SciCombinator

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Concept: Ricin

174

Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.-Gunning, A. P., Kirby, A. R., Fuell, C., Pin, C., Tailford L. E., Juge, N. Mining the “glycocode”-exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy.

Concepts: DNA, Proteins, Mass, Ricin, Peanut, Faboideae, Glycomics, Ulex europaeus

146

Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant-containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.

Concepts: Immune system, Antibody, Protein, Immunology, Escherichia coli, Castor oil plant, Ricin, Castor oil

28

Bioenergy production combined with phytoremediation has been suggested to help in solving two critical world problems: the gradual reduction of fossil fuels and soil contamination. The aim of this research was to investigate the potential for the use of Ricinus communis L. (castor oil plant) as an energy crop and plant species to remediate metal-polluted sites. This study was performed in mine tailings containing high concentrations of Cu, Zn, Mn, Pb and Cd. Physico-chemical characterization, total, DTPA-extractable and water-soluble metals in rhizospheric tailings heap samples were carried. Metal concentrations in plant tissues and translocation factors (TFs) were also determined. The Ricinus seed-oil content was high between 41 and 64%, seeds from San Francisco site 6 had the highest oil content, while these from site 7 had the lowest. No trend between oil yield vs seed origin site was observed. Seed-oil content was negatively correlated with root concentration of Cu, Zn, Pb and Cd, but no correlation was observed with the extractable-metals. According to its shoot metal concentrations and TFs, castor bean is not a metal accumulator plant. This primary colonizing plant is well suited to cope with the local toxic conditions and can be useful for the stabilization of these residues, and for then decreasing metal bioavailability, dispersion and human health risks on these barren tailings heaps and in the surrounding area. Our work is the first report regarding combined oil production and a phytostabilization role for Ricinus plants in metal mine tailings and may give a new value to suitable metal-polluted areas.

Concepts: Castor oil plant, Ricin, Euphorbiaceae, Acalypheae

27

OBJECTIVE: In immunotherapy of HPV-16-associated cervical cancers, the E7 protein is considered as a prime candidate. However, it is a poor inducer of a cytotoxic T-cell response when used as a singular antigen in protein vaccination. Therefore, to design effective cancer vaccines, the best tumor antigens should be combined with the most effective immunogens or drug delivery tools to achieve positive clinical results. In this study, we fused HPV-16 E7 with the lectin subunit of ricin toxin (RTB) from castor plant as a vaccine adjuvant/carrier. MATERIALS AND METHODS: After reaching the soluble form of the recombinant protein, we designed 2 preventive and inhibition tumor models for investigation of the prevention and rejection of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-RTB or E7 without any adjuvant. RESULTS: We demonstrated that prophylactic immunization of E7-RTB protected mice against challenge from TC-1 cells. Also in the therapeutic model, E7-RTB could inhibit TC-1 tumor growth in the lung. The results were significant compared with the immunization of E7 singularly. CONCLUSIONS: We concluded that immunization with E7-RTB protein without any adjuvant could generate antitumor effects in mice challenged with TC-1 cells. This research verifies the clinical applications and the future prospects for development of HPV-16 E7 therapeutic vaccines fused to immunoadjuvants.

Concepts: Immune system, HPV vaccine, Vaccine, Vaccination, Immunology, Adaptive immune system, Antigen, Ricin

2

In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism, seed storage proteins, toxins and allergens. Additionally, we have used an off-line Hydrophilic Interaction Chromatography (HILIC) as a step of peptide fractionation preceding the Reverse Phase nanoLC coupled with a LTQ Orbitrap. We were able to identify a total of 1875 proteins and from these 1748 could be mapped to extant castor gene models, expanding considerably the number proteins so far identified from developing castor seeds. Cluster validation and statistical analysis resulted in 975 protein trend patterns and the relative abundance of 618 proteins. The results presented in this work give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism and catabolism of fatty acid and the pattern of deposition of reserve proteins, toxins and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of reserve proteins that are differentially expressed during seed development.

