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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 Although originally deemed successful, Xigris was ultimately withdrawn due to lack of efficacy and an unacceptable bleeding risk.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)
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Taoka, Yuji, Mitsuhiro Uchiba, Masayoshi Johno, and Kenji Okajima. "Neuroprotection by Recombinant Thrombomodulin." Thrombosis and Haemostasis 83, no. 03 (2000): 462–68. http://dx.doi.org/10.1055/s-0037-1613837.
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SummaryWe examined whether recombinant human soluble thrombomodulin (rhs-TM) reduces compression trauma-induced spinal cord injury through protein C activation in rats. Administration of rhs-TM, either before or after the induction of spinal cord injury (SCI), markedly reduced the resulting motor disturbances. However, neither rhs-TM pretreated with an anti-rhs-TM monoclonal antibody (MAb) F2H5, which inhibits thrombin binding to rhs-TM, nor those pretreated with MAb R5G12, which selectively inhibits protein C activation by rhsTM, prevented the motor disturbances. Intramedullary hemorrhages, observed 24 h after trauma, were significantly reduced in animals given rhs-TM. The increase in the tissue levels of tumor necrosis factor-α (TNF-α), TNF-α mRNA expression, and the accumulation of leukocytes in the damaged segment of the spinal cord were significantly inhibited in animals receiving rhs-TM, but these effects were not observed following administration of rhs-TM pretreated with MAb R5G12 or MAb F2H5. Leukocytopenia and activated protein C all produced effects similar to those of rhs-TM.These findings suggest that rhs-TM prevents compression traumainduced SCI by inhibiting leukocyte accumulation by reducing the expression of TNF-α mRNA and such therapeutic effects of rhs-TM could be dependent on its protein C activation capacity. Findings further suggest that thrombomodulin can be implicated not only in the coagulation system but in regulation of the inflammatory response.
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Hobden, A. N., and T. J. R. Harris. "The impact of biotechnology and molecular biology on the pharmaceutical industry." Proceedings of the Royal Society of Edinburgh. Section B. Biological Sciences 99, no. 1-2 (1992): 37–45. http://dx.doi.org/10.1017/s0269727000013038.
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Synopsis:Biotechnology had its initial impact on the pharmaceutical industry well before the perceived time. The use of fermentation technology to produce antibiotics was a cornerstone for the development of the industry. This event was both before cloning (BC) and before DNA (rather than after DNA – AD). Even now the antibiotic market, which is worth over 10 billion U.S. dollars a year, is the most valuable segment of the total market, (c.200 billion dollars per year). Nevertheless the impact of biotechnology in drug discovery was until recently perceived solely to be the use of recombinant DNA techniques to produce therapeutic proteins and modified versions of them by protein engineering.There are several other places where genetic engineering is influencing drug discovery. The expression of recombinant proteins in surrogate systems (e.g. in E. coli, yeast or via baculovirus infection or in mammalian cells) provides materials for structure determination (e.g. HIV protease) and structure/function studies (e.g. various receptors). Recombinant DNA techniques are influencing assay technology by allowing access to proteins in sufficient quantity for high throughput screening.In addition, screening organisms can be constructed where a particular protein function can be measured in a microorganism by complementation or via reporter gene expression.Transgenic animals also illustrate the power of the technology for drug discovery. Not only will transgenic rats and mice be used as models of disease but also for efficacy and toxicological profiling. What is learned in transgenic rodents may well set the scene for somatic cell gene therapy in humans.
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Zadravec, Petra, Borut Štrukelj, and Aleš Berlec. "Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species." Applied and Environmental Microbiology 81, no. 6 (January 9, 2015): 2098–106. http://dx.doi.org/10.1128/aem.03694-14.
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ABSTRACTSafety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface ofLactococcus lactisby fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genusLactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control.Lactobacillus salivariusATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides ofLb. salivariusas a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display onL. lactiswas demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selectingLactobacillus salivariusATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display onL. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.
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Esland, Lola, Marco Larrea-Alvarez, and Saul Purton. "Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii." Biology 7, no. 4 (October 10, 2018): 46. http://dx.doi.org/10.3390/biology7040046.
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Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.
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Lotem, J., and L. Sachs. "In vivo control of differentiation of myeloid leukemic cells by recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3." Blood 71, no. 2 (February 1, 1988): 375–82. http://dx.doi.org/10.1182/blood.v71.2.375.375.
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Abstract The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (colony-stimulating factors [CSF] and interleukin 3 [IL-3], macrophage and granulocyte inducing proteins, type 7 [MGI-1]) and another class (called MGI-2) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemic cells can differ in their response to these regulatory proteins. One type of leukemic clone can be differentiated in vitro to mature cells by incubating with the growth-inducing proteins granulocyte-macrophage (GM) CSF or IL-3, and another type of clone can be differentiated in vitro to mature cells by the differentiation-inducing protein MGI-2. We have now studied the ability of different myeloid regulatory proteins to induce the in vivo differentiation of these different types of mouse myeloid leukemic clones in normal and cyclophosphamide-treated mice. The results show that in both types of mice (a) the in vitro GM-CSF- and IL- 3-sensitive leukemic cells were induced to differentiate to mature cells in vivo in mice injected with pure recombinant GM-CSF and IL-3 but not with G-CSF, M-CSF, or MGI-2; (b) the in vitro MGI-2-sensitive leukemic cells differentiated in vivo by injection of MGI-2 and also, presumably indirectly, by GM-CSF and IL-3 but not by M-CSF or G-CSF; (c) in vivo induced differentiation of the leukemic cells was associated with a 20- to 60-fold decrease in the number of blast cells; and (d) all the injected myeloid regulatory proteins stimulated the normal myelopoietic system. Different normal myeloid regulatory proteins can thus induce in vivo terminal differentiation of leukemic cells, and it is suggested that these proteins can have a therapeutic potential for myeloid leukemia in addition to their therapeutic potential in stimulating normal hematopoiesis.
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Lotem, J., and L. Sachs. "In vivo control of differentiation of myeloid leukemic cells by recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3." Blood 71, no. 2 (February 1, 1988): 375–82. http://dx.doi.org/10.1182/blood.v71.2.375.bloodjournal712375.
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The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (colony-stimulating factors [CSF] and interleukin 3 [IL-3], macrophage and granulocyte inducing proteins, type 7 [MGI-1]) and another class (called MGI-2) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemic cells can differ in their response to these regulatory proteins. One type of leukemic clone can be differentiated in vitro to mature cells by incubating with the growth-inducing proteins granulocyte-macrophage (GM) CSF or IL-3, and another type of clone can be differentiated in vitro to mature cells by the differentiation-inducing protein MGI-2. We have now studied the ability of different myeloid regulatory proteins to induce the in vivo differentiation of these different types of mouse myeloid leukemic clones in normal and cyclophosphamide-treated mice. The results show that in both types of mice (a) the in vitro GM-CSF- and IL- 3-sensitive leukemic cells were induced to differentiate to mature cells in vivo in mice injected with pure recombinant GM-CSF and IL-3 but not with G-CSF, M-CSF, or MGI-2; (b) the in vitro MGI-2-sensitive leukemic cells differentiated in vivo by injection of MGI-2 and also, presumably indirectly, by GM-CSF and IL-3 but not by M-CSF or G-CSF; (c) in vivo induced differentiation of the leukemic cells was associated with a 20- to 60-fold decrease in the number of blast cells; and (d) all the injected myeloid regulatory proteins stimulated the normal myelopoietic system. Different normal myeloid regulatory proteins can thus induce in vivo terminal differentiation of leukemic cells, and it is suggested that these proteins can have a therapeutic potential for myeloid leukemia in addition to their therapeutic potential in stimulating normal hematopoiesis.
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Liaw, Patricia C. "Mediators of Disseminated Intravascular Coagulation: Molecular Mechanisms." Blood 130, Suppl_1 (December 7, 2017): SCI—2—SCI—2. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-2.sci-2.
