Ricin isolation

Other Lethal Agents. There are a number of substances, many found in nature, which are known to be more toxic than nerve agents (6). None has been weaponized. Examples of these toxic natural products include shellfish poison, isolated from toxic clams puffer fish poison, isolated from the viscera of the puffer fish the active principle of curare "heart poisons" of the digitaUs type the active principle of the sea cucumber active principles of snake venom and the protein ricin, obtained from castor beans (See Castor oil). 

Ricin, an extremely toxic molecule isolated from the castor bean, inactivates eukaryotic 28S tibosomal RNA by providing the N-glycolytic cleavage or removal of a single adenine. 

Biological characterization of the nanoparticles was carried out by monitoring in vitro interactions with hepatocytes isolated from rat liver [12], Haemagglutination inhibition test of erythrocytes with ricine agglutinin (RCA120) was carried out [13]. The fluorescence of the hepatocytes incubated with the FlTC-labeled nanoparticles was determined by means of a FACS Star Becto-Dickinson instmment. 

Toxin A chains are isolated from ricin and abrin by reductive cleavage of the toxin, followed by separation of the chains These procedures are hazardous and should not be undertaken without the proper safeguards (4). 

Extracts from the castor bean can be used to isolate ricin, an extremely potent poison that is one of the leading candidates for use by terrorists. Ricin is a large-molecule, heterogeneous protein. A chemical marker for ricin is the hydrolysis product alkaloid ricinine, shown in Figure 19.2. 

Multiple purification-schemes have been applied to the separation of the toxin and the hemagglutinin.144,146,147,150,194,64,-651 Fractionation using salt and ethanol precipitation led to crystallization844,645 of the toxin known as ricin or ricin D. The hemagglutinin was isolated, free from toxic activity, by ion-exchange chromatography and gel filtration.642,646-648 With the introduction of affinity chromatography on Sepharose 4B, to which both proteins bind, purification of the two R. 

Funatsu, G., Funatsu, M. (1970). Isolation and chemical properties of various types of ricin. Jpn. J. Med. Sci. Biol. 23 342-4 Gareth, D., Griffiths, G.D., Rice, P., Allenby, A.C., Bailey, S.C., Upshall, D.G. (1995). Inhalation toxicology and histopa-thology of ricin and abrin toxins. Inhal. Toxicol. 7 269-88 Gill, D.M. (1982). Bacterial toxins a table of lethal amounts. Microbiol. Rev. 46 86-94. 

Waller, G.R., Negi, S.S. (1958). Isolation of ricin, ricinine, and the allergenic fraction from castor seed pomace from two different sources. J. Am. Oil Chem. Soc. 35 409-12. 

RIPs should generally appear as a single band on SDS-PAGE. The presence of additional bands is a clear indication of impurity, and may complicate the interpretation of electrophoretic analysis of conjugate products. The exception is ricin A chain that has been isolated from the native toxin. Native ricin A chain exists as a mixture of two differently... 

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

Identification of the residues of ricin involved in the covalent linkage to the affinity ligand is important for complete characterization of the cytotoxic effector moiety and to ensure consistency of the immunoconjugate product. However, due to the intrinsic heterogeneity of the ligand, isolation of individual species of ligand-bound B-chain peptides by traditional peptide mapping has not been possible. 

In order to test the specificity of the thiol labeling by MIANS and the general efficacy of this method to subsequently isolate and identify reactive thiols, the A-chain of ricin was used as a model protein. Although the A-chain contains two cysteine (Cys) residues, only the C-terminal Cys that forms the intermolecular disulfide linkage to the B-chain is accessible upon reduction of the native protein under the conditions employed (Montfort et al., 1987). If MIANS is indeed specific for reactive thiols and the anti-MIANS affinity column is effective, only MIANS-labeled peptides corresponding to the C-terminal sequence of ricin A-chain should be obtained (with a blank cycle at the Cys position upon automated Edman sequencing, due to its derivatization). 

Here we report the results obtained using MIANS-labeling in conjunction with affinity chromatography to map free thiols in reduced, native ricin A-chain. We further describe the results obtained when utilizing this method to isolate ligand-bound ricin B-chain peptides. 

There is now a substantial literature on the synthesis and testing of chimeric molecules. These substances are each composed of at least two functional domains, such as a tissue-targeting domain and a pharmacologically active domain. One common strategy in constructing chimeric toxins is to isolate the binding domain of one toxin (e.g., diphtheria toxin) and attach this to the poisoning domain of another (e.g., ricin). This chimeric molecule attaches only to cells that have the diphtheria toxin receptor, and it expresses only the intracellular effects of ricin. 

