Ricin blocking

Lectin toxicity - nausea, diarrhea, headache, confusion, dehydration, death Wisteria, castor bean (Ricinus communis) Lectins bind to cell surfaces Ricin - blocks protein synthesis, very toxic, 5 to 6 beans can kill a child... 

Complete elucidation of the mechanisms by which ricin kills target cells remains an area of active study, but it is clear that the A -glycosidase activity of RTA is the essential triggering event. Inhibition of protein synthesis precedes other detectable alterations in target cell biochemistry. Ricin blocks amino acid incorporation in cmde microsome preparations before changes occur in energy metabohsm or oxidative phosphorylation the toxin has essentially no effect on mitochondrial respiration in isolated mitochondria or tumor cells (Waller et al., 1966 Dirheimer et al., 1968 Lin et al., 1971). Likewise, the first observable cytotoxic effect of ricin in cell culture is typically the inhibition of protein synthesis, followed by a reduction in DNA synthesis (Lin et al., 1970 Lin et al., 1971 Onozaki et al., 1972 Refsnes et al., 1974 Nicolson et al., 1975 Olsnes et al., 1976 Refsnes et al., 1977). 

The ricin and abnn A chains used to block high affinity binding sites on the column should be alkylated to remove the free sulfhydryl group that could interfere with conjugate purification... 

The natural chemical ricin, which occurs in the castor bean, is a deadly poison and it takes only one molecule to kill a cell. Its first action on encountering a cell is to bind to a carbohydrate on the cell surface and wait. It doesn t have to wait for long before the cell decides to investigate and takes the ricin molecule inside it thereby seals its fate. The ricin migrates to the only site in the cell where proteins are made and once there it blocks it. Eventual cell death is ensured. The deadly toxin released by E.coli Oiff. Hj plays a similar trick to gain access to cells. Viruses also dock onto external carbohydrates and the influenza virus is particularly good at doing this. But what Nature does, humans can also do, and we can do it to defeat these natural enemies. 

The primary clinical targets of immunotoxins are tumors, based on the principle that the MAb will target the toxin to the tumor cells and the highly toxic moiety will then kill the cancer cells. Examples of toxins are ricin, diphtheria toxin and abrin, which are all glycoproteins. Their toxicity is based on their ability to block protein synthesis at the ribosomal protein assembly site. They are normally extremely toxic and not suitable for therapeutic purposes because they induce liver and vascular toxicity, even at low dose levels. 

Ricin toxin consists of two protein moieties connected by a disulphide bridge. Chain A (Mw 32 kD) blocks the ribosomal activity, and chain B (Mw 34 kD) is responsible for cell entry of the A chain. 

Fidias P, Grossbard ML, Lynch T. A phase II study of the immunotoxin N901-blocked ricin in small-cell lung cancer. Clin Lung Cancer 2002 3 219-22. 

The toxic ricin is a small protein molecule consisting of two parts, chains A and B. The B chain is similar to proteins called lectins which recognize and bind to the membranes surrounding the cells in our bodies. The B chain attaches the ricin to the cell membrane which then folds inwards so that the ricin molecule is taken inside the cell inside a bag called a vacuole. There is only one bond between the A and B chains and this now breaks. The B chain then makes a hole in the vacuole through which the A chain passes into the cell. Here it heads straight for structures called ribosomes, where proteins, many of which are vital for the functioning of our bodies, are made. The A chain then selectively removes a specific molecule (the base adenine) from the RNA in the ribosomes. RNA contains the information required to make proteins, and removal of part of the information blocks the synthesis of proteins. The cell therefore dies. One molecule of ricin may be sufficient to kill one cell. This makes it the most potent toxin known. 

The mechanisms of toxic action of abrin and ricin are similar. The B-chain attains cell recognition and binding function to facilitate toxin transport across the cell membrane, whereas the A-chain, once internalized by the cell, blocks protein synthesis by catalytically modifying the ribosomes. Both toxins ultimately kill target cells in animal or cell culture models by both necrosis and apoptosis. 