Concepts: Protein, Gene, Amino acid, Metabolism, Nutrition, Fat, Castor oil plant, Ricin

1

Context. Ingestions of the seed of the castor bean plant (Ricinus communis) carries the risk of toxicity from ricin, a potent inhibitor of protein synthesis. Objective. We sought to describe characteristics of castor bean seed exposures reported to a state-wide poison control system. Methods. This was an observational case series. A state-wide poison control system’s database was reviewed for exposures to castor bean plant seeds from 2001 to 2011. Case notes were reviewed and data collected, when available, included age, gender, circumstances surrounding exposure, number of castor beans consumed, whether beans were chewed or crushed, symptoms described, laboratory values (aspartate aminotransferase [AST], alanine aminotransferase [ALT], prothrombin time [PT] and international normalized ratio [INR]), duration of follow-up, treatment, and patient outcomes. Results. Eighty-four cases were identified. Ingestions were unintentional in 50 cases (59%) cases and intentional in 34 (40%) cases. A median of 10 seeds (range: 1-20) were ingested in intentional cases versus 1 seed (range: 1-40) in unintentional cases. In 49 (58%) of cases the seeds were reported to have been chewed or crushed. Gastrointestinal symptoms were the most commonly reported symptoms. Vomiting (n = 39), nausea (n = 24), diarrhea (n = 17), and abdominal pain (n = 16) predominated. One patient developed hematochezia and vomiting after reportedly ingesting and intravenously injecting castor bean seeds. Laboratory values were documented in 17 (20%) cases. Only one abnormality was noted; an asymptomatic patient one week following ingestion had AST/ALT of 93 U/L and 164 U/L, respectively. Ricinine was confirmed in the urine of two patients. Twenty-three (27%) cases received activated charcoal. Seventy-two (86%) of cases were calls from health care facilities or referred to health care facilities by the poison control center. Twenty-two (26%) cases were admitted for a median of 2 days (range: 1-10). Admitted cases ingested a median of 8.5 seeds (range: 1-20). Intentional ingestions were followed for median of 37.5 h (range: 0.5-285.5) while unintentional cases were followed for 14 h (range: 1-182). No delayed symptoms, serious outcomes, or deaths were reported. Discussion. Due to the presence of ricin, there is concern for serious outcomes after ingestions of the seeds of the castor bean plant. In this study GI symptoms were most commonly reported but serious morbidity or mortality was not present. The true risk of castor bean plant seed ingestions should continue to be re-evaluated. Conclusion. In this retrospective review, gastrointestinal symptoms were the most common symptoms described after reported exposures to castor bean seeds. These exposures were not associated with serious morbidity, mortality, or delayed symptoms.

Concepts: Ingestion, Alanine transaminase, Seed, Castor oil plant, Ricin, Euphorbiaceae, Acalypheae, Bean

0

The sucrose synthase (SUS) interactome of developing castor oilseeds (COS; Ricinus communis) was assessed using coimmunoprecipitation (co-IP) with anti-(COS RcSUS1)-IgG followed by proteomic analysis. A 41-kDa polypeptide (p41) that coimmunoprecipitated with RcSUS1 from COS extracts was identified as reversibly glycosylated polypeptide-1 (RcRGP1) by LC-MS/MS and anti-RcRGP1 immunoblotting. Reciprocal Far-Western immunodot blotting corroborated the specific interaction between RcSUS1 and RcRGP1. Co-IP using anti-(COS RcSUS1)-IgG and clarified extracts from other developing seeds as well as cluster (proteoid) roots of white lupin and harsh hakea consistently recovered 90 kDa SUS polypeptides along with p41/RGP as a SUS-interactor. The results suggest that SUS interacts with RGP in diverse sink tissues to channel UDP-glucose derived from imported sucrose into hemicellulose and/or glycoprotein/glycolipid biosynthesis. This article is protected by copyright. All rights reserved.

