Browsing by Subject "Ricin"
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Item Development of castor (Ricinus communis) var. brigham with ultra low ricin content by analyzing soluble seed proteins(2012-08) Wettasinghe, Ruwanthi; Auld, Dick L.; Allen, Randy D.; Fokar, Mohamed; Abidi, Noureddine; Payton, Paxton R.Castor (Ricinus communis L.) is one of the oldest cultivated oil crops in the world. The unique oil produced in castor seeds contains ricinoleic acid, an essential specialty product that is widely used as a raw material in numerous commercial applications. In addition, the pressed cake of oil extraction is a value added by-product of animal feed and organic fertilizer industries. Also, castor seeds contain storage proteins that include ricin, a potent cytotoxin found only in the endosperm of the seed. Following oil extraction, ricin remains in the pressed cake which constitutes up to 5% of the dry matter. Ricin has been categorized as a potential bioterrorism agent by the United States Centers for Disease Control and Prevention hence, the presence of residual toxin in the byproduct hinders its usage as an animal feed and organic fertilizer in commercial applications. The purpose of this study was to identify castor var. Brigham seeds with low ricin content in each seed. This goal was accomplished by partial seed analysis and by image processing of an identified band that was recognized as ricin. Advance biotechnological strategies and tools utilized in this study, incorporated into castor breeding programs, may play a pivotal role in the development of castor var. Brigham with ultra low ricin content. Low ricin castor cultivars would eliminate the cost and time needed in ricin detoxification of pressed cake therefore, contributing to change human perception of the toxicity of castor and providing a global prospective to cultivate castor as an agronomically important crop.Item Extraction of ricin from castor(Texas Tech University, 1999-12) Ammatanda, Muthanna NischalNot availableItem Intradermal Administration of RiVax, a Ricin Vaccine(2010-11-02T18:18:52Z) Selvaduray, Praveena; Vitetta, EllenRicin toxin is a CDC Level B Biothreat due to its extreme toxicity and ease of production. The most effective method for minimizing ricin toxicity in humans is prophylactic vaccination. We have previously described the efficacy and safety of RiVax, a recombinant mutant of ricin A chain (RTA). RiVax has no residual toxicity from either its ribotoxic site or its vascular leak-inducing site. When administered by intramuscular (IM) injection, it was safe and immunogenic in mice, rabbits, and humans. A three dose regimen of IM administered RiVax also protected mice from an LD50dose of ricin delivered by injection, gastric gavage or aerosol. In this study we have attempted to increase the utility and immunogenicity of RiVax. To this end, we have compared intradermal (ID) vs. IM administration of RiVax by evaluating the following parameters of vaccine efficacy: (1) short-term antibody responses and protection of mice from a 10X LD50of ricin following a three dose vaccine regimen; (2) long-term antibody responses and protection of mice from a 10X LD50of ricin following a three dose vaccine regimen; (3) protective effect of a single high dose of RiVax from a 10X LD50dose of ricin; (4) the minimum dose of ricin at which fully vaccinated animals are no longer protected; (5) the rate of antigen trafficking to draining lymph nodes (DLN) following administration of RiVax. In the short term, when RiVax was delivered with alum, very low doses of vaccine administered ID were superior to the same low doses administered IM, with regard to both antibody production and protection against ricin delivered by injection, gavage, or aerosol. Low doses of ID vaccine were also superior in maintaining lung function in mice exposed to aerosolized ricin. Comparing the same parameters in the long term or after a single dose of RiVax, ID and IM vaccinations were equally effective. Both ID and IM vaccination were also similar in their ability to protect mice from a supra-lethal challenge with injected ricin. One possible explanation for the improved efficacy of low doses of RiVax administered ID was that the vaccine trafficked more effectively to the DLNs. This appeared to be the trend, albeit not a statistically significant one. Given the increased efficacy of low doses of ID vaccine in protecting mice against ricin delivered to the lung and gut, we suggest that it should be considered for testing in humans.Item Quantifying ricin in agricultural soils(Texas Tech University, 2002-12) Boroda, EliTo date there are no reports of extensive scientific investigations on the analysis, determination or content of ricin, RCAeo (Ricinus communis agglutinin - 60 kDa) in agricultural soils. To accurately quantify the amount of ricin available within the soil, a modified ELISA (Enzyme Linked Immunosorption Assay) was developed. The ELISA followed an extraction process using an isotonic saline solution as a ricin carrier. The range of ricin detection was between 0.010 ^g ricin/g soil to 0.300 |jg ricin/g soil as contrasted with the previously employed radial immunodiffusion assays with a sensitivity limited tol.O mg ricin/gram soil. Using the modified ELISA method, the ricin content in the surrounding soil of germinating castor seeds planted 0.5 cm deep in 25 g of soil was detennined in Olton and Amarillo soils. These two soils are common agricultural soils in Lubbock, Texas area. Ricin was detected in both soils washed off the seeds of germination days four, six and eight days after seeding. Ricin was not detected in days two or ten after seeding. In addition, ricin was not found in any of the remaining soil. An investigation quantifying ricin within a field (30 meters by 152 meters) that had supported a crop of castor (Ricinus communis) in a previous growing season with an intermediate crop of cotton (Gossypium hirsutum), ricin was detected. In a separate field study, ricin was also only detected during