org/10.1099/0022-1317-83-5-1173.
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The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains a high proportion of arginine and lysine residues, is responsible for highly efficient protein transduction through the plasma membrane. To identify the role of the PTD sequence motif in transduction, various deletions and substitutions were introduced into the PTD. Tat–green fluorescent protein (GFP) fusion proteins, containing various lengths of the Tat PTD, were expressed and the extent of their transduction into mammalian cells was analysed by Western blot analysis and fluorescence microscopy. Deletion analysis of PTD mapped to a nine amino acid motif (residues 49–57: RKKRRQRRR) sufficient for transduction. Further deletion of this Tat basic domain either at the N terminus or at the C terminus significantly decreased transduction efficiency. The transduction efficiencies of GFPs fused to nine consecutive lysine (9Lys–GFP) or arginine (9Arg–GFP) residues were similar to that of Tat(49–57)–GFP. The transduced proteins localized to both the nucleus and the cytosol, as assessed by confocal microscopy and Western blot analysis of subcellular fractions from transduced cells. Thus, the availability of recombinant GFP fusion proteins facilitates the simple and specific identification of protein transduction mediated by these peptide sequences. The modified PTD sequences designed in this study may provide useful tools necessary for delivering therapeutic proteins/peptides into cells.
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Cherian, Reeja Maria, Chunsheng Jin, Jining Liu, Niclas G. Karlsson, and Jan Holgersson. "Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors." Infection and Immunity 84, no. 10 (July 25, 2016): 2842–52. http://dx.doi.org/10.1128/iai.00341-16.
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The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibitClostridium difficiletoxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterizeO-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminatedO-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models ofC. difficileinfection will reveal its TcdA-inhibitory effect and therapeutic potential inC. difficile-associated diseases.
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Ningrum, Ratih Asmana, Widdya Kusuma Wardhani, Ike Wahyuni, and Apon Zaenal Mustopa. "Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms." ANNALES BOGORIENSES 22, no. 2 (December 31, 2018): 57. http://dx.doi.org/10.14203/ann.bogor.2018.v22.n2.57-64.
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Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C.
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Mosnier, Laurent O., Andrew J. Gale, Subramanian Yegneswaran, and John H. Griffin. "Activated protein C variants with normal cytoprotective but reduced anticoagulant activity." Blood 104, no. 6 (September 15, 2004): 1740–44. http://dx.doi.org/10.1182/blood-2004-01-0110.
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Abstract Recombinant activated protein C (APC), a well-defined anticoagulant enzyme, reduced mortality in severe sepsis patients in a phase 3 trial. However, 2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, failed to do so, implying the physiologic relevance of APC's less well-defined anti-inflammatory and antiapoptotic activities. Recombinant APC therapy conveys an increased risk of serious bleeding complications due to APC anticoagulant activity. To generate recombinant APC variants with reduced risk of bleeding due to reduced anticoagulant activity, we dissected APC's anticoagulant activity from its cytoprotective activity by site-directed mutagenesis. Using staurosporine-induced endothelial cell apoptosis assays, we show here that Ala mutations (RR229/230AA and KKK191_ 193AAA) in 2 APC surface loops that severely reduce anticoagulant activity result in 2 APC variants that retain normal antiapoptotic activity that requires protease activated receptor-1 and endothelial cell protein C receptor. Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of such APC variants may reduce serious bleeding risks while providing the beneficial effects of APC acting directly on cells. (Blood. 2004;104: 1740-1744)
Dissertations / Theses on the topic "Therapeutic proteins; Recombinant; Protein C"
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O'Hara, John F. "An investigation of post-translational processing in the transgenic mammary gland." Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365215.
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Thirumangalathu, Renuka. "Understanding physical and chemical stability of proteins in solution : relevance to therapeutic protein and monoclonal antibody formulations /." Connect to abstract via ProQuest. Full text is not available online, 2007.
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Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 133-143). Online version available via ProQuest Digital Dissertations.
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Precht, Thomas A. "Regulation of neuronal apoptosis by the mitochondria /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008.
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Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 112-125). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Kinkade, Rebecca. "Rb-Raf-1 interaction as a therapeutic target for proliferative disorders." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002426.
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Simms, Amy Nicole. "Examination of Neisseria gonorrhoeae opacity protein expression during experimental murine genital tract infection /." Download the dissertation in PDF, 2005. http://www.lrc.usuhs.mil/dissertations/pdf/Simms2005.pdf.
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Jimenez, Maria Carolina Sarti. "Estudos biofísicos, estruturais e imunológicos de proteínas recombinantes correspondentes a antígenos de superfície de merozoítas de Plasmodium vivax." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-15052017-171133/.