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Abstract Disseminated intravascular coagulation (DIC) is characterized by a spectrum of haemorrhage and microvascular thrombosis complicating many conditions including sepsis and trauma. In recent years, there is growing evidence that damage associated molecular patterns (DAMPs) play a crucial role in the pathogenesis of DIC. Upon cell death and/or cell activation, extracellular DNA as well as DNA binding proteins (e.g. histones and high mobility group box 1 protein) are released into the circulation. These molecules can influence hemostasis by promoting coagulation via the contact pathway, inducing platelet aggregation, activating endothelial cells, and inhibiting fibrinolysis. Extracellular HMGB1 also activates neutrophils to induce the release of neutrophil extracellular traps, which may further contribute to tissue injury and organ dysfunction. Cell-free DNA (cfDNA) from nuclear, mitochondrial, and bacterial sources has varying pro-inflammatory effects, although all three have similar procoagulant and platelet-stimulating potential. Elevated levels of cfDNA and histones are predictive of poor outcome in sepsis and trauma, with neutrophils being the major source of DNA released from whole blood in vitro. In septic patients, endogenous cfDNA correlates positively with thrombin generation potential, and addition of recombinant DNase attenuates thrombin generation. In a cecal ligation and puncture (CLP) model of sepsis, plasma cfDNA levels rise within a few hours and are accompanied by elevations in IL-6 and thrombin-antithrombin complexes. Delayed (ie. 6 hours post-CLP surgery) but not early administration of recombinant DNase decreases bacterial load in the lungs, blood, and peritoneal cavity, and attenuates organ damage. Thus, the timing of DNase administration may be a crucial element in future investigations of the therapeutic potential of DNase in sepsis. With respect to potential therapeutic inhibitors of histones, activated protein C (APC) cleaves histones H2A, H3, and H4. Co-injection of APC with histones rescues mice from death. C-reactive protein (CRP) is an acute phase protein that reduces histone-induced endothelial cell damage and platelet aggregation. Administration of histones and CRP to mice reduces endothelial damage, alleviates thrombocytopenia, and attenuates coagulation activation. Heparin can also bind histones and prevent histone-mediated cytotoxicity of endothelial cells. In vivo, non-anticoagulant heparin reduces mortality from sterile inflammation and from sepsis in mouse models. Translational studies of septic patients have shown that the prognostic utility of clinical scores can be enhanced by combining it with cfDNA and protein C levels, suggesting that inclusion of cfDNA and protein C in risk stratification tools may be valuable for monitoring response to treatment, enhancing confidence in clinical decision making, or for inclusion in trials of new anti-sepsis therapies. In summary, cfDNA and DNA-binding proteins are critically involved in the pathogenesis of DIC. Strategies that inhibit or neutralize the harmful effects of cfDNA and histones may have great therapeutic potential. Disclosures No relevant conflicts of interest to declare.
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Sadowski, Julia, Ulrike Erben, Jan F. Richter, Wilfrid Bleiss, Alexandra Bittroff-Leben, Eckhard Thiel, Michael Krauss, and Michael Notter. "Selective Uptake and Intracellular Distribution of Covalently Multimerized Human Stem Cell Factor (SCF)-Fusion Proteins into Human c-kit+ Acute Myeloid Leukemia (AML) Cells." Blood 110, no. 11 (November 16, 2007): 1594. http://dx.doi.org/10.1182/blood.v110.11.1594.1594.
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Abstract C-kit expression has been detected in primary blasts of human AML patients frequently at high levels, but to a much lower extent in normal hemopoietic progenitor cells. As previously shown, a trimeric SCF-immunoglobulin (Ig)G1 fusion protein induced more profound modulation of c-kit compared to native SCF (Erben et al Cancer Res59: 2924–2930, 1999). In this study we investigated the life span and distribution of recombinant multimeric SCF proteins intracellularly. Furthermore, we asked if c-kit modulation by SCF is useful to selectively target therapeutics into AML blasts. The CS-1 cell line established from a patient with monoblastic AML relapsing after allogeneic stem cell transplantation showed increasing c-kit expression over time in vivo and in vitro and was used as a model system for high-risk AML. Sorted CD34+ hemopoietic progenitor cells served as non-malignant control. In addition to SCF-IgG1, further recombinant SCF fusion proteins were generated by eukaryotic expression: SCF linked to an enhanced green fluorescent protein (SCF-EGFP reporter protein) and myc-tagged SCF dimerized via the hinge-region of human IgG1 (SCF-μ transporter protein). SCF-μ was bound to the surface of myc antibody-coated nanoparticles (SCF-μ/B model therapeutic carrier). Corresponding to initial surface c-kit expression levels, SCF-EGFP (10 nM) modulated c-kit on the total population of CS-1 AML blasts and on only a minor subfraction of non-malignant CD34+ progenitor cells with a maximum observed at 90 min as determined by flow cytometry. SCF-IgG1 fusion protein but not human IgG1 inhibited SCF-EGFP binding in a dose-dependent manner indicating receptor specificity. Confocal laser scanning microscopy revealed initial focal aggregation of SCF-EGFP localized to one cell pole of the surface of CS-1 followed by apparent internalization and intracellular redistribution represented by circumferential multiple microspots at 20 minutes following incubation. SCF-μ showed a comparable cellular distribution pattern over time. Intracellular staining signals increased in intensity when CS-1 cells were incubated with SCF-μ/B compared to free SCF-μ indicating increased delivery of SCF-μ when attached to nanoparticles. SCF-μ/B clearly co-localized with internalized red fluorescent transferrin within CD71+ CS-1 cells in co-incubation experiments consistent with their strong association with the endosomal compartment. Internalization and subcellular distribution was confirmed by transmission electron microscopy demonstrating nanoparticles being localized in both endosomes and cytoplasm. In summary, augmented c-kit modulation on leukemia cells by covalently dimerized SCF-μ freely or bound to nanoparticles is a new transport mechanism resulting in their intracellular delivery. Since nanoparticles can be loaded with bioactive compounds, e.g. toxins or small molecules, this approach may offer new options for targeted therapy of c-kithigh AML.
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Groener, Albrecht, and Christine Dreier. "UV-C Treatment Of Intermediates Of Biologicals To Inactivate Viruses Without Denaturing Desired Protein." Blood 122, no. 21 (November 15, 2013): 4822. http://dx.doi.org/10.1182/blood.v122.21.4822.4822.
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Dedicated virus reduction steps implemented in the manufacturing process of biologicals, either isolated from human plasma or produced as recombinant proteins, are essential safety measures to assure that a potential virus contamination of the source material will not be transmitted to patients requiring these therapeutic proteins. Currently applied virus reduction steps as solvent/detergent treatment and virus filtration are very effective virus reduction methods with inherent method-dependent gaps regarding the reduction capacity for a very wide range of viruses of diverse physico-chemical characteristics: solvent/detergent treatment does not inactivate non-enveloped viruses and, depending on the pore size of the virus filter, small viruses are not removed when the desired protein is large and has to pass the filter. Therefore, another virus inactivation method was studied which is considered especially effective for small viruses: UV-C treatment using the UVivatec system provided by Sartorius Stedim Biotech GmbH, Göttingen, Germany. Experiments were performed to study the impact of UV-C treatment on the integrity of proteins employing fibrinogen as an example for a large protein and on the inactivation capacity for poliovirus (a small non-enveloped virus). The integrity of fibrinogen was assessed by comparing the untreated fibrinogen with the UV-C treated fibrinogen using HPLC, Clauss assay and thromboelastometry. Virus inactivation was studied in a bioassay using a sensitive cell culture infectivity assay employing a cynomolgus cell line. The results show that UV-C treatment inactivates viruses and modifies fibrinogen in a dose dependent manner; the monomer, dimer and polymer peak in the fibrinogen preparation studied changed from approx. 75% to 60%, 17% to 25% and 8% to 13%, respectively, at a UV-C intensity of 400 J/m² demonstrated by HPLC measurement. In order to protect fibrinogen from modifications, the antioxidant glutathione was added to the fibrinogen preparation. At an UV-C intensity of approx. 300 J/m², sufficient to effectively inactivate viruses studied, a modification of fibrinogen was not any longer detectable. Disclosures: Groener: CSL Behring: Employment.
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Shibata, Yasuko, Kimiyo Kurihara, Hisashi Takiguchi, and Yoshimitsu Abiko. "Construction of a Functional Single-Chain Variable Fragment Antibody against Hemagglutinin from Porphyromonas gingivalis." Infection and Immunity 66, no. 5 (May 1, 1998): 2207–12. http://dx.doi.org/10.1128/iai.66.5.2207-2212.1998.
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ABSTRACT Hemagglutinin is a major glycoprotein of Porphyromonas gingivalis vesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by using P. gingivalis vesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against theP. gingivalis hemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791–2799, 1992) were allowed to react with protein blots from P. gingivalis vesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating from P. gingivalisvesicles. In addition, the ScFv antibody inhibited theP. gingivalis vesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vκ12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.
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Shapiro, Gary S., Crystal Van Peursem, David A. Ornelles, Jerome Schaack, and James DeGregori. "Recombinant Adenoviral Vectors Can Induce Expression of p73 via the E4-orf6/7 Protein." Journal of Virology 80, no. 11 (June 1, 2006): 5349–60. http://dx.doi.org/10.1128/jvi.02016-05.