Concepts: Protein, Amino acid, Castor oil plant, Ricin, Euphorbiaceae, Acalypheae, All rights reserved, Copyright

0

Ricin is a highly toxic ribosome-inactivating lectin occurring in the seeds of castor bean (Ricinus communis L.). Castor bean grows throughout tropical and sub-tropical regions and is a very important crop due to its high seed content of ricinoleic acid, an unusual fatty acid, which has several industrial applications. However, due to the presence of the toxin, castor bean can cause death after the exposure of animals to low doses of ricin through skin contact, injection, inhalation or oral routes. Aiming to generate a detoxified genotype, we explored the RNAi concept in order to silence the ricin coding genes in the endosperm of castor bean seeds. Results indicated that ricin genes were effectively silenced in genetically modified (GM) plants, and ricin proteins were not detected by ELISA. Hemagglutination activity was not observed with proteins isolated from GM seeds. In addition, we demonstrated that seed proteins from GM plants were not toxic to rat intestine epithelial cells or to Swiss Webster mice. After oil extraction, bio-detoxified castor bean cake, which is very rich in valuable proteins, can be used for animal feeding. Gene silencing would make castor bean cultivation safer for farmers, industrial workers and society.

Concepts: DNA, Protein, Gene, Genetics, RNA, Seed, Castor oil plant, Ricin

0

Ricin, a highly toxic protein, is considered as a potential biowarfare agent and homicidal weapon. Therefore, fast precaution of potential ricin toxin plays an important role in national security and public safety. Herein, a simple, sensitive and accu-rate visual detection of active ricin in complex samples is presented by combining magnetic affinity enrichment with a spe-cific AuNP probe. On one hand, a dual-recognition magnetic absorbent was fabricated by simultaneously incorporating two different affinity ligands (Concanavalin A and galactosamine) on low-foul polymer brushes grafted magnetic beads, which show stronger multivalent synergy binding capacity for ricin even under complex interfering environments. On the other hand, a homo-adenine-constituted oligodeoxynucleotide named poly(21dA) was conjugated to AuNPs (the poly(21dA)-AuNPs), which could serve as a specific depurination substrate of active ricin. Based on the fact that coralyne could trigger the intact poly(21dA)-AuNPs aggregate by forming non-Watson-Crick homo-adenine/coralyne complex, but the poly(21dA)-AuNPs after reacting with active ricin might fail to form this complex due to the loss of adenines, a sensitive visual assay for active ricin was developed. By adopting this “two-step” strategy, we could detect active ricin as low as 10 ng mL-1 with the naked eyes or 3.8 ng mL-1 aided by a UV/vis spectrometer. This stratagy could well apply in various ricin-spiked complex matrices. The features of this strategy such as readily operation, facile readout and easy accessibility make it a better choice for fast on-field active ricin detection.

Concepts: Ricin, Lectin

0

Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these concerns, there are no licensed therapies available for treating ricin intoxication. Here, we describe the development of a F(ab')₂ polyclonal ovine antitoxin against ricin and demonstrate the efficacy of a single, post-exposure, administration in an in vivo murine model of intoxication against aerosolised ricin. We found that a single dose of antitoxin afforded a wide window of opportunity for effective treatment with 100% protection observed in mice challenged with aerosolised ricin when given 24 h after exposure to the toxin and 75% protection when given at 30 h. Treated mice had reduced weight loss and clinical signs of intoxication compared to the untreated control group. Finally, using imaging flow cytometry, it was found that both cellular uptake and intracellular trafficking of ricin toxin to the Golgi apparatus was reduced in the presence of the antitoxin suggesting both actions can contribute to the therapeutic mechanism of a polyclonal antitoxin. Collectively, the research highlights the significant potential of the ovine F(ab')₂ antitoxin as a treatment for ricin intoxication.

Concepts: Medicine, Cell, Golgi apparatus, Organelle, Flow cytometry, Castor oil plant, Ricin, Castor oil