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Diversas proteínas de superfície de merozoítas (MSPs) de Plasmodium têm sido consideradas candidatas a compor uma vacina contra a malária. Nos últimos anos estudamos diversos aspectos da resposta imune naturalmente adquirida contra proteínas recombinantes baseadas nas MSPs de P. vivax. Estes estudos demonstraram que estas proteínas recombinantes mantêm suas funções imunológicas, podendo servir como base para a caracterização de suas estruturas tridimensionais. Com o objetivo de obter informações estruturais sobre as MSPs de P. vivax, 10 proteínas recombinantes, correspondentes à região C-terminal da MSP-1 (MSP119), e diferentes regiões da MSP-3α e MSP-3β foram expressas em Escherichia coli. Os dados estruturais da MSP119 foram obtidos por modelagem molecular com base nas coordenadas cristalográficas da MSP19 de P. cynomolgi. Por outro lado, existem poucas informações estruturais sobre as proteínas da família MSP-3 de Plasmodium. A análise da estrutura primária dessas proteínas indica que elas apresentam um domínio central rico em alaninas que estão organizadas como motivos \"heptads\". Esse tipo de estrutura primária favorece a formação de estruturas do tipo α-hélices e \"coiled-coil\" (CC). No presente estudo, a composição da estrutura secundária de cada proteína recombinante foi caracterizada preliminarmente por ensaio de Dicroísmo Circular, realizado na região do UV distante. Com base nos resultados obtidos, selecionamos duas proteínas recombinantes baseadas na região C-terminal da MSP-3α (CC4 e CC5) para análises biofísicas mais detalhadas. Inicialmente, demonstramos por espectrometria de massa que ambas as proteínas têm massa molecular esperada. Entretanto, os dados obtidos por cromatografia em gel filtração sugeriram que essas proteínas formam oligômeros. No caso específico da proteína CC5, estes dados foram confirmados por ultracentrifugação analítica que indicou a formação de tetrâmeros elongados, corroborando com a formação de estrutura do tipo \"coiled-coil\". Como o papel biológico dessas proteínas não é conhecido, os dados estruturais obtidos neste estudo podem servir como base para o entendimento da função dessas proteínas. Na segunda parte deste projeto, selecionamos cinco proteínas recombinantes para estudos comparativos de reconhecimento por anticorpos IgG de indivíduos procedentes de áreas endêmicas de malária vivax. Tais estudos confirmaram dados prévios que as MSPs são imunogênicas em infecções naturais. Em conjunto, nossos resultados sugerem que, assim como a MSP119, proteínas recombinantes baseadas na MSP-3a e MSP-313 podem ser exploradas em futuros estudos de indução de imunidade protetora contra a malária vivax em primatas não humanos.Several merozoite surface proteins (MSPs) of Plasmodium have been considered candidates to compose a vaccine against malaria. In the last years, we have studied severaI aspects of the natural/y acquired immune response against recombinant proteins based on MSPs of P. vivax. These studies demonstrated that the recombinant proteins maintain their immunological functions and could be used for the characterization of their three-dimensional structure. To gain structural information on the MSPs of P. vivax, 10 recombinant proteins corresponding to the C-terminal region of MSP-1 (MSP119) and to different regions of the MSP-3α and MSP-3β were expressed in Escherichia coli. The structural data of the MSP119 were obtained by molecular modeling based on the crystallographic coordinates of the P. cynomolgi MSP119. On the other hand, there is limited structural information available for MSP-3 family of Plasmodium. The analysis of the primary structure of these proteins indicates that they present a central alanine-rich domain organized as heptads repeats. This type of primary structure favors the formation of α-helices and coiled-coil (CC) structures. In the present study, the composition of the secondary structure of each recombinant protein was characterized preliminarily by circular dichroism monitored in the far-UV region. On the basis of the obtained results, we selected two recombinant proteins based on C-terminal region of the MSP-3α (CC4 and CC5) for detailed biophysical analyses. Initially, we demonstrated that the monomer mass assigned for the two recombinant proteins corresponded exactly to those predicted from the primary sequence. However, during size exclusion chromatography, the proteins eluted at volumes corresponding to molecular weights that were much larger than their monomeric masses, suggesting that both proteins are oligomeric molecules. Interestingly, analytical ultracentrifugation experiments showed that the CC5 oligomers are elongated molecules. As the function of these proteins is not known, the structural data obtained in this study can be used to understand the function of these proteins. In the second part of this study, we selected five recombinant proteins for comparative recognition by IgG antibodies of the individuais from endemic areas of malaria vivax. These studies confirmed previous data that the MSPs are imunogenic in natural infections. Together, our results suggest that, as well as the MSP119, that recombinant proteins based on the MSP-3α and MSP-3β can be explored in future studies for the induction of protective immunity against malaria vivax.
