The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Kirsten Sandvig - One of the best experts on this subject based on the ideXlab platform.
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intracellular transport and cytotoxicity of the protein toxin Ricin
Toxins, 2019Co-Authors: Natalia Sowarogozinska, Kirsten Sandvig, Hanna Sominka, Jowita Nowakowskagolacka, Monika SlominskawojewodzkaAbstract:Ricin can be isolated from the seeds of the castor bean plant (Ricinus communis). It belongs to the ribosome-inactivating protein (RIP) family of toxins classified as a bio-threat agent due to its high toxicity, stability and availability. Ricin is a typical A-B toxin consisting of a single enzymatic A subunit (RTA) and a binding B subunit (RTB) joined by a single disulfide bond. RTA possesses an RNA N-glycosidase activity; it cleaves ribosomal RNA leading to the inhibition of protein synthesis. However, the mechanism of Ricin-mediated cell death is quite complex, as a growing number of studies demonstrate that the inhibition of protein synthesis is not always correlated with long term Ricin toxicity. To exert its cytotoxic effect, Ricin A-chain has to be transported to the cytosol of the host cell. This translocation is preceded by endocytic uptake of the toxin and retrograde traffic through the trans-Golgi network (TGN) and the endoplasmic reticulum (ER). In this article, we describe intracellular trafficking of Ricin with particular emphasis on host cell factors that facilitate this transport and contribute to Ricin cytotoxicity in mammalian and yeast cells. The current understanding of the mechanisms of Ricin-mediated cell death is discussed as well. We also comment on recent reports presenting medical applications for Ricin and progress associated with the development of vaccines against this toxin.
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Ricin and Ricin-Containing Immunotoxins: Insights into Intracellular Transport and Mechanism of action in Vitro
Antibodies, 2013Co-Authors: Monika Słomińska-wojewódzka, Kirsten SandvigAbstract:Ricin is a type II ribosome inactivating protein (RIP) isolated from castor beans. Its high toxicity classifies it as a possible biological weapon. On the other hand, Ricin linked to specific monoclonal antibodies or used in other conjugates has powerful medical applications. Ricin consists of an A-chain (RTA) that damages ribosomes and inhibits protein synthesis, and a B-chain that plays a role in binding and cellular uptake. A number of recent studies have demonstrated that Ricin-induced inhibition of protein synthesis is not the only mechanism responsible for cell death. It turns out that Ricin is able to induce apoptosis in different cell lines and multiple organs in animals. However, the molecular link between protein synthesis inhibition and Ricin-dependent triggering of apoptotic cell death is unclear. This review describes the intracellular transport of Ricin and Ricin-based immunotoxins and their mechanism of action in different non-malignant and cancer cell lines. Moreover, various Ricin-containing immunotoxins, their composition, medical applications and side-effects will be described and discussed. Understanding the mechanism of action of Ricin-based immunotoxins will facilitate construction of effectively acting immunotoxins that can be used in the clinic for cancer treatment
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Role of lipids in the retrograde pathway of Ricin intoxication.
Traffic (Copenhagen Denmark), 2003Co-Authors: Bjørn Spilsberg, Gerrit Van Meer, Kirsten SandvigAbstract:The plant toxin Ricin binds to both glycosphingolipids and glycoproteins with terminal galactose and is transported to the Golgi apparatus in a cholesterol-dependent manner. To explore the question of whether glycosphingolipid binding of Ricin or glycosphingolipid synthesis is essential for transport of Ricin from the plasma membrane to the Golgi apparatus, retrogradely to the endoplasmic reticulum or for translocation of the toxin to the cytosol, we have investigated the effect of Ricin and the intracellular transport of this toxin in a glycosphingolipid-deficient mouse melanoma cell line (GM95), in the same cell line transfected with ceramide glucosyltransferase to restore glycosphingolipid synthesis (GM95-CGlcT-KKVK) and in the parental cell line (MEB4). Ricin transport to the Golgi apparatus was monitored by quantifying sulfation of a modified Ricin molecule, and toxicity was studied by measuring protein synthesis. The data reveal that Ricin is transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum and translocated to the cytosol equally well and apparently at the same rate in cells with and without glycosphingolipids. Importantly cholesterol depletion reduced endosome to Golgi transport of Ricin even in cells without glycosphingolipids, demonstrating that cholesterol is required for Golgi transport of Ricin bound to glycoproteins. The rate of retrograde transport of Ricin was increased strongly by monensin and the lag time for intoxication was reduced both in cells with and in those without glycosphingolipids. In conclusion, neither glycosphingolipid synthesis nor binding of Ricin to glycosphingolipids is essential for cholesterol-dependent retrograde transport of Ricin. Binding of Ricin to glycoproteins is sufficient for all transport steps required for Ricin intoxication.