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ABSTRACT Despite the utility of recombinant adenoviral vectors in basic research, their therapeutic promise remains unfulfilled. Most engineered adenoviral vectors use a heterologous promoter to transcribe a foreign gene. We show that adenoviruses containing the cytomegalovirus immediate-early promoter induce the expression of the proapoptotic cellular protein TAp73 via the cyclin-dependent kinase-retinoblastoma protein-E2F pathway in murine embryonic fibroblasts. Cells transduced with these vectors also expressed high levels of the adenoviral E4-orf6/7 and E2A proteins. By contrast, adenoviruses containing the ubiquitin C promoter failed to elicit these effects. E4-orf6/7 is necessary and sufficient for increased TAp73 expression, as shown by using retrovirus-mediated E4-orf6/7 expression and adenovirus with the E4-orf6/7 gene deleted. Activation of TAp73 likely occurs via E4-orf6/7-induced dimerization of E2F and subsequent binding to the inverted E2F-responsive elements within the TAp73 promoter. In addition, adenoviral vectors containing the cytomegalovirus immediate-early promoter, but not the ubiquitin C promoter, cooperated with chemotherapeutic agents to decrease cellularity in vitro. In contrast to murine embryonic fibroblasts, adenoviruses containing the ubiquitin C promoter, but not the cytomegalovirus immediate-early promoter, induced both E4-orf6/7 and TAp73 in human foreskin fibroblasts, emphasizing the importance of cellular context for promoter-dependent effects. Because TAp73 is important for the efficacy of chemotherapy, adenoviruses that increase TAp73 expression may enhance cancer therapies by promoting apoptosis. However, such adenoviruses may impair the long-term survival of transduced cells during gene replacement therapies. Our findings reveal previously unknown effects of foreign promoters in recombinant adenoviral vectors and suggest means to improve the utility of engineered adenoviruses by better controlling their impact on viral and cellular gene expression.
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Luo, Yongneng, Limin Jiang, and Zi'an Mao. "Recent Advances in Clinical Trials of HCV Vaccines." Journal of Applied Virology 3, no. 1 (January 20, 2014): 10. http://dx.doi.org/10.21092/jav.v3i1.48.
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<p> Hepatitis C virus infects nearly 3% of the global population, and spreads to 3-4 million new people annually. HCV infection is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases and causes liver-related death in more than 300,000 people each year. Unfortunately, there is currently no vaccine for HCV prevention (prophylactic vaccine) or treatment (therapeutic vaccine). Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus and induce strong cross-reactive CD4+/CD8+ T-cell and neutralizing antibody responses in preventing or clearing HCV infection. So far, a few of vaccine development approaches are successful and some of the HCV vaccine candidates have reached human clinical trials, including those modalities mainly based on recombinant proteins (envelope proteins and core protein subunit), synthetic peptides, DNA (plasmid) and viral vectors (virosome). Encouraging results were obtained for those HCV vaccine formulations consisting of prime-boost regimen involving a live recombinant viral vector vaccine alone or in combination with DNA or subunit vaccine. Among several other vaccine strategies under preclinical development, the most promising one is virus like particle based vaccine that will be moving into human studies soon.</p>
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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.
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Zhydzetski, A. V., M. I. Patapovich, I. V. Kudina, U. A. Prakulevich, and M. V. Sholukh. "Selection conditions of the recombinant bovine α-interferon from E. coli inclusion bodies." Proceedings of the National Academy of Sciences of Belarus, Biological Series 64, no. 1 (February 18, 2019): 7–17. http://dx.doi.org/10.29235/1029-8940-2019-64-1-7-17.
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Like other proteins of the cytokine family, bovine α-interferon activates and modulates antiviral state of the target cells and inhibits division and growth of the infected cells which makes it an excellent candidate as a new antiviral therapeutic agent.This study is concerned with the determination of the optimal isolation, purification and refolding conditions of the recombinant bovine interferon-α (rbIFN-α) from inclusion bodies (IBs). Main methods used were UV/Visible spectroscopy, electrophoresis, liquid chromatography and refolding by dilution.It was found that two step IBs washing with solutions containing 50 mmol/l Tris, 50 mmol/l NaCl and 3.5 mol/l urea and their subsequent solubilization in 50 mmol/l Tris-HCl, pH 9.8 mol/l Urea and 20 mmol/l β-mercaptoethanol allow us to receive the target protein in monomeric form and 53.18 ± 9.3 % purity. Further application of the anion-exchange tandem chromatography on DE 52 cellulose and toyopearl DEAE-650 M gives a possibility to remove the major impurities and obtain rbIFN-α with 80.7 ± 8.6 % purity. Refolding by dilution in the buffer containing 20 mmol/l NaPB, рН 7.4, 0.4 mol/l sucrose, 1 mmol/l L-Cys, 0.1 mmol/l L-Cystine, 1 mmol/l EDTA, 0.05 % Kolliphor EL at 10 °C followed by the protein collection allows to get the recombinant rbIFN-α in homogeneous state, with 98.43 % purity and antiviral activity about (5 ± 3.6)•106 U/mg.
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Pawar, Pritish S., Keith J. Micoli, Haitao Ding, William J. Cook, John C. Kappes, Yabing Chen, and Jay M. McDonald. "Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling." Biochemical Journal 412, no. 3 (May 28, 2008): 459–68. http://dx.doi.org/10.1042/bj20071507.
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We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1β-converting enzyme]-like inhibitory protein}. A Ca2+-dependent direct interaction between CaM and FLIPL, but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3±5.7% increase (n=6, P=0.001) in CaM–FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca2+ that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM–FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIPL was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197–213 of FLIPL. Compared with overexpression of wild-type FLIPL that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIPL with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM–FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.
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Nurdiani, Dini, Hariyatun Hariyatun, and Wien Kusharyoto. "Secretory expression of human insulin precursor in Pichia pastoris employing truncated α-factor leader sequence and a short C-peptide." Indonesian Journal of Biotechnology 23, no. 2 (December 24, 2018): 102. http://dx.doi.org/10.22146/ijbiotech.38958.
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In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.
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Evans, Robert P., Kim Laginha, Linda M. Pilarski, Andrew Belch, and Tony Reiman. "Analysis of Protein Biology to Increase Efficacy of Aurora Kinase Inhibitors for Multiple Myeloma Therapy." Blood 112, no. 11 (November 16, 2008): 1699. http://dx.doi.org/10.1182/blood.v112.11.1699.1699.
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Abstract Despite improvement in therapeutic regimes which have extended survival of patients with multiple myeloma, the search for novel targets to further advance treatment options continues. It is critical to the success of targeted cancer therapy that the rational selection of drugs be derived from the molecular and cellular biology of protein targets. Furthermore, studies have indicated that focused combination drug therapy could produce remarkable improvement in patient outcomes. Aurora kinases A/B/C (AURK), components of the centrosome and important for cell cycle function, are promising new targets in the treatment of numerous types of cancer. We have previously validated AURKs as a therapeutic target in pre-clinical models of multiple myeloma. In this study our aim is to better define the biology of AURKs in both normal and malignant cells, with the goal of identifying rational strategies for improving the therapeutic potential of Aurora kinase inhibitors (AKIs) as anti-cancer agents. We use a systematic two-pronged approach to map post-translational modifications and novel protein-protein interactions that define the interactome of AURKs. We also examine whether Aurora kinase interactions and posttranslational modifications are significantly different in cancer cells compared to normal cells. Increased understanding of Aurora kinase biology will help develop mechanisms to intensify the anti-cancer effects of AKIs. SUMOylation is a recently discovered posttranslational modification of numerous proteins that has been linked to many cancer types. A small ubiquitin-like modifier (SUMO) group is conjugated at the canonical ubiquitin site of a protein to alter its function, stability or sub-cellular localization. We believe that Aurora kinases may be differentially SUMOylated in cancer cells compared to normal cells, and that SUMOylation of Aurora kinases in cancer cells may alter their function and their susceptibility to AKI therapy. Here we have determined putative SUMOylation sites of all three AURKs in silico and also have evidence of AURK SUMOylation using an in vitro assay and also in human HEK293 cells. Furthermore, the gene encoding the central SUMO conjugating enzyme - UBC9 - is expressed at higher levels in CD138+ malignant plasma cells compared to other CD138−bone marrow cells in myeloma patients. As part of our strategy, we have also used Tandem Affinity Purification (TAP) technology to identify novel protein-protein interactions of Aurora kinases. In the initial stage, we have established stable expressing clones in a human cancer cell line and a normal line that express TAP-tagged AURKA/B or C. In pilot experiments we have successfully isolated pure recombinant AURKA along with interacting proteins using the TAP method. Using mass spectrometry (MS/MS) protein ID, we have identified putative protein interaction partners with AURKA which have implications in general cancer biology and centrosomal structure. In the future, these interactions will be validated using conventional biochemical methods before being tested in myeloma cell lines.