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Sung, Meng-Chen, and 宋孟真. "The Biological Effects of Human Recombinant Thrombomodulin Proteins Independent of Protein C Activation Pathway." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/30446275161382588406.
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碩士國立成功大學
生物化學研究所
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Thrombomodulin (TM) is a vascular endothelial cell receptor and cofactor in the clinically important protein C anticoagulant system. TM contains five structure domains: N-termianal lectin-like domain (TMD1), EGF-like domain (TMD2), serine and threonine-rich domain (TMD3), transmembrane domain (TMD4), and C-terminal cytoplasmic domain (TMD5). In recent studies, TM domains were shown to have several biological functions beyond anticoagulation including mitogenic effect on various cells, angiogenic activity and the possible participation in the embryogenesis. In our previous studies, the novel angiogenic effects of TM domains 2 and 3 (TMD23) were discovered both in vitro and in vivo. However, the detailed mechanism of TMD23 modulating angiogenesis still remained to be solved. In this study, the Pichica pastoris protein expression system was used to express the recombinant TMD23 and three protein C activation-defected TMD23 mutant proteins using site-direct mutagenesis. The recombinant TMD23 proteins were purified by affinity nickel-chelating column chromatography. TM cofactor activity assay showed that these site-direct mutated proteins lost their protein C activation activity. We further demonstrated that the biological function of the three mutated proteins was similar to that of the wild type TMD23. These three mutants also stimulated proliferation, migration and tube formation of human umbilical vein endothelial cell (HUVEC) in vitro and induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. We showed that the angiogenic activity of TM is independent of protein C activation pathway. In addition, the function of many candidate mediators was investigated. By modifying far Western blotting assay and immunoprecipitation - Western blotting analysis, we discovered that the recombinant TMD23 protein may interact with fibroblast growth factor receptor 1 (FGFR1; Flg) in HUVECs. These results suggested that TMD23 might act through tyrosyl kinase-like receptors such as FGFR1 to modulate angiogenesis.
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Manoharan, Simna. "Engineering the N-Glycosylation Pathway in Pichia Pastoris for the Expression of Glycoprotein Hormones." Thesis, 2016. http://hdl.handle.net/2005/3017.
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Proteins, participating in a myriad of biological function, are at the core of all cellular activities occurring within living organisms. Therapeutic proteins, hence constitute a major part of the pharmaceutical industry. The glycoprotein hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and human chorionic gonadotropin (CG) regulate various reproductive and metabolic functions in humans and hence have high therapeutic potentials. The increasing demand of recombinant proteins for therapeutic uses drives the development of better expression systems.
The methylotrophic yeast Pichia pastoris, has been termed as an industrial workhorse for heterologous protein expression. However, the N-glycosylation in yeast is of the high mannose type, resulting in a reduced serum half-life of the recombinant proteins. In the current work, we have re-engineered the Pichia N-glycosylation pathway to mimic the human type of N-glycosylation. Towards this end, we abolished the yeast native N-glycosylation and introduced enzymes from various eukaryotic sources into the system. These modifications resulted in the conversion of the yeast Man9-20GlcNAc2 glycan structure to a more human like GlcNAc2Man3GlcNAc2 form on over 70 % of the heterologous expressed proteins.
In order to demonstrate the application of these strains as efficient protein expression hosts, the glycoengineerd Pichia was used for large scale expression of the glycoprotein hormones, hCG and FSH. The purified recombinant hormones were found to have binding affinities and structure similar to that of the natural hormones. These recombinant hormones were also able to elicit over two fold responses in animal models compared to buffer controls and the activity was comparable to the natural counterparts. Thus, we report the generation of a glycoengineered Pichia pastoris, which can be considered as a serious contender for the expression of glycosylated proteins of therapeutic importance.
Books on the topic "Therapeutic proteins; Recombinant; Protein C"
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Niazi, Sarfaraz. Handbook of biogeneric therapeutic proteins: Regulatory, manufacturing, testing, and patent issues. Boca Raton: Taylor & Francis, 2006.
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International Workshop on NovoSeven (1998 Frankfurt am Main, Germany). Recombinant factor VIIa: RFVIIa : update on clinical experiences. Neckargemünd: Weller Verlag, 1999.
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1961-, Castro Fidel O., and Jänne Juhani, eds. Mammary gland transgenesis: Therapeutic protein production. Berlin: Springer, 1998.
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Schmidt, Stefan R. Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges. Wiley & Sons, Incorporated, John, 2013.
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Schmidt, Stefan R. Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges. Wiley & Sons, Incorporated, John, 2013.