A group of plant lectins, such as abrin, ricin, and mod-eccin, are highly toxic to eukaryotic cells. Their mode of action consists of inhibition of protein synthesis by enzymatically inactivating the EF-2 binding region of the 60S ribosomal subunit, whereas the diphtheria toxin inactivates the EF-2 protein itself. Ricin is isolated from castor beans and has a molecular weight of 66,000. Like most plant and bacterial toxic proteins, ricin contains two... 

The oxidation of thiol groups disrupts redox balance (deflned later in this chapter), which can set into motion a cascade of events, such as apoptosis, oxidant production, and increased activity of redox-regulated transcription factors (e.g., nuclear factor kappa beta [NF-kB]). The occurrence of OS is not isolated to the vesicant class of weapons of mass destmction (WMD). Radiation (Kang et al., 2006), bacterial infections (e.g., anthrax) (Hanna et al., 1994 Kuhn et al., 2006), viral infections (e.g., influenza) (Ghezzi and Ungheri, 2004), and ricin (Kumar et al., 2003 Suntres et al., 2005) exposures also induce OS as part of the host pathogen response, which is acute inflammation. 

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 B-lymphocytes may represent one of the preferential targets of ricin toxicity in vivo. Lymphatic tissues from animals exposed to ricin show extensive hyperplasia and cellular necrosis with edema, hyperemia, and hemorrhage (Waller et al., 1966). Rats injected (i.m.) with ricin or abrin develop numerous apoptotic-like bodies in ileal crypts, para-aortic lymph nodes, and Peyer s patches (Griffiths et al., 1987). The finding of apoptosis in whole animals may be due to a direct effect of ricin on cells of the lymphatic tissue, as is observed with isolated cells in vitro, or it may partly reflect the numerous pathological sequelae of toxin exposure, including severe shock (Griffiths et al., 1988 Howat, 1988). 

Production of toxin chimeras is possible by applying similar technology to recombine isolated A- and B-chains from closely related type 2 RlPs (Olsnes and Pihl, 1986). For example, hybrid toxins composed of the modeccin A-chain and RTB have been produced and found to be even more toxic to Vero cells than are native ricin or modeccin (Sundan et al., 1983). Isolated abrin A-chain can be combined with RTB, or abrin B-chain with RTA, to produce chimeras with almost the same toxicity as the native toxins (Olsnes et al., 1974a). Similar experiments have been conducted combining viscumin A-chain and RTB to produce a chimera with cytotoxicity intermediate between that of viscumin and ricin (Tonevitskii et al., 1994). 

Sublethal effects of injected ricin have been documented in isolated case reports, and are difficult to distinguish from those of many other toxic or infectious agents. A 36 year old chemist who allegedly injected himself (i.m.) with an unknown amount of ricin prepared from homogenized castor seed, for example, complained of headache and rigors approximately 10 h later, then developed anorexia and nausea, a sinus tachycardia, erythematous areas around the puncture wounds, and local lymphadenopathy at the injection sites (Fine et al., 1992). 

Ricin is not inactivated significantly by freezing, chilling, or storage at ambient temperature. Ricin stored at 4°C in the liquid state with a suitable preservative (e.g., sodium azide) retains significant potency for years. Isolated RTA is less stable in solution at 4°C than is the holotoxin, but either can be stored below —20°C for long periods in 10% glycerol. 

Heating a 1% (w/v) solution of ricin to >85°C for 30 min results in complete inactivation as judged by toxicity in laboratory mice (Hunt et al., 1918). Dry heat of >100°C for 60 min in an ashing oven or steam autoclave treatment at >121 °C for 1 h reduces the activity of pure ricin by >99% (Wannemacher et al., 1989). Heat inactivation of impure toxin preparations (e.g., crude ricin plant extracts) may vary. Heat-denatured ricin can undergo limited refolding (<1%) to yield active toxin. Isolated RTA and RTB are more easily inactivated by heating than is the holotoxin (Olsnes et al., 1975 Taira et al., 1978). 

A ricin-resistant mouse lymphoma cell line has been isolated which was shown to possess a normal number of ricin-binding sites [63-66]. These cells internalized reduced amounts of ferritin-ricin complexes at low toxin concentrations compared with the parent cells. Resistance in this cell line is therefore due to a defect in transferring ricin from the cell surface to the cytoplasm, since... 

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