The Identification and Isolation of Reactive Thiols in Ricin A-Chain and Blocked Ricin Using 2-(4 -Maleimidylanilino)naphthalene-6-sulfonic... 

The identification of reactive or chemically modified residues of proteins is often extremely important for the characterization of proteins and their activity. Peptide mapping in conjunction with Edman sequencing and/or mass spectrophotometric analysis has been the method of choice to accomplish this characterization. However, this approach alone may not be sufficient or optimal for every situation as was the case when trying to identify the affinity ligand attachment sites on the B-chain of blocked ricin (Lambert et al., 1991a). 

Extinction coefficients used for ricin A-chain and blocked ricin B-chain at 280 nm for 0.1% solutions were 0.765 and 1.48, respectively (Olsnes and Pihl, 1973). The extinction coefficient for MIANS is 20,000 M cm" at 322 nm (Gupte and Lane, 1979). The empirically determined contribution of MIANS to the absorbance of the labeled protein at 280 nm was 0.9 x A320. 

Abbreviations MIANS, 2-(4 -maleimidylanilino)naphthalene-6-sulfonic acid PBS, phosphate buffered saline 20 mM sodium phosphate containing 150 mM sodium chloride, pH 7.2 EDTA, ethylene diamine tetraacetic acid DTT, dithiothreitol GuHCl, guanidine hydrochloride 2-ME, 2-mercaptoethanol RB2L-MIANS, ricin B-chain covalently blocked by two affinity ligands and MIANS labeled TEA, Trifluoroacetic Acid. 

Identification of A-Chain and Blocked Ricin Using MIANS 247... 

The reduction of ricin A-chain yielded 0.96 mole SH/mole protein. Following the labeling step, the ratio of MIANS/A-chain was 0.70 as determined by measurement of absorbance at 280 nm and 320 nm. The reduction of blocked ricin yielded 1.3 mole SH/mole protein. Following labeling and separation of the blocked B-chain, the ratio of MIANS/blocked B-chain was 0.97. 

How does toxalbumin relate to ricin Toxalbumins consist of two subunits (A and B) which are joined by a disulfide bond. The A subunit irreversibly binds the 60S ribosomal subunit, which blocks protein synthesis. The B subunit allows for binding and penetration across the gastrointestinal cell wall. Sufficient doses of ricin can cause cell death due to continued inhibition of protein synthesis. 

Shiga toxin produced by Shigella dysenteriae has similar structural features. The toxin binds to a glycolipid (Gb3), undergoes endocytosis, and the enzymatie Ai fragment, which is a specific N-glycosidase, removes adenine from one particular adenosine residue in the 28S RNA of the 60S ribosomal subunit. Removal of the adenine inactivates the 60S ribosome, blocking protein synthesis. Ricin, abrin, and a number of related plant proteins inhibit eukaryotic protein synthesis in a similar manner (Chapter 25). 

The use of antibody- or aptamer-based approaches to protect against ricin holds potential as a possible pretreatment for armed forces, or as an adjunct to PPE for civilian first-responders and other special populations required to enter contaminated areas. However, extracellular antitoxin would probably be of limited use in a bioterrorist or civilian mass casualty scenario, because the therapeutic window for administration is likely to be short (a few hours), the delay to onset of symptoms is relatively long, and there presently is no method of immediately detecting ricin exposure. It also remains to be determined whether specific combinations of MAbs are required for optimal in vivo protection against the toxin. Moreover, in some cases, MAbs that bind toxin with high avidity and block enzymatic activity of RTA in vitro actually enhance the toxicity of ricin in vivo (Maddaloni et al., 2004). 

This chapter presents methods for preparing ITs with disulfide-linked toxins as exemplified by RTA, PAP, and a truncated recombinant Pseudomonas exotoxin (PE35) and with thioether-linked toxins exemplified by blocked ricin (bRT) and truncated recombinant Pseudomonas exotoxin (PE38). 

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