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Schmidt, Stefan R. Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges. Wiley & Sons, Incorporated, John, 2013.
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Schmidt, Stefan R. Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges. Wiley & Sons, Incorporated, John, 2013.
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1948-, Dierauf Leslie A., ed. CRC handbook of marine mammal medicine: Health, disease, and rehabilitation. Boca Raton, Fla: CRC Press, 1990.
Find full textBook chapters on the topic "Therapeutic proteins; Recombinant; Protein C"
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"Recombinant Protein Subunit Vaccines and Delivery Methods." In Therapeutic Peptides and Proteins, 358–93. CRC Press, 2015. http://dx.doi.org/10.1201/b18392-13.
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"Recombinant Protein Subunit Vaccines and Delivery Methods." In Therapeutic Peptides and Proteins, 347–70. CRC Press, 2005. http://dx.doi.org/10.1201/9781420039832-15.
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"Recombinant Protein Subunit Vaccines and Delivery Methods." In Therapeutic Peptides and Proteins, 327–50. CRC Press, 2005. http://dx.doi.org/10.1201/9781420039832.ch8.
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Becker, Richard C., and Frederick A. Spencer. "Anticoagulants." In Fibrinolytic and Antithrombotic Therapy. Oxford University Press, 2006. http://dx.doi.org/10.1093/oso/9780195155648.003.0036.
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Because of the narrow therapeutic index of warfarin and unfractionated heparin (UFH), monitoring their anticoagulant effects is required. On the other hand, lowmolecular- weight heparin (LMWH) and fibrinolytic agents need to be monitored only under certain circumstances. Although newer anticoagulants will not require routine monitoring for dose titration, a means to determine their systemic effects and individual (patient-specific) response to administration will likely have roles in clinical practice. The prothrombin time is used to monitor vitamin K antagonist therapy. This test is sensitive to the plasma concentrations (activity) of clotting factors II (prothrombin), V, VII, and X. Vitamin K antagonists affect the vitamin K–dependent factors II, VII, IX, and X, as well as proteins C, S, and Z. Thus, the prothrombin time does not reflect the effect of vitamin K antagonists on some factors (IX and proteins C, S, and Z) and is sensitive to others (factor V) (not directly influenced by treatment). The prothrombin time is not an ideal test for monitoring vitamin K antagonists; however, its simplicity and widespread availability have established its place in clinical practice. By convention, prothrombin times are now reported as international normalized ratios (INRs). This is the ratio of the patient’s prothrombin time to a control prothrombin time, raised to a power—the international sensitivity index (ISI). The latter reflects the calibration of the thromboplastin used for the prothrombin time testing to an internationally agreed upon standard. In many laboratories the reagent currently used is a recombinant thromboplastin, which has an ISI of 1.0 There are several cautions related to interpreting the results of prothrombin time tests that are worth monitoring. Since the test is sensitive to the level of factor V in the plasma, improper sample storage or delayed testing may cause loss of factor V (activity) and yield prothrombin time values above the expected range. High concentrations of heparin may also prolong the prothrombin time; this usually occurs when the sample is obtained within a few minutes of administering a bolus dose. Direct thrombin inhibitors, such as hirudin, bivalirudin, argatroban, and ximelagatran, may also prolong the prothrombin time to a variable degree.
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Hasan, Mohammad Raghibul, Bader Saud Alotaibi, Sultan F. Alnomasy, and Khalid Umar Fakhri. "Cancer Immunotherapy." In Handbook of Research on Advancements in Cancer Therapeutics, 1–41. IGI Global, 2021. http://dx.doi.org/10.4018/978-1-7998-6530-8.ch001.
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Cancer immunotherapy has become a powerful clinical strategy as well as an established pillar for the treatment of cancers to improving the prognosis of many cancer patients with a broad variety of solid tumors as well as blood cancers. The primary goals of immunotherapy are (a) to increase anti-tumor response, (b) decrease the immune suppression, and (c) to enhance the immunogenicity of tumors. This chapter aims to discuss the mechanism and different types of immunotherapies used for different cancers. It will also focus on recombinant products including immunostimulants, immunotoxins, antibodies, fusion proteins, engineered cytotoxic T cells, engineered immunocytokines, vaccines, checkpoint inhibitors, CAR T-cell therapy, and nanomedicine. Although immunotherapy has a rare side effect, it is not fully understood. The development of new strategies has been on the clinical trial to enhance the benefit of cancer patients to meet with challenges of limited efficacy and/or toxicity.
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Stabel, Silvia, Marek Liyanage, and David Frith. "Expression of Protein Kinase C Isozymes in Insect Cells and Isolation of Recombinant Proteins." In Methods in Neurosciences, 154–73. Elsevier, 1993. http://dx.doi.org/10.1016/b978-0-12-185285-6.50022-1.
