A case of accidental Ricin poisoning

A case report of accidental, mild ricin poisoning by ingestion of ten Castor beans by a 70 year old man. The clinical presentation is followed by a discussion about the toxicity of ricin, its use and misuse and the contrast between the toxicity of pure ricin and the ingestion of castor beans. Management of castor bean poisoning is also discussed.peer-reviewe

Ricin B chain targeted to the endoplasmic reticulum of tobacco protoplasts is degraded by a CDC48- and vacuole-independent mechanism

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway

Methods for nanoparticle labeling of ricin and effect on toxicity

The unique optical properties associated with nanostructured materials that support the excitation of surface plasmons offer many new opportunities for the enhanced optical investigation of biological materials that pose a security threat. In particular, ricin is considered a significant bioterrorism risk due to its high toxicity combined with its ready availability as a byproduct in castor oil production. Therefore, the development of optical techniques capable of rapid on-site toxin detection with high molecular specificity and sensitivity continues to be of significant importance. Furthermore, understanding of the ricin cell entry and intracellular pathways remains poor due to a lack of suitable bioanalytical techniques. Initial work aimed at simultaneously tackling both these issues is described where different approaches for the nanoparticle labeling of ricin are investigated along with changes in ricin toxicity associated with the labeling process

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states

A human embryonic kidney 293T cell line mutated at the Golgi -mannosidase II locus

Disruption of Golgi -mannosidase II activity can result in type II congenital dyserythropoietic anemia and can induce lupus-like autoimmunity in mice. Here, we isolate a mutant human embryonic kidney (HEK) 293T cell line, called Lec36, that displays sensitivity to ricin that lies between the parental HEK 293T cells, whose secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which only produce oligomannose-type N-linked glycans. The stem cell marker, 19A, was transiently expressed in the HEK 293T Lec36 cells, and in parental HEK 293T cells with and without the potent Golgi -mannosidase II inhibitor, swainsonine. Negative-ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi -mannosidase II: a point mutation in one allele mapping to the active site and an in-frame deletion of twelve-nucleotides in the other. Expression of wild-type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and provides a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia

A double safety lock tumor-specific device for suicide gene therapy in breast cancer

Producción CientíficaThe complexity and continuous evolution of cancer make the design of novel strategies of treatment a constant challenge in biomedicine. Moreover, most of cancer treatments are still not tumor-specific and provoke high systemic toxicity. Herein we have developed a novel selective nanodevice to eliminate tumor cells while leaving healthy ones intact. To achieve this objective, a polyplex carrier, comprising an elastin like-recombinamer covalently conjugated to an aptamer and complexed with therapeutic DNA, was tested. This carrier forms a double-lock multifunctional device due to specific binding to a tumor cell marker and the selective expression of therapeutic DNA inside human breast-cancer cells. Due to the stability provided by ELRs, the homogeneous population of polyplexes obtained showed selective toxicity against cancer cells in in vitro and in vivo assay. Inhibition of tumor progression was detected early being very significant at the end point, with a dose-dependent reduction in tumor mass. Histological studies revealed a specific reduction in tumor parenchyma and in specific tumor cell markers. These results represent an important step toward the rational development of an efficient, safe and more specialized gene-delivery device for tumor therapy.Fondo Social Europeo - FEDER- Unión Europea (project MP-2014-646075Ministerio de Economía, Industria y Competitividad (project PCIN-2015-010 / MAT2015-68901-R / MAT2016-78903-R)Junta de Castilla y León (project VA317P18

The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. Our results implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states

Retrograde transport pathways utilised by viruses and protein toxins

A model has been presented for retrograde transport of certain toxins and viruses from the cell surface to the ER that suggests an obligatory interaction with a glycolipid receptor at the cell surface. Here we review studies on the ER trafficking cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin, and compare the retrograde routes followed by these protein toxins to those of the ER trafficking SV40 and polyoma viruses. We conclude that there is in fact no obligatory requirement for a glycolipid receptor, nor even with a protein receptor in a lipid-rich environment. Emerging data suggests instead that there is no common pathway utilised for retrograde transport by all of these pathogens, the choice of route being determined by the particular receptor utilised

The isolation and characterisation of temperature-dependent Ricin A chain molecules in Saccharomyces cerevisiae.

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum (ER) membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the ER of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperaturedependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37°C while the same cells did not grow at 23ºC. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23°C but not at 37°C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterisation of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three dimensional structure. The potential utility of these mutants is discussed