George P. Anderson - One of the best experts on this subject based on the ideXlab platform.
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ultrasensitive detection of Ricin toxin in multiple sample matrixes using single domain antibodies
Analytical Chemistry, 2015Co-Authors: Shonda T Gaylord, Ellen R. Goldman, George P. Anderson, Trinh L Dinh, Kevin C Ngan, David R WaltAbstract:Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although Ricin intoxication is not transmittable from person to person, even a single Ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for Ricin intoxication, ultrasensitive analytical assays capable of detecting Ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of Ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of Ricin toxin. The Simoa was able to detect Ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using ...
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Binding Kinetics of AntiRicin Single Domain Antibodies and Improved Detection Using a B Chain Specific Binder
Analytical Chemistry, 2010Co-Authors: George P. Anderson, Rachael D. Bernstein, Marla D. Swain, Dan Zabetakis, Ellen R. GoldmanAbstract:Single domain antibodies are the recombinantly expressed binding fragments derived from heavy chain antibodies found in camels and llamas. These unique binding elements offer many desirable properties such as their small size (∼15 kDa) and thermal stability, which makes them attractive alternatives to conventional monoclonal antibodies. We created a phage display library from llamas immunized with Ricin toxoid and selected a number of single domain antibodies. Phage selected on Ricin were found to bind to either Ricin A chain or the intact molecule; no Ricin B chain binders were identified. By panning on B chain, we identified binders and have characterized their binding to the Ricin B chain. While they have a poorer affinity than the previously described A chain binders, it was found that they performed dramatically better as capture reagents for the detection of Ricin, providing a limit of detection in enzyme linked immunosorbent assay (ELISA) below 100 pg/mL and excellent specificity for Ricin versus t...
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Fiber optic-based biosensor for Ricin.
Biosensors and Bioelectronics, 1997Co-Authors: Upvan Narang, George P. Anderson, Frances S. Ligler, James BuransAbstract:We report on an evanescent wave fiber-optic biosensor for detecting a potently toxic protein, Ricin, in the picograms per milliliter range. A sandwich immunoassay scheme was used to detect Ricin. First, an anti-Ricin IgG was immobilized onto the surface of an optical fiber in two different ways. In the first method, the antibody was directly coated to the silanized fiber using a crosslinker. Second, avidin-coated fibers were incubated with biotinylated anti-Ricin IgG to immobilize the antibody using an avidin-biotin bridge. The assay using the avidin-biotin linked antibody demonstrated higher sensitivity and wider linear dynamic range than the assay using antibody directly conjugated to the surface. The linear dynamic range of detection for Ricin in buffer using the avidin-biotin chemistry is 100 pg/ml-250 ng/ml. The limits of detection for Ricin in buffer solution and river water are 100 pg/ml and 1 ng/ml, respectively. At higher concentrations of Ricin (> 50 ng/ml), we observe a strong interaction of Ricin with the avidin coated on the surface of the fibers. We have demonstrated that this interaction is primarily due to the lectin activity of Ricin and is significantly reduced using fibers coated with neutravidin or by adding galactose to the Ricin samples.
Ellen R. Goldman - One of the best experts on this subject based on the ideXlab platform.