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Mani, Hemalatha, Yi-Cheng Chen, Yen-Kai Chen, Wei-Lin Liu, Shih-Yen Lo, Shu-Hsuan Lin, and Je-Wen Liou. "Nanosized Particles Assembled by a Recombinant Virus Protein Are Able to Encapsulate Negatively Charged Molecules and Structured RNA." Polymers 13, no. 6 (March 11, 2021): 858. http://dx.doi.org/10.3390/polym13060858.
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RNA-based molecules have recently become hot candidates to be developed into therapeutic agents. However, successful applications of RNA-based therapeutics might require suitable carriers to protect the RNA from enzymatic degradation by ubiquitous RNases in vivo. Because of their better biocompatibility and biodegradability, protein-based nanoparticles are considered to be alternatives to their synthetic polymer-based counterparts for drug delivery. Hepatitis C virus (HCV) core protein has been suggested to be able to self-assemble into nucleocapsid-like particles in vitro. In this study, the genomic RNA-binding domain of HCV core protein consisting of 116 amino acids (p116) was overexpressed with E. coli for investigation. The recombinant p116 was able to assemble into particles with an average diameter of approximately 27 nm, as visualized by electron microscopy and atomic force microscopy. Measurements with fluorescence spectroscopy, flow cytometry, and fluorescence quenching indicated that the p116-assembled nanoparticles were able to encapsulate small anionic molecules and structured RNA. This study demonstrates methods that exploit the self-assembly nature of a virus-derived protein for nanoparticle production. This study also suggests that the virus-derived protein-assembled particles could possibly be developed into potential carriers for anionic molecular drugs and structured RNA-based therapeutics.
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Ahmad Fuad, Fazia Adyani, and Suriyea Tanbin. "Enhanced Expression and Purification Strategy for Recombinant Bacterially-Expressed Human Hexokinase II." Molekul 16, no. 1 (March 28, 2021): 82. http://dx.doi.org/10.20884/1.jm.2021.16.1.709.
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Dengue virus hijacks the host cellular mechanism to propagate and survive during viral infection, in which the central carbon mechanism plays a crucial role to upregulate DENV infection through the increase of human hexokinase II (HKII) activity. Since the enzyme governs the glycolytic pathway, it has potentials as a target for anti-dengue (DENV) drug development. In this study, the production of human hexokinase II protein has been enhanced by using bacterial system for anti-dengue therapeutic purpose. The HKII gene was cloned into pET28b vector and transformed into the E. coli strain BL21 (DE3) for HKII expression. In order to obtain soluble recombinant HKII in an active form, we optimized protein expression under specific conditions at 18°C for 19 hours using Terrific Broth media, in the presence of 0.5 mM isopropyl-2-D-thiogalactopyranoside (IPTG). The pET28b-HKII construct expressed in BL21(DE3) system exhibited adequate protein expression, thus, this construct was subsequently proceeded to purification process. The expressed protein was purified to homogeneity by a combination of Immobilized Metal Ion Affinity Chromatography (IMAC)and size exclusion chromatography (SEC), resulting in pure, active bacterially-expressed HKII with a specific activity of 56. 67U.mg-1. The amount of HKII obtained from 2 L culture is 80 mg, with a yield percentage of 10.5%. Hence in this study, human HKII has successfully been cloned and expressed as a soluble protein that can be utilized for further therapeutic studies.
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Kataoka, Kosuke, Atsuo Amano, Shigetada Kawabata, Hideki Nagata, Shigeyuki Hamada, and Satoshi Shizukuishi. "Secretion of Functional Salivary Peptide by Streptococcus gordonii Which Inhibits Fimbria-Mediated Adhesion ofPorphyromonas gingivalis." Infection and Immunity 67, no. 8 (August 1, 1999): 3780–85. http://dx.doi.org/10.1128/iai.67.8.3780-3785.1999.
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ABSTRACT Porphyromonas gingivalis, a putative periodontopathogen, can bind to human salivary components with its fimbriae. We have previously shown that fimbriae specifically bind to a peptide domain shared by a major salivary component, i.e., proline-rich (glyco)proteins (PRPs). The synthetic domain peptide PRP-C (pPRP-C) significantly inhibits the fimbrial binding to PRPs. In this study, a recombinant strain of Streptococcus gordonii secreting pPRP-C was generated as a model of a possible approach to prevent the oral colonization by the pathogen. A duplicate DNA fragment (prpC) encoding pPRP-C was obtained by self-complementary annealing of synthetic oligonucleotides. prpC was connected downstream to a promoter and a gene encoding a signal peptide ofStreptococcus downei glucosyltransferase I in frame. The linked fragments were inserted into the plasmid pMNK-4 derived from pVA838. The constructed plasmid was inserted to produce the transformant S. gordonii G9B, which then successfully secreted recombinant pPRP-C (r-pPRP-C) of the expected size. The concentrated bacterial culture supernatant containing r-pPRP-C inhibited the binding of P. gingivalis cells and fimbriae to PRP1 in a dose-dependent manner up to 72 and 77%, respectively. The r-pPRP-C concentrate also inhibited the coaggregation of P. gingivalis with various streptococcal strains as effectively as synthetic pPRP-C in a dose-dependent manner. Collectively, pPRP-C was found to be able to prevent P. gingivalis adherence to salivary receptor protein and plaque-forming bacteria. These results suggest that this recombination approach with a nonperiodontopathic bacterium may be suitable for the therapeutic prevention of P. gingivalis adherence to the oral cavity.
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Dettenmeier, Patricia, Bridget Swindell, Mary Stroud, Nancy Arkins, and April Howard. "Role of Activated Protein C in the Pathophysiology of Severe Sepsis." American Journal of Critical Care 12, no. 6 (November 1, 2003): 518–24. http://dx.doi.org/10.4037/ajcc2003.12.6.518.
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Sepsis is a complex syndrome that can lead to multiple organ failure and death. Severe sepsis has been associated with mortality rates ranging from 28% to 50% and is the most common cause of death in the noncardiac intensive care unit. Despite advances in both antibiotic therapy and supportive care, the mortality rate due to severe sepsis has remained fundamentally unchanged in the past several decades. With increased understanding of the pathophysiology of sepsis, particularly the intricate interplay between activation of coagulation and inflammation, novel therapeutic agents that may improve clinical outcomes are being researched and developed. The epidemiology, pathophysiology, and treatment of severe sepsis are reviewed. Also discussed are the recently published results from a multicenter, randomized, placebo-controlled phase 3 clinical trial of drotrecogin alfa (activated), a recombinant form of human activated protein C, in patients with severe sepsis. The nursing implications of this new approved therapy are discussed.
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Liu, Franklin, Qing Dallas-Yang, Gino Castriota, Paul Fischer, Francesca Santini, Marc Ferrer, Jing Li, et al. "Development of a novel GLUT4 translocation assay for identifying potential novel therapeutic targets for insulin sensitization." Biochemical Journal 418, no. 2 (February 11, 2009): 413–20. http://dx.doi.org/10.1042/bj20082051.
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GLUT4 (glucose transporter 4) plays important roles in glucose homoeostasis in vivo. GLUT4 expression and function are diminished in diabetic human and animal subjects. The goal of the present study is to develop a cell-based assay for identifying negative regulators of GLUT4 translocation as potential targets for the treatment of Type 2 diabetes. Traditional GLUT4 translocation assays performed in differentiated myocytes or adipocytes are difficult to perform, particularly in HTS (high-throughput screening) mode. In the present study, we stably co-expressed c-Myc and eGFP [enhanced GFP (green fluorescent protein)] dual-tagged recombinant GLUT4 with recombinant IRS1 (insulin receptor substrate 1) in HEK-293 cells (human embryonic kidney cells) (HEK-293.IRS1.GLUT4 cells). Insulin treatment stimulated both glucose uptake and GLUT4 translocation in these cells. GLUT4 translocation is quantified by a TRF (time-resolved fluorescence) assay in a 96-well HTS format. TRF assays confirmed insulin-stimulated GLUT4 translocation, which can be inhibited by PI3K (phosphoinositide 3-kinase) or Akt [also called PKB (protein kinase B)] inhibitors. Treatment with palmitate increased IRS1 serine phosphorylation and reduced insulin-stimulated Akt phosphorylation and GLUT4 translocation, indicating insulin resistance. Knockdown of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and PTP1B (protein tyrosine phosphatase 1B) gene expression by siRNA (small interfering RNA) treatment significantly increased GLUT4 translocation only in cells treated with palmitate but not in untreated cells. Similar results were obtained on treatment with siRNA of JNK1 (c-Jun N-terminal kinase 1), S6K1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and PKCθ (protein kinase C θ). In summary, we have established and validated a novel GLUT4 translocation assay that is optimal for identifying negative regulators of GLUT4 translocation. In combination with more physiologically relevant secondary assays in myotubes and adipocytes, this assay system can be used to identify potential novel therapeutic targets for the treatment of Type 2 diabetes.