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Dascher, Christiane, Ellen J. Tisdale, and William E. Balch. "[20] Transient expression of small GTPases to study protein transport along secretory pathway in Vivo using recombinant T7 vaccinia virus system." In Small GTPases and Their Regulators Part C: Proteins Involved in Transport, 165–73. Elsevier, 1995. http://dx.doi.org/10.1016/s0076-6879(95)57022-5.
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Baines, Dev. "Analysis of purity." In Protein Purification Techniques. Oxford University Press, 2001. http://dx.doi.org/10.1093/oso/9780199636747.003.0007.
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In approaches to protein purification, the concepts of yield and purity are routinely used, but are often difficult to define in absolute terms. To some extent the purity of a protein sample will be defined by the final designated use of the product. In most cases, analyses will involve measurement of the mass of the protein in the sample and quantitation of specific property of the target molecule (e.g. activity) to provide values for the yield. Thus, the calculation of specific activity of given fractions through the purification process provides a valuable indication of the level of the purity attained. Although, conventionally, specific activity is used in the purification of enzymes, due to the availability of sensitive and specific assays, recent developments in fast chromatographic separations and protein mass spectrometry have led to application of these techniques to address the purity of a wider variety of biomolecules. The level of purity for any protein product requires several factors to be taken into consideration. Besides the intended use, the source of the protein will dictate the extent of analyses required, since the level of impurities present in the final product will depend not only on the purification process used but also on the starting source material. For bulk enzyme preparations (for use e.g. in biotransformations or related applications) it may only be necessary to ensure the product is free of any contaminating activities which could effect the outcome of these types of applications. For proteins required for physical studies (protein crystallography, primary sequence analysis), the purity criteria are more stringent, particularly, since the lack of purity (including sample microheterogeneity) can drastically influence the outcome of such studies. Alternatively, proteins intended for therapeutic use will have purity considerations significantly different, constrained not only by regulatory requirements but also by clinical responses that may arise from the presence of any contaminants. The nature of these contaminants, as mentioned earlier, will depend on the starting source of the target molecule (i.e. animal tissue, human serum, recombinant micro-organisms [prokaryotes, eukaryotes], and hybridomas).
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Chen, Minyong, Steven J. Dupard, Colleen M. McClung, Cristian I. Ruse, Mehul B. Ganatra, Saulius Vainauskas, Christopher H. Taron, and James C. Samuelson. "Improving the Study of Protein Glycosylation with New Tools for Glycopeptide Enrichment." In Fundamentals of Glycosylation [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.97339.
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High confidence methods are needed for determining the glycosylation profiles of complex biological samples as well as recombinant therapeutic proteins. A common glycan analysis workflow involves liberation of N-glycans from glycoproteins with PNGase F or O-glycans by hydrazinolysis prior to their analysis. This method is limited in that it does not permit determination of glycan attachment sites. Alternative proteomics-based workflows are emerging that utilize site-specific proteolysis to generate peptide mixtures followed by selective enrichment strategies to isolate glycopeptides. Methods designed for the analysis of complex samples can yield a comprehensive snapshot of individual glycans species, the site of attachment of each individual glycan and the identity of the respective protein in many cases. This chapter will highlight advancements in enzymes that digest glycoproteins into distinct fragments and new strategies to enrich specific glycopeptides.
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Wong, Janica C., Priyatham Gorjala, Benjamin Costantino, and Ronald R. Fiscus. "Anti-Angiogenic and Anti-Cancer Effects by Targeting the Protein Kinase G Type-Iα (PKG-Iα) Signaling Pathway and its Downstream Effects on Expression of Inhibitor of Apoptosis Proteins, C-IAP1, Livin and Survivin." In Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives. InTech, 2016. http://dx.doi.org/10.5772/60774.
Full textConference papers on the topic "Therapeutic proteins; Recombinant; Protein C"
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Giannelli, B. F. "MOLECULAR GENETICS OF HAEMOPHILIA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643981.