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ultrasensitive detection of Ricin toxin in multiple sample matrixes using single domain antibodies
Analytical Chemistry, 2015Co-Authors: Shonda T Gaylord, Ellen R. Goldman, George P. Anderson, Trinh L Dinh, Kevin C Ngan, David R WaltAbstract:Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although Ricin intoxication is not transmittable from person to person, even a single Ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for Ricin intoxication, ultrasensitive analytical assays capable of detecting Ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of Ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of Ricin toxin. The Simoa was able to detect Ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using ...
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Binding Kinetics of AntiRicin Single Domain Antibodies and Improved Detection Using a B Chain Specific Binder
Analytical Chemistry, 2010Co-Authors: George P. Anderson, Rachael D. Bernstein, Marla D. Swain, Dan Zabetakis, Ellen R. GoldmanAbstract:Single domain antibodies are the recombinantly expressed binding fragments derived from heavy chain antibodies found in camels and llamas. These unique binding elements offer many desirable properties such as their small size (∼15 kDa) and thermal stability, which makes them attractive alternatives to conventional monoclonal antibodies. We created a phage display library from llamas immunized with Ricin toxoid and selected a number of single domain antibodies. Phage selected on Ricin were found to bind to either Ricin A chain or the intact molecule; no Ricin B chain binders were identified. By panning on B chain, we identified binders and have characterized their binding to the Ricin B chain. While they have a poorer affinity than the previously described A chain binders, it was found that they performed dramatically better as capture reagents for the detection of Ricin, providing a limit of detection in enzyme linked immunosorbent assay (ELISA) below 100 pg/mL and excellent specificity for Ricin versus t...
David R Walt - One of the best experts on this subject based on the ideXlab platform.
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ultrasensitive detection of Ricin toxin in multiple sample matrixes using single domain antibodies
Analytical Chemistry, 2015Co-Authors: Shonda T Gaylord, Ellen R. Goldman, George P. Anderson, Trinh L Dinh, Kevin C Ngan, David R WaltAbstract:Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although Ricin intoxication is not transmittable from person to person, even a single Ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for Ricin intoxication, ultrasensitive analytical assays capable of detecting Ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of Ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of Ricin toxin. The Simoa was able to detect Ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using ...
Brigitte G. Dorner - One of the best experts on this subject based on the ideXlab platform.
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Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.
Toxins, 2021Co-Authors: Maria Lucia Orsini Delgado, Arnaud Avril, Julie Prigent, Julie Dano, Audrey Rouaix, Sylvia Worbs, Brigitte G. Dorner, Clémence Rougeaux, François Becher, François FenailleAbstract:Ricin, a highly toxic protein from Ricinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different Ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti Ricin A-chain) and RB34 and RB37 (anti Ricin B-chain)) against Ricin D. In this study, we evaluated their protective capacity against both Ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively Ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with Ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different Ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.
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characterization of Ricin and r communis agglutinin reference materials
Toxins, 2015Co-Authors: Sylvia Worbs, Martin Skiba, Martin Soderstrom, Marjaleena Rapinoja, Reinhard Zeleny, Heiko Russmann, Heinz Schimmel, Paula Vanninen, Stenake Fredriksson, Brigitte G. DornerAbstract:Ricinus communis intoxications have been known for centuries and were attributed to the toxic protein Ricin. Due to its toxicity, availability, ease of preparation, and the lack of medical countermeasures, Ricin attracted interest as a potential biological warfare agent. While different technologies for Ricin analysis have been established, hardly any universally agreed-upon "gold standards" are available. Expert laboratories currently use differently purified in-house materials, making any comparison of accuracy and sensitivity of different methods nearly impossible. Technically challenging is the discrimination of Ricin from R. communis agglutinin (RCA120), a less toxic but highly homologous protein also contained in R. communis. Here, we established both highly pure Ricin and RCA120 reference materials which were extensively characterized by gel electrophoresis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS), and matrix-assisted laser desorption ionization-time of flight approaches as well as immunological and functional techniques. Purity reached >97% for Ricin and >99% for RCA120. Different isoforms of Ricin and RCA120 were identified unambiguously and distinguished by LC-ESI MS/MS. In terms of function, a real-time cytotoxicity assay showed that Ricin is approximately 300-fold more toxic than RCA120. The highly pure Ricin and RCA120 reference materials were used to conduct an international proficiency test.