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Fibriansah, Guntur, Elisa X. Y. Lim, Jan K. Marzinek, Thiam-Seng Ng, Joanne L. Tan, Roland G. Huber, Xin-Ni Lim, et al. "Antibody affinity versus dengue morphology influences neutralization." PLOS Pathogens 17, no. 2 (February 23, 2021): e1009331. http://dx.doi.org/10.1371/journal.ppat.1009331.
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Different strains within a dengue serotype (DENV1-4) can have smooth, or “bumpy” surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.
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David, Victor, Barbara Barbosa Succar, João Alfredo de Moraes, Roberta Ferreira Gomes Saldanha-Gama, Christina Barja-Fidalgo, and Russolina Benedeta Zingali. "Recombinant and Chimeric Disintegrins in Preclinical Research." Toxins 10, no. 8 (August 7, 2018): 321. http://dx.doi.org/10.3390/toxins10080321.
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Disintegrins are a family of small cysteine-rich peptides, found in a wide variety of snake venoms of different phylogenetic origin. These peptides selectively bind to integrins, which are heterodimeric adhesion receptors that play a fundamental role in the regulation of many physiological and pathological processes, such as hemostasis and tumor metastasis. Most disintegrins interact with integrins through the RGD (Arg-Gly-Asp) sequence loop, resulting in an active site that modulates the integrin activity. Some variations in the tripeptide sequence and the variability in its neighborhood result in a different specificity or affinity toward integrin receptors from platelets, tumor cells or neutrophils. Recombinant forms of these proteins are obtained mainly through Escherichia coli, which is the most common host used for heterologous expression. Advances in the study of the structure-activity relationship and importance of some regions of the molecule, especially the hairpin loop and the C-terminus, rely on approaches such as site-directed mutagenesis and the design and expression of chimeric peptides. This review provides highlights of the biological relevance and contribution of recombinant disintegrins to the understanding of their binding specificity, biological activities and therapeutic potential. The biological and pharmacological relevance on the newest discoveries about this family of integrin-binding proteins are discussed.
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Veldwijk, Marlon R., Carsten Herskind, Stephanie Laufs, Marius Stiefelhagen, W. Jens Zeller, Stefan Fruehauf, and Frederik Wenz. "An Novel Approach in Protective Gene Therapy: Recombinant Adeno-Associated Virus 2-Mediated Transfer of the Human Superoxide-Dismutase Gene." Blood 104, no. 11 (November 16, 2004): 5265. http://dx.doi.org/10.1182/blood.v104.11.5265.5265.
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Abstract Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue such as hematopoietic cells without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against superoxide-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). Oxidative stress-induced apoptosis plays a role in both radiation- and chemotherapy-induced tissue damage. Relevant cytostatic drugs for which oxidative stress as one mechanism of action has been described are e.g. anthracyclins, platinum analogues, etoposide and ara-C. For gene therapeutic approaches, recombinant adeno-associated virus 2 (rAAV-2)-based vectors offer attractive advantages over other vector systems: low immunogenicity, possibility of in vivo application, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. Here, we report the production and testing of novel rAAV-2-SOD vectors with the goal of normal tissue protection. Material and methods: Various rAAV-2 vectors containing CuZnSOD, MnSOD and fusion proteins of both with the enhanced green fluorescent protein (eGFP) gene were cloned and vector stocks were produced. Human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. Cells were seeded and transduced with the rAAV-2-SOD vectors. Gene transfer and transgene expression were investigated using FACS and an SOD-activity assay. Results: Over 70% of all HeLa cells expressed SOD and significant amounts of functional SOD protein were detected (table 1). Conclusion: These results forms the basis to evaluate the radio- and chemoprotective effects of AAV-mediated SOD gene therapy in hematopoietic and non-hematopoietic (e.g. mucosal) cells. Vector % GFP+ cells SOD activity (U/mg) Mock control - 435 ± 37 rAAV-2-CuZnSOD N/A 965 ± 112 rAAV-2-CuZnSOD/eGFP 78 ± 2 1093 ± 178 rAAV-2-MnSOD N/A 1516 ± 191 rAAV-2-MnSOD/eGFP 77 ± 3 1204 ± 124
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Finetti, Federica, Andrea Moglia, Irene Schiavo, Sandra Donnini, Giovanni Berta, Federica Di Scipio, Andrea Perrelli, Claudia Fornelli, Lorenza Trabalzini, and Saverio Retta. "Yeast-Derived Recombinant Avenanthramides Inhibit Proliferation, Migration and Epithelial Mesenchymal Transition of Colon Cancer Cells." Nutrients 10, no. 9 (August 24, 2018): 1159. http://dx.doi.org/10.3390/nu10091159.
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Avenanthramides (Avns), polyphenols found exclusively in oats, are emerging as promising therapeutic candidates for the treatment of several human diseases, including colon cancer. By engineering a Saccharomyces cerevisiae strain, we previously produced two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, YAvnI) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, YAvnII), which are endowed with a structural similarity to bioactive oat avenanthramides and stronger antioxidant properties. In this study, we evaluated the ability of these yeast-derived recombinant avenanthramides to inhibit major hallmarks of colon cancer cells, including sustained proliferation, migration and epithelial-mesenchymal transition (EMT). Using the human colon adenocarcinoma cell line HT29, we compared the impact of YAvns and natural Avns, including Avn-A and Avn-C, on colon cancer cells by performing MTT, clonogenic, adhesion, migration, and anchorage-independent growth assays, and analyzing the expression of EMT markers. We found that both YAvns and Avns were able to inhibit colon cancer cell growth by increasing the expression of p21, p27 and p53 proteins. However, YAvns resulted more effective than natural compounds in inhibiting cancer cell migration and reverting major molecular features of the EMT process, including the down-regulation of E-cadherin mRNA and protein levels.
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Okamoto, Takayuki, Hironobu Tanigami, Koji Suzuki, and Motomu Shimaoka. "Thrombomodulin: A Bifunctional Modulator of Inflammation and Coagulation in Sepsis." Critical Care Research and Practice 2012 (2012): 1–10. http://dx.doi.org/10.1155/2012/614545.
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Deregulated interplay between inflammation and coagulation plays a pivotal role in the pathogenesis of sepsis. Therapeutic approaches that simultaneously target both inflammation and coagulation hold great promise for the treatment of sepsis. Thrombomodulin is an endogenous anticoagulant protein that, in cooperation with protein C and thrombin-activatable fibrinolysis inhibitor, serves to maintain the endothelial microenvironment in an anti-inflammatory and anticoagulant state. A recombinant soluble form of thrombomodulin has been approved to treat patients suffering from disseminated intravascular coagulation (DIC) and has thus far shown greater therapeutic potential than heparin. A phase II clinical trial is currently underway in the USA to study the efficacy of thrombomodulin for the treatment of sepsis with DIC complications. This paper focuses on the critical roles that thrombomodulin plays at the intersection of inflammation and coagulation and proposes the possible existence of interactions with integrins via protein C. Finally, we provide a rationale for the clinical application of thrombomodulin for alleviating sepsis.
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Spek, C. Arnold, and Valder Arruda. "The Role of Activated Protein C in Cancer." Blood 118, no. 21 (November 18, 2011): SCI—18—SCI—18. http://dx.doi.org/10.1182/blood.v118.21.sci-18.sci-18.