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Haemophilia B, an X-linked recessive disease with an incidence of 1/30,000 newborn males, is due to defects in the gene for coagulation factor IX, which is on the long am of the X chromosome at band Xq27.1. This gene consists of approximately 34 Kb and contains 8 exons which specify a mRtfc of 2803 residues coding for a protein of 415 aa preceded by a prepro signal peptide of 46 aa. Coripanson of the functional domains of the factor IX protein with the exon structure of the gene supports the exon/protein domain hypothesis of gene evolution. The factor IX gene seems to be formed by a number of functionally and evolutionally independent modules. The signal peptide and the gla (γcarboxy-glutamic) region encoded in the first three exons are homologous to those of factor X, protein C and prothrombin. Thevfourth and fifth exons which code for the connecting peptide are homologous to one another and to the epidermal growth factor, a module that has been used in the construction of a great variety of proteins including different members of the coagulation and fibrinolytic pathways. The sixth exon encodes the activation peptide region, while the catalytic region of factor IX is coded by the seventh and eighth exon. This is at variance with other serine protease genes that have different exons for the segments containing the cardinal ami no-acids of the active centre (histidine, aspartic acid and serine).Natural selection acts against detrimental mutations of the factor IX gene and at each generation a proportion of haemophilia B genes is eliminated, as a significant number of patients does not reproduce. There appears to be no selective advantage to the heterozygote and therefore haemophilia B is maintained in the population by new mutations. Consequently, a significant proportion of patients should be born to non-carrier mothers, and unrelated patients should carry different gene defects, as recently verified by detailed analysis of individual haemophilia B genes.The defects of factor IX described so far comprise both point mutations and gene deletions. The latter affect either part or the whole of the gene and are often associated with the development of antibodies against therapeutically adninistered factor IX (the inhibitor complication). Since gene deletions may result in the complete absenceof factor IX synthesis or in the production of an extremely abnormal product, it has been suggested that mutationspreventing the synthesis of a factor IX gene product capable of inducing immune tolerance to normal factor IX is important in predisposing to the inhibitor complication.Among the point mutations described so far, those affecting the signal peptide are of particular interest. Substitutions of the arginine at positions -4 and -1 cause failure of propeptide cleavage. Thus they indicate that the propeptide consists of 18 aa an(lthat lts excision is necessary for factor IX function. It appears also that the propeptide contains a signal for γcarboxylation which has been conserved during the evolution of different γcarboxylated proteins.In spite of coagulant treatment, haemophilia B is a serious disease and one for which genetic counselling is required. Paramount for this is the detection of carriers and the diagnosis ofaffected male fetuses. DNA probes derived from the cloned factor IX gene have been used for this purpose. Carrier and first or second trimester prenatal diagnoses have been done using factors IX gene markers to follow the transmission of haemophilia B genes. Six sequence variations causing restriction fragment length polymorphisms (RFLP) in the factor IX gene have been detected and used as markers for such indirect diagnoses The efficiency of the above markers is reduced by linkage disequilibrium but, nevertheless, they offer definite carrier and nremtal diagnoses in 75-80% of the relatives of familial cases of haemophilia B.The indirect detection of gene defects is of modest help in the counselling of individuals from the families of isolated patients, but new methods for the direct detection of gene mutations promise better results in such families and also the attainment of % diagnostic success in relatives of familial cases.Finally the successful expression of recombinant factor IX genes in tissue culture and transgenic mammals raises hopes of therapeutic advances.
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Cuppoletti, John. "Composite Synthetic Membranes Containing Native and Engineered Transport Proteins." In ASME 2008 Conference on Smart Materials, Adaptive Structures and Intelligent Systems. ASMEDC, 2008. http://dx.doi.org/10.1115/smasis2008-449.
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Our membrane transport protein laboratory has worked with material scientists, computational chemists and electrical and mechanical engineers to design bioactuators and sensing devices. The group has demonstrated that it is possible to produce materials composed native and engineered biological transport proteins in a variety of synthetic porous and solid materials. Biological transport proteins found in nature include pumps, which use energy to produce gradients of solutes, ion channels, which dissipate ion gradients, and a variety of carriers which can either transport substances down gradients or couple the uphill movement of substances to the dissipation of gradients. More than one type of protein can be reconstituted into the membranes to allow coupling of processes such as forming concentration gradients with ion pumps and dissipating them with an ion channel. Similarly, ion pumps can provide ion gradients to allow the co-transport of another substance. These systems are relevant to bioactuation. An example of a bioactuator that has recently been developed in the laboratory was based on a sucrose-proton exchanger coupled to a proton pump driven by ATP. When coupled together, the net reaction across the synthetic membrane was ATP driven sucrose transport across a flexible membrane across a closed space. As sucrose was transported, net flow of water occurred, causing pressure and deformation of the membrane. Transporters are regulated in nature. These proteins are sensitive to voltage, pH, sensitivity to a large variety of ligands and they can be modified to gain or lose these responses. Examples of sensors include ligand gated ion channels reconstituted on solid and permeable supports. Such sensors have value as high throughput screening devices for drug screening. Other sensors that have been developed in the laboratory include sensors for membrane active bacterial products such as the anthrax pore protein. These materials can be self assembled or manufactured by simple techniques, allowing the components to be stored in a stable form for years before (self) assembly on demand. The components can be modified at the atomic level, and are composed of nanostructures. Ranges of sizes of structures using these components range from the microscopic to macroscopic scale. The transport proteins can be obtained from natural sources or can be produced by recombinant methods from the genomes of all kingdoms including archea, bacteria and eukaryotes. For example, the laboratory is currently studying an ion channel from a thermophile from deep sea vents which has a growth optimum of 90 degrees centigrade, and has membrane transport proteins with very high temperature stability. The transport proteins can also be genetically modified to produce new properties such as activation by different ligands or transport of new substances such as therapeutic agents. The structures of many of these proteins are known, allowing computational chemists to help understand and predict the transport processes and to guide the engineering of new properties for the transport proteins and the composite membranes. Supported by DARPA and USARMY MURI Award and AFOSR.