the last December sampling of the same year crop. Soil in this study was the Amarillo fine sandy loam. Half of a castor field (121 meters by 121 meters) had been pivot irrigated. The other half had been furrow irrigated. Ricin detected was significantly less in the furrow irrigated half when compared to the center pivot irrigation. Ricin was also noted in response to a controlled stress trial only when the stress consisted of a combination of saline irrigation water EC (Electroconductivity) of 10 dS/m and drought. No other stresses, such as heat (35.0 °C to 38 °C), saline irrigation or drought, alone resulted in any detectable ricin within the sampled the soil. This data suggests that a combination of growth conditions combined with a specific set of external stress factors and human cultivation methods may play a determining role in the natural deposition ricin in agricultural soil as well as its long-term retention.Item Selection of castor with divergent concentrations of ricin and RCA using radial immunodiffusion(Texas Tech University, 1997-12) Pinkerton, Scott DavisOne of the most limiting factors in the production of castor (Ricinus communis L.) is the presence of two toxic proteins, ricin and RCA. Ricin and RCA's presence in the endosperm of the seed creates potential health hazards to both producers and processors. The objectives of this research were to determine the range of ricin/RCA concentration in the USDA castor collection and to determine the inheritance of ricin/RCA concentrations in segregating populations. The 263 accessions from 34 countries of the USDA collection screened for ricin/RCA concentration ranged from 1.9 to 16.0 mg/g. Only 1% of the accessions had ricin/RCA concentrations less than 3.0 or greater than 13.0 mg/g. In 1994, two accessions from the USDA germplasm collection PI 257 654 and PI 258 368 reported to have low ricin/RCA concentration were crossed with the commercial oil producing cultivar 'Hale'. In 1996, Fg seed were harvested and evaluated to determine inheritance of ricin/RCA concentration using a radial immunodiffusion (RID) assay which utilized ricin specific antibodies. Five of six F^ populations fit a 15:1 (high:low ricin/RCA) phenotypic ratio and five of six F3 populations fit a 55:9 (high:low ricin/RCA) phenotypic ratio. Heterogeneity X^2 for the F^2 and F^3 generations showed that all six segregating populations did not significantly differ in their segregation patterns. Low ricin/RCA content appeared to be controlled by two recessive alleles. Average ricin/RCA concentration of individual Fg plants ranged from 2.1 to 17.9 mg/g and in the F^3 generation ranged from 0.1 to 20.1 mg/g. Use of the RID assay provided an effective method to screen castor lines for divergent concentrations of ricin/RCA. These results will allow development of non-toxic oilseed cultivars and cultivars with extremely high concentrations of ricin/RCA for pharmaceutical production.Item Structure based design of a ricin antidote(2012-12) Jasheway, Karl Richard; Robertus, Jon D.; Hackert, Marvin LRicin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10-4 M range, leaving significant room for improvement. This thesis discusses the development of a subset of inhibitors belonging to the pterin family in which amino acids have been utilized as building blocks. Inhibitors in this family have achieved a significant increase in potency, and have provided valuable structural information for further development.Item Synthesis and optimization of a library of small molecule inhibitors of ricin toxin A(2012-05) Pruet, Jeffrey Michael; Anslyn, Eric V., 1960-Ricin is a potent cyctotoxin with no known antidote. Chapter 1 provides background and context for this thesis, which is primarily focused on probing the active site of Ricin toxin A (RTA). Relevant information about Ricin, its use, method of action, and noteworthy contributions towards the discovery of Ricin A chain inhibitors are provided. Furthermore, a brief description of the assays used by our collaborators to monitor RTA inhibition is provided. Additionally, a great deal of this thesis pertains to a particular heterocycle, pterin, and thus the remainder of Chapter 1 is dedicated to pterins, their physical properties, biological relevance, and selected reports of pterin chemistry. Chapter 2 details preliminary research focused on the use of nucleic acid-based platforms as RTA inhibitors. Two specific nucleic acids were chosen, adenine and guanine, and the chapter is split to address them individually. Rational for their use is provided, as well as the synthetic strategies investigated. Both platforms showed significant interference with the analysis assay, most pronounced for the adenine series. A primary goal throughout this thesis is the identification of a simple, rapid method to provide a library of new compounds. To this end, discussion of improved synthetic routes are provided within the section dedicated to guanines. Initial investigation into pterins as a platform for RTA inhibitors is provided in Chapter 3. Much of this chapter is concerned with hurtles encountered while dealing with the poor solubility of pterins, purification, and limits in reaction scope. Finally this chapter details a significant discovery in pterin's utility, both in terms of synthetic ease and preference towards one regioisomer over another. A variety of amides are initially used to probe the active site for significant interactions to the pterin pendents. Chapter 4 builds off the discoveries detailed within the previous chapter. Efforts to optimize the preliminary amide series from Chapter 3 are described, leading to a significant enhancement in activity. Additionally, Chapter 4 describes a synthetic breakthrough which greatly enhanced the speed of synthesis and complexity of the designed pterin inhibitors. Building upon the goal to map the RTA active site, a description of various peptide conjugated pterins is provided, as well as efforts to arrive at optimized isosteres of the most promising peptide derivatives.