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Abstract Abstract SCI-18 Activated protein C (APC) is a natural anticoagulant that blocks the amplification of the coagulation cascade via inactivation of factors Va and VIIIa. The APC/PC pathway is initiated by complex formation of thrombin, thrombomodulin (TM), and the endothelial protein C receptor (EPCR) allowing the conversion of zymogen protein C into its activated form. Based on the well-accepted view that coagulation contributes to cancer progression and that anticoagulant treatment may benefit some cancer patient groups, it has been hypothesized that the natural anticoagulant protein C pathway may also play a role in cancer progression. Interestingly, it has recently been shown that endogenous APC limits experimental metastasis of B16 melanoma cells in mouse lungs. Notably, an APC-blocking antibody dramatically increased the number of experimental lung metastasis although not due to diminished anticoagulant activity of APC but largely due to reduced APC-driven S1P1-mediated VE-cadherin-dependent vascular barrier enhancement. In line with these findings repeated administration of recombinant human APC as well as transgenic overexpression of EPCR also diminished experimental metastasis of B16 melanoma cells. It may thus be tempting to speculate that recombinant APC could be a novel treatment strategy to limit cancer progression. However, APC has however a short half-life, needs intravenous administration, and is associated with severe bleeding complications, complicating the potential clinical application of these findings. In contrast to APC, zymogen PC has a longer half-life and is associated with significantly less bleeding complications. To prove or refute the hypothesis that zymogen PC may be an attractive alternative treatment option for APC in cancer patients, we recently compared the effect of continuous overexpression of murine APC or zymogen PC in the liver by viral-mediated gene transfer in experimental metastasis. Interestingly, both APC and zymogen PC overexpression was highly effective in limiting experimental metastasis. An APC variant (APC-5A) with reduced anticoagulant but normal signaling properties did not limit experimental metastasis, whereas the protective effect of zymogen PC remained even in the absence of protease activated receptor-1 (PAR-1), which is the main mediator of APCs cytoprotective effect. Zymogen PC may thus be a novel therapeutic target to limit cancer progression. In conclusion, the natural anticoagulant APC pathway may play an important role in limiting cancer cell extravasation and interventions seeking to modulate the PC system may ultimately benefit the cancer patient. The challenge is however to confirm these findings in alternative preclinical cancer models and eventually to translate our findings into a clinical setting. Disclosures: No relevant conflicts of interest to declare.
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Saumet, Anne, Mouna Ben Slimane, Michel Lanotte, Jack Lawler, and Véronique Dubernard. "Type 3 repeat/C-terminal domain of thrombospondin-1 triggers caspase-independent cell death through CD47/αvβ3 in promyelocytic leukemia NB4 cells." Blood 106, no. 2 (July 15, 2005): 658–67. http://dx.doi.org/10.1182/blood-2004-09-3585.
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Abstract By means of its antiangiogenic activity, thrombospondin-1 (TSP-1) exerts indirect antitumoral action on solid tumors. Here, we investigated potential antitumor action in an in vitro cell model for promyelocytic leukemia (NB4-LR1), resistant to retinoid maturation. Purified soluble TSP-1 added to cultures induced a strong dose-dependent growth inhibition and a slowly developing maturation-independent cell death. Recombinant fragments of TSP-1 allowed mapping of these activities to its type 3 repeat/C-terminal domain, features that are distinct from those of TSP-1 action on solid tumors, previously ascribed to the type 1 repeat domain. Cell death in leukemia was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization followed by membrane permeabilization. Mitochondria membrane depolarization was inherent to TSP-1 action but did not produce release of death-promoting proteins (eg, noncaspase apoptosis regulators, apoptosis-induced factor [AIF], endonuclease G, or Omi/HtrA2 or the caspase regulators, cytochrome c or second mitochondrial activator of caspase/direct inhibitor of apoptosis protein-binding protein with low isoelectric point [Smac/DIABLO]). Although detected, reactive oxygen species (ROS) production was likely not involved in the death process. Finally, receptor agonist RFYVVM and RGD peptides indicated that TSP-1 death effects are mediated by membrane receptors CD47 and αvβ3. These results demonstrated a new domain-specific antitumoral activity of TSP-1 on a leukemia cell line, which extends TSP-1 therapeutic potential outside the area of vascularized solid tumors. (Blood. 2005;106:658-667)
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François, Catherine, Isabelle Bernard, Sandrine Castelain, Bryan Charleston, Martin D. Fray, Jean-Claude Capiod, and Gilles Duverlie. "Quantification of Different Human Alpha Interferon Subtypes and Pegylated Interferon Activities by Measuring MxA Promoter Activation." Antimicrobial Agents and Chemotherapy 49, no. 9 (September 2005): 3770–75. http://dx.doi.org/10.1128/aac.49.9.3770-3775.2005.
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ABSTRACT Alpha interferons (α-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of α-IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of α-IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by α-IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other α-IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.
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Cornelius, Claire A., Lauriane E. Quenee, Katie A. Overheim, Frederick Koster, Trevor L. Brasel, Derek Elli, Nancy A. Ciletti, and Olaf Schneewind. "Immunization with Recombinant V10 Protects Cynomolgus Macaques from Lethal Pneumonic Plague." Infection and Immunity 76, no. 12 (September 15, 2008): 5588–97. http://dx.doi.org/10.1128/iai.00699-08.
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ABSTRACT Vaccine and therapeutic strategies that prevent infections with Yersinia pestis have been sought for over a century. Immunization with live attenuated (nonpigmented) strains and immunization with subunit vaccines containing recombinant low-calcium-response V antigen (rLcrV) and recombinant F1 (rF1) antigens are considered effective in animal models. Current antiplague subunit vaccines in development for utilization in humans contain both antigens, either as equal concentrations of the two components (rF1 plus rLcrV) or as a fusion protein (rF1-rLcrV). Here, we show that immunization with either purified rLcrV (a protein at the tip of type III needles) or a variant of this protein, recombinant V10 (rV10) (lacking amino acid residues 271 to 300), alone or in combination with rF1, prevented pneumonic lesions and disease pathogenesis. In addition, passive immunization studies showed that specific antibodies of macaques immunized with rLcrV, rV10, or rF1, either alone or in combination, conferred protection against bubonic plague challenge in mice. Finally, we found that when we compared the reactivities of anti-rLcrV and anti-rV10 immune sera from cynomolgus macaques, BALB/c mice, and brown Norway rats with LcrV-derived peptides, rV10, but not rLcrV immune sera, lacked antibodies recognizing linear LcrV oligopeptides.
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Tazzari, PierLuigi, Veronica Papa, Francesca Ricci, Francesca Chiarini, Camilla Evangelisti, Giovanni Martinelli, Andrea Bontadini, James McCubrey, and Alberto M. Martelli. "Synergistic Proapoptotic Activity of Recombinant TRAIL Plus the Akt Inhibitor Perifosine in Acute Myelogenous Leukemia Cells-a Novel Therapeutic Approach for Leukemia Displaying Elevated Akt Signaling." Blood 112, no. 11 (November 16, 2008): 957. http://dx.doi.org/10.1182/blood.v112.11.957.957.
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Abstract The Tumor Necrosis Factor (TNF) family member TNF-Related Apoptosis Inducing Ligand (TRAIL) was originally reported to induce apoptosis in many tumor cells but not in normal cells in vivo and thus represents a promising anticancer cytokine. The in vitro cytotoxic response of acute myelogenous leukemia (AML) cell lines to recombinant TRAIL varies from good to moderate, however, a number of in vitro studies have convincingly demonstrated that AML primary cells are resistant to the proapoptotic activity of TRAIL used as a single agent. To potentiate the response of AML cells to TRAIL cytotoxicity, we have adopted a strategy of combining perifosine, a novel Akt inhibitor, with recombinant TRAIL. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease cellular FLICEinhibitory (c-FLIP, an inhibitor of caspase-8 activation) protein in human lung cancer cells. Both perifosine and TRAIL, when used alone, induced cell death by apoptosis in THP- 1 AML cells, which normally express constitutively active Akt and nonfunctional p53. Perifosine treatment, at concentrations well below the IC50 (0.5 μM), dephosphorylated Akt on Ser473 and increased TRAIL-R2 levels, as demonstrated by flow cytometry, western blot, and RT-PCR. Perifosine also downmodulated cFLIP-L and XIAP levels. However, perifosine did not affect expression of TRAIL-R1 and TRAIL-R4 receptors, or of other proteins which are critical for TRAIL-mediated proapoptotic signaling, including FADD and Mcl-1. Perifosine and TRAIL strongly synergized to induce cytotoxicity as suggested by calculation of the combination index (CI range: 0.15–0.37). The combined treatment resulted in upregulation of caspase-8 activity and apoptosis which was markedly reduced by a selective caspase-8 inhibitor (Z-IETD-FMK). While cFLIP-L and XIAP downregulation was dependent on inhibition of NF-κB activity caused by perifosine, upregulation of TRAIL-R2 expression was dependent on generation of reactive oxygen species (ROS) by perifosine which in turn sequentially activated protein kinase C (PKC)α, JNK2, and c-Jun. A ROS scavenger (N-acetylcysteine), siRNA downregulation of either PKCα or c-Jun, or a JNK1/2 selective pharmacological inhibitor (SP600125), all markedly impaired perifosine-dependent TRAIL-R2 upregulation. Perifosine synergized with TRAIL by inducing apoptosis exclusively in primary AML cells displaying constitutive activation of the Akt pathway. Also in primary AML blasts, perifosine upregulated TRAIL-R2 levels, downmodulated the expression of both c-FLIP and XIAP, and increased Ser 63 p-c-Jun levels, without affecting the expression of FADD. Remarkably, perifosine increased p-JNK2 levels and TRAIL-R2 expression in primary AML patient blasts (CD34+, CD38Low/Neg, CD123+) enriched in putative leukemic stem cells. Perifosine and TRAIL combined treatment was effective in inducing apoptosis (55–60%) in this immature blast population, as documented by a quadruple staining flow cytometric technique for CD34+, CD38Low/Neg, CD123+, Annexin V+ blast cells. The combined treatment negatively affected the clonogenic activity of CD34+ cells from AML patients with Akt activation. In contrast, CD34+ cells from healthy donors and AML patients without Akt activation were resistant to perifosine plus recombinant TRAIL treatment. Our findings suggest that a combination consisting of perifosine plus recombinant TRAIL might offer a novel therapeutic strategy for AML displaying enhanced Akt signaling by overcoming critical mechanisms of apoptosis resistance. Moreover, this kind of combination therapy could be effective also in AML cases exhibiting nonfunctional p53 or low levels of p53.