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Beardsley, D. S. "IMMUNE THROMBOCYTOPENIA (ITP) : PLATELET TARGET ANTIGENS OF THE ANTIBODIES IN DIFFERENT CLINICAL SETTINGS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644757.
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Autoantibodies against platelet antigens are important in the pathogenesis of ITP. We have studied the antigenic targets of these autoantibodies by immunoblotting using electrophoretically separated proteins from normal, Glanzmann's Thrombasthenic, and Bernard-Soulier Syndrome platelets immobilized on nitrocellulose paper. Incubation of these proteins with ITP patient serum, immunoglobulin, or F(ab*>2, followed by labeled antiglobulin, allowed identification of the antigenic target glycoproteins (GPfs). Patients with ITP of several categories were studied: A) Chronic ITP (> 1 yr duration) including a subset of patients who hadmild thrombocytopenia but significant hemorrhagic manifestations. B) Acute ITP (<6 mo) following varicella infectionAcute onset ITP unresponsive to any of the usual therapeutic modalities. A majority of patients in group A (32/48) had antibodies directed against a protein with a MW of lOOkD. In some cases, the target antigen was localized to GPIIIa specifically by absence of reactivity with Type I Glanzmann's thrombasthenic platelets known to be totally deficient in GPIIb and Ilia. Two individuals in the subset with unexpectedly severe hemorrhagic symptoms also had anti-GPIIIa antibodies, but the antigenic target was different since only these two antibodies could be shown to interfere with binding of 125j_fibrinogen to normal plateletsIn contrast to the individuals with anti-GPIIIa antibodies, all group B patients (7/7) with acute post-varicella ITP had antibodies directed against an 85kD thrombin sensitive protein, and two of the three group C individuals studied showed antibodies directed against GPIb. These studies indicate that a number of different platelet antigens can be the targets of platelet autoantibodies in ITP. Results suggest that antibodies against a particular antigen may correlate with a similar clinical setting in different patients. Further work will determine whether antigen identification at the time of diagnosis can be used to predict the clinical course for an individual patient
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Chen, Kok Hao, and Jong Hyun Choi. "DNA Oligonucleotide-Templated Nanocrystals: Synthesis and Novel Label-Free Protein Detection." In ASME 2009 International Mechanical Engineering Congress and Exposition. ASMEDC, 2009. http://dx.doi.org/10.1115/imece2009-11958.
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Semiconductor and magnetic nanoparticles hold unique optical and magnetic properties, and great promise for bio-imaging and therapeutic applications. As part of their stable synthesis, the nanocrystal surfaces are usually capped by long chain organic moieties such as trioctylphosphine oxide. This capping serves two purposes: it saturates dangling bonds at the exposed crystalline lattice, and it prevents irreversible aggregation by stabilizing the colloid through entropic repulsion. These nanocrystals can be rendered water-soluble by either ligand exchange or overcoating, which hampers their widespread use in biological imaging and biomedical therapeutics. Here, we report a novel scheme of synthesizing fluorescent PbS and magnetic Fe3O4 nanoparticles using DNA oligonucleotides. Our method of PbS synthesis includes addition of Na2S to the mixture solution of DNA sequence and Pb acetate (at a fixed molar ratio of DNA/S2−/Pb2+ of 1:2:4) in a standard TAE buffer at room temperature in the open air. In the case of Fe3O4 particle synthesis, ferric and ferrous chloride were mixed with DNA in DI water at a molar ratio of DNA/Fe2+/Fe3+ = 1:4:8 and the particles were formed via reductive precipitation, induced by increasing pH to ∼11 with addition of ammonium hydroxide. These nanocrystals are highly stable and water-soluble immediately after the synthesis, due to DNA termination. We examined the surface chemistry between oligonucleotides and nanocrystals using FTIR spectroscopy, and found that the different chemical moieties of nucleobases passivate the particle surface. Strong coordination of primary amine and carbonyl groups provides the chemical and colloidal stabilities, leading to high particle yields (Figure 1). The resulting PbS nanocrystals have a distribution of 3–6 nm in diameter, while a broader size distribution is observed with Fe3O4 nanoparticles as shown in Figure 1b and c, respectively. A similar observation was reported with the pH change-induced Fe3O4 particles of a bimodal size distribution where superparamagnetic and ferrimagnetic magnetites co-exist. In spite of the differences, FTIR measurements suggest that the chemical nature of the oligonucleotide