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Miller, Jason, Min Luo, Hua Wang, Zhaohui Wang, Xinliang Ding, Ashley Campbell, Jonathan Almazan, Zhijian Chen, Jinming Gao, and Tian Zhao. "P857 ONM-500 – a novel STING-activating therapeutic nanovaccine platform for cancer immunotherapy." Journal for ImmunoTherapy of Cancer 8, Suppl 1 (April 2020): A7—A8. http://dx.doi.org/10.1136/lba2019.11.
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BackgroundEfficacy of cancer vaccines requires the induction of tumor antigen-specific cytotoxic T-lymphocytes (CTL) to effectively clear established tumors. Orchestration of antigen presentation, co-stimulatory signaling, and innate cytokine signals are necessary steps for tumor-specific T-cell activation. The ONM-500 nanovaccine platform1-2 utilizes a novel pH-sensitive polymer that forms an antigen-encapsulating nanoparticle and functions both as a carrier for antigen delivery of both peptide and protein antigens to dendritic cells and acts as an adjuvant, activating the stimulator on interferon genes (STING) pathway and generating a CD8+ CTL response. Peptide antigens have translational challenges due to complex formulations and/or HLA-type-specific antigen sequence recognition, processing and presentation. Full-length protein antigens alleviate HLA subtype limitation, allowing coverage of multi-immunogenic T cell epitopes in patients. Pairing ONM-500 adjuvant with the full-length E6 and E7 oncoproteins from human papillomavirus (HPV) cancers shows great potential to treat HPV-associated cancer in patients.MethodsBased on the previously demonstrated STING-dependent T cell activation by ONM-500 [1], the nanovaccine was formulated with full-length HPV16 E6 and E7 proteins (recombinant), and the nanoparticle properties and antigen loading were characterized. In vivo lymph node accumulation following subcutaneous administration was evaluated using fluorescent nanovaccines. Direct binding of ONM-500 to recombinant human STING (CTD) was evaluated using isothermal titration calorimetry (ITC) compared to the endogenous ligand 2’,3’-cGAMP. Antitumor efficacy was evaluated in multiple syngeneic tumor models, including the TC-1 model which overexpresses HPV16 E6 and E7 with the ONM-500 vaccine in combination with anti-PD-1 checkpoint inhibitor. Long-term anti-tumor memory was evaluated in a follow-up rechallenge study after 60 days in tumor-free animals.ResultsCharacterization of ONM-500 nanovaccine shows reproducible particle chemi-physical properties and antigen loading. The nanoparticle size substantiates the effective lymph node accumulation for antigen cross-presentation in dendritic cells following subcutaneous administration. ITC studies with human STING demonstrated effective binding by ONM-500 adjuvant. The nanovaccine anti-tumor efficacy was previously demonstrated in melanoma, colorectal, and HPV-associated syngeneic tumor models. In TC-1 tumors, ONM-500 nanovaccine containing full-length E6/E7 protein showed 100% overall survival at 55 days (figure 1). Tumor growth inhibition was also improved over E7 antigen peptide formulated nanovaccine. A rechallenge study demonstrated long-term antigen-specific anti-tumor memory response.Abstract P857 Figure 1ConclusionsONM-500 STING-activating nanovaccines effectively deliver antigens in vivo to lymph nodes to elicit antigen-specific CTL response. The anti-tumor efficacy in multiple tumor models demonstrates the potential of ONM-500 as a general STING agonist cancer vaccine platform, and full-length E6/E7 incorporated ONM-500 is being developed for HPV-associated cancers.Ethics ApprovalAll animal procedures were performed with ethical compliance and approval by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center (Protocol No. 2017-101954) and Pennsylvania State College of Medicine (Protocol No. 47682).ReferencesLuo M, Wang H, Wang Z, Cai H, Lu Z, Li Y, Du M, Huang G, Wang C, Chen X, Porembka MR, Lea J, Frankel AE, Fu YX, Chen ZJ, Gao J. A STING-activating nanovaccine for cancer immunotherapy. Nat Nanotechnol 2017; 12:648–654.Luo M, Liu Z, Zhang X, Han C, Samandi LZ, Dong C, Sumer BD, Lea J, Fu YX, Gao J. Synergistic STING activation by PC7A nanovaccine and ionizing radiation improves cancer immunotherapy. J Control Release 2019; 28:154–160.
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Zivny, Jan, Pavel Klener, Ondrej Toman, Petr Halada, Robert Ivanek, Tereza Simonova, Ladislav Andera, Emanuel Necas, and Jiri Petrak. "Proteomic and mRNA Expression Chip Analysis of Acquired TRAIL-Resistance in Human HL60 Myeloid Leukemia Cells." Blood 110, no. 11 (November 16, 2007): 4155. http://dx.doi.org/10.1182/blood.v110.11.4155.4155.
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Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a proapoptotic cytokine implicated in cancer cell surveillance. TRAIL induces apoptosis of target cells by the extrinsic, receptor-mediated, apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed, either as a single agent or in combination with chemotherapy agents. Development of TRAIL-resistant clones in the originally TRAIL-sensitive tumor cell population may be a critical complication of TRAIL based cancer therapy. Prolonged exposure of TRAIL-sensitive leukemia cell line, “wild-type” (WT) HL60 cells, to recombinant soluble TRAIL resulted in the establishment of resistant subclones. To get deeper insight into the molecular mechanism of acquired TRAIL resistance we compared expression profiles of TRAIL-sensitive HL-60 cells and TRAIL-resistant HL-60 subclones using microarray mRNA gene expression chips and 2D-PAGE followed by MS and MS/MS analysis. Using cluster analysis of microarray mRNA gene expression profiles of 6 individual HL60 resistant subclones we identified two molecular signatures of TRAIL resistance. When compared to WT HL60 TRAIL-sensitive mRNA expression the group of HL60 subclones designed as phenotype 1 (P1, n=3) had 1122 significantly upregulated and 985 significantly downregulated mRNAs and subclones designed as phenotype 2 (P2, n=3) had 1583 significantly upregulated and 1798 significantly downregulated mRNAs (p<0.05). The subclones of P1 and P2 phenotype shared 533 upregulated and 422 downregulated genes compared to HL60 WT cells. To identify differentially expressed proteins we performed 2D-PAGE of two representative subclones of P1 and P2 phenotypes (in five replicates) and compared them with HL60 WT cells. We detected and identified differentially expressed proteins involved in various aspects of cellular metabolism. Except for downregulation of chromobox protein homolog 5, a protein involved in the regulation of gene expression, and downregulation of Annexin A6 and protein disulfide-isomerase A3 precursor the TRAIL-resistant P1 and P2 subclones showed different protein expression profile. In P1 TRAIL-resistant cells we identified downregulation of proteins involved in energy metabolism, including pyruvate kinase, enolase, cytochrome c-reductase, NADH dehydrogenase and downregulation of proteins involved in proteasome activity, such as proteasome activator complex subunits 1 and 2. In P2 TRAIL-resistant cells we detected downregulation of important regulatory proteins, such as DNA replication licensing factor MCM7, proliferation-associated protein 2G4, replication protein A 32 kDa subunit. In both, P1 and P2, subclones we identified significant changes in cytoskeletal rearrangement proteins. In addition, several chaperones were differentially expressed, namely protein-disulfide isomerase, GrpE, HSC71 and HSP27, 78 kDa glucose-regulated protein precursor. Our data show two HL60 cell line-derived TRAIL resistant subclones, P1 and P2, have specific molecular signatures suggesting distinct mechanisms of TRAIL resistance. These distinct pathways for development of resistance to TRAIL-induced apoptosis are relevant for design of more effective strategies for leukemia therapy.
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van ’t Veer, Cornelis, Joris Roelofs, Bruce Gerlitz, Brian Grinnell, Marcel Levi, Tom der Poll, and Marcel Schouten. "Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia." Thrombosis and Haemostasis 106, no. 12 (2011): 1189–96. http://dx.doi.org/10.1160/th11-06-0438.
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SummaryRecombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associated with a better therapeutic response as compared to later treatment. In a mouse model it was recently confirmed that recombinant murine (rm-)APC decreases coagulation activation and improves survival in pneumococcal pneumonia; however, APC did not impact on the inflammatory response. The aim of this study was to determine the effect of APC treatment instigated early in infection on activation of coagulation and inflammation after induction of pneumococcal pneumonia. Mice were infected intranasally with viable S. pneumoniae. Mice were treated with rm-APC (125 μg) or vehicle intraperitoneally 12 hours after infection and were sacrificed after 20 hours, after which blood and organs were harvested for determination of bacterial outgrowth, coagulation activation and inflammatory markers. In this early treatment model, rm-APC treatment inhibited pulmonary and systemic activation of coagulation as reflected by lower levels of throm-bin-antithrombin complexes and D-dimer. Moreover, rm-APC reduced the levels of a large number of cytokines and chemokines in the lung. When administered early in pneumococcal pneumonia, rm-APC inhibits systemic and pulmonary activation of coagulation and moreover exerts various anti-inflammatory effects in the lung.
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Fumian, Tulio M., Daniel Enosi Tuipulotu, Natalie E. Netzler, Jennifer H. Lun, Alice G. Russo, Grace J. H. Yan, and Peter A. White. "Potential Therapeutic Agents for Feline Calicivirus Infection." Viruses 10, no. 8 (August 16, 2018): 433. http://dx.doi.org/10.3390/v10080433.
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Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 μM and 2.7 μM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2′-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 μM and 2.5 μM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.
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Báez-Astúa, Andrés, Elsa Herráez-Hernández, Natalio Garbi, Hilda A. Pasolli, Victoria Juárez, Harald zur Hausen, and Angel Cid-Arregui. "Low-Dose Adenovirus Vaccine Encoding Chimeric Hepatitis B Virus Surface Antigen-Human Papillomavirus Type 16 E7 Proteins Induces Enhanced E7-Specific Antibody and Cytotoxic T-Cell Responses." Journal of Virology 79, no. 20 (October 15, 2005): 12807–17. http://dx.doi.org/10.1128/jvi.79.20.12807-12817.2005.
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ABSTRACT Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (106 infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8+-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein.
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Pérez, Yolanda, Roman Bonet, Miriam Corredor, Cecilia Domingo, Alejandra Moure, Àngel Messeguer, Jordi Bujons, and Ignacio Alfonso. "Semaphorin 3A—Glycosaminoglycans Interaction as Therapeutic Target for Axonal Regeneration." Pharmaceuticals 14, no. 9 (September 7, 2021): 906. http://dx.doi.org/10.3390/ph14090906.
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Semaphorin 3A (Sema3A) is a cell-secreted protein that participates in the axonal guidance pathways. Sema3A acts as a canonical repulsive axon guidance molecule, inhibiting CNS regenerative axonal growth and propagation. Therefore, interfering with Sema3A signaling is proposed as a therapeutic target for achieving functional recovery after CNS injuries. It has been shown that Sema3A adheres to the proteoglycan component of the extracellular matrix (ECM) and selectively binds to heparin and chondroitin sulfate-E (CS-E) glycosaminoglycans (GAGs). We hypothesize that the biologically relevant interaction between Sema3A and GAGs takes place at Sema3A C-terminal polybasic region (SCT). The aims of this study were to characterize the interaction of the whole Sema3A C-terminal polybasic region (Sema3A 725–771) with GAGs and to investigate the disruption of this interaction by small molecules. Recombinant Sema3A basic domain was produced and we used a combination of biophysical techniques (NMR, SPR, and heparin affinity chromatography) to gain insight into the interaction of the Sema3A C-terminal domain with GAGs. The results demonstrate that SCT is an intrinsically disordered region, which confirms that SCT binds to GAGs and helps to identify the specific residues involved in the interaction. NMR studies, supported by molecular dynamics simulations, show that a new peptoid molecule (CSIC02) may disrupt the interaction between SCT and heparin. Our structural study paves the way toward the design of new molecules targeting these protein–GAG interactions with potential therapeutic applications.
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Yang, Darong, Xianghe Meng, Qinqin Yu, Li Xu, Ying Long, Bin Liu, Xiaohong Fang, and Haizhen Zhu. "Inhibition of Hepatitis C Virus Infection by DNA Aptamer against Envelope Protein." Antimicrobial Agents and Chemotherapy 57, no. 10 (July 22, 2013): 4937–44. http://dx.doi.org/10.1128/aac.00897-13.
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ABSTRACTHepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients.
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Jiang, Jie, Daryl Cole, Nigel Westwood, Lee Macpherson, Farzin Farzaneh, Ghulam J. Mufti, Mahvash Tavassoli, and Joop Gaken. "Apoptin Induced Tumour Specific Killing Via Protein Kinase C Isoforms: A Potential Therapeutic Strategy In Plasma Cell Dyscrasias." Blood 116, no. 21 (November 19, 2010): 5039. http://dx.doi.org/10.1182/blood.v116.21.5039.5039.
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Abstract Abstract 5039 There is mounting evidence that malignant cells have an intrinsic ability to prevent apoptosis. In the present study we provide evidence that the ectopic expression of Apoptin can restore the failing apoptosis program in myeloma cells via protein kinase C b (PKCb) and overcome intrinsic or acquired resistance to cell death. Apoptin (VP3), a chicken anemia virus (CAV)-derived protein has been shown to possess tumor specific cytotoxicity; its expression induces apoptosis in human tumor and transformed cells but there is little or no cytotoxic effect in normal human cells or cell lines derived from different tissues including peripheral blood mononuclear cells, fibroblast and epithelial cells. Several studies have shown that the tumor specific killing of Apoptin correlates with its phosphorylation and its subcellular localization. In cancer cells, Apoptin is localized in the nucleus and is phosphorylated on threonine108 by an as yet unknown kinase, whereas in normal cells Apoptin is detected in the cytoplasm and is essentially unphosphorylated. We developed a lentiviral vector encoding a GFP-Apoptin fusion gene (LV-GFP-AP), which delivers the Apoptin gene efficiently to haematopoietic cells. Apoptin significantly and selectively killed a number of leukemia cell lines including K562, HL60, U937, KG1 and NB4. In particular, the dexamethasone resistant multiple myeloma cell line MM1.R and the dexamethasone sensitive cell line MM1.S were efficiently killed by Apoptin. In contrast normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, we showed that the dexamethasone resistant MM1.R cells were considerably more susceptible to Apoptin induced cell death than the parental matched MM1.S cells. This correlated with increased phosphorylation and activation of the Apoptin protein in MM1.R cells. Expression profiling of MM1.R and MM1.S cells identified a number of differentially expressed kinases. PKCb was over-expressed 9 fold in MM1.R cells and we showed, by immunoprecipitation and in vivo kinase studies, that this kinase was responsible for Apoptin phosphorylation. Analysis of the Apoptin amino acid sequence for potential phosphorylation sites indicated seven putative phosphorylation sites corresponding to the PKC kinase consensus motifs (S/TXK/R or S/TXXK/R). These sites included Thr-108, which has been previously shown to be phosphorylated in tumor cells, but not in normal cells. In vitro studies showed that recombinant Apoptin protein was phosphorylated by recombinant GST-PKCb protein at the Thr-108 site. Addition of a PKCb specific inhibitor resulted in diminished Apoptin phosphorylation whilst an unrelated inhibitor had no such effect. Furthermore, shRNA knockdown or drug mediated inhibition of PKCb in vivo significantly reduced Apoptin phosphorylation. Finally, we found that Apoptin mediated cell death proceeded via the up-regulation of PKCb, activation of caspase-9/3, cleavage of the PKCd catalytic domain and down-regulation of MERTK and AKT protein kinases. Collectively these results demonstrate a novel pathway for Apoptin activation involving PKCb and PKCd. Our results show that Apoptin is able to effectively eliminate multiple myeloma cells which have become resistant to dexamethasone. In addition, this study has led to the identification of tumor specific cellular targets such as PKCb, whose modulation by shRNAs and small molecule drugs can induce strong anti-myeloma effects. Importantly, the evidence from our data suggests that protein kinase C inhibitors may have an important therapeutic role in plasma cell neoplasia. Disclosures: No relevant conflicts of interest to declare.