stabilization in this case is identical to the PbS system. As a particular application, we demonstrate that aptamer-capped PbS QD can detect a target protein based on selective charge transfer, since the oligonucleotide-templated synthesis can also serve the additional purpose of providing selective binding to a molecular target. Here, we use thrombin and a thrombin-binding aptamer as a model system. These QD have diameters of 3∼6 nm and fluoresce around 1050 nm. We find that a DNA aptamer can passivate near IR fluorescent PbS nanocrystals, rendering them water-soluble and stable against aggregation, and retain the secondary conformation needed to selectively bind to its target, thrombin, as shown in Figure 2. Importantly, we find that when the aptamer-functionalized nanoparticles binds to its target (only the target), there is a highly systematic and selective quenching of the PL, even in high concentrations of interfering proteins as shown in Figure 3a and b. Thrombin is detected within one minute with a detection limit of ∼1 nM. This PL quenching is attributed to charge transfer from functional groups on the protein to the nanocrystals. A charge transfer can suppress optical transition mechanisms as we observe a significant decrease in QD absorption with target addition (Figure 3c). Here, we rule out other possibilities including Forster resonance energy transfer (FRET) and particle aggregation, because thrombin absorb only in the UV, and we did not observe any significant change in the diffusion coefficient of the particles with the target analyte, respectively. The charge transfer-induced photobleaching of QD and carbon nanotubes was observed with amine groups, Ru-based complexes, and azobenzene compounds. This selective detection of an unlabeled protein is distinct from previously reported schemes utilizing electrochemistry, absorption, and FRET. In this scheme, the target detection by a unique, direct PL transduction is observed even in the presence of high background concentrations of interfering negatively or positively charged proteins. This mechanism is the first to selectively modulate the QD PL directly, enabling new types of label free assays and detection schemes. This direct optical transduction is possible due to oligonucleotidetemplated surface passivation and molecular recognition. This chemistry may lead to more nanoparticle-based optical and magnetic probes that can be activated in a highly chemoselective manner.
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Newman, J., and D. Farb. "LARGE SCALE PREPARATION OF VON WILLEBRAND FACTOR BY AFFINITY CHROMATOGRAPHY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644093.
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Treatment of bleeding in von Willebrand's disease usually consists of the infusion of cryoprecipitate or plasma. DDAVP is effective in some patients. Commercial concentrates for the treatment of Hemophilia A are ineffective as a source of von Willebrand Factor (vWF) replacement in von Willibrand's disease presumedly because of the absence of higher molecular weight forms of vWF protein. A vWF concentrate obtained during the course of preparation of an affinity purified Factor VIII may provide an alternative therapeutic agent without impacting the available Factor VIII supplies. The process used for the preparation of a highly purified Factor VIII concentrate (MonoclateTM, Armour Pharmaceutical Co.) from cryoprecipitate includes an affinity chromatography step which separates vWF/Factor VIII complex from other proteins in cryoprecipitate using an anti-vWF monoclonal antibody (C. A. Fulcher & T. S. Zimmerman, 1982). Factor VIII is then dissociated from the vWF remaining on the column and is eluted immediately by 3M sodium thiocyanante (NaSCN) as a step in the regeneration of the column. Unless the NaSCN is rapidly removed from the elutriant, the vWF activity as measured by platelet agglutination is destroyed. We have taken the NaSCN eluate and processed it immediately over an in-line G-10 or G-25 Sephadex column which removes the NaSCN while the vWF is eluted with a buffered isotonic solution. Alternatively, the vWF has been precipitated from the NaSCN by ammonium sulfate or polyethylene glycol. VWF prepared by any of these methods retains platelet agglutinating activity and has a distribution of vWF multimers similar to those of vWF in normal plasma.The potency of vWF prepared from cryoprecipitate by this process is 20 units/ml and the specific activity is 20 units/mg. In several fractionations of kilogram amounts of cryoprecipitate, vWF was isolated and subjected to lyophilization and heating at 68° for 30 hours without loss of bioactivity. Drug development in progress suggest that the combined effect of affinity chromatography and exposure to NaSCN may negate the need for a heating procedure to reduce the risk of viral transmittance.