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Title: Ricin
Date: 2012
Source: Wikipedia

Ricin from the castor oil plant Ricinus communis, is a highly toxic, naturally occurring protein. A dose as small as a few grains of salt can kill an adult. The LD of ricin is around 22 micrograms per kilogram (1.76 mg for an average adult, around 1/228 of a standard aspirin tablet (0.4 g gross)) in humans if exposure is from injection or inhalation. Oral exposure to ricin is far less toxic and lethal dose can be up to 20–30 milligrams per kilogram.

Ricin is poisonous if inhaled, injected, or ingested, acting as a toxin by the inhibition of protein synthesis. It is resistant, but not impervious, to digestion by peptidases. By ingestion, the pathology of ricin is largely restricted to the gastrointestinal tract where it may cause mucosal injuries; with appropriate treatment, most patients will make a full recovery. Because the symptoms are caused by failure to make protein, they emerge only after a variable delay from a few hours to a full day after exposure. An antidote not yet tested on humans has been developed by the UK military, and a vaccine has been developed by the US military, and has had some human testing, and so far shown to be safe, and effective when lab mice were injected with ricin-antibody rich blood mixed with ricin. Symptomatic and supportive treatment is available. Long term organ damage is likely in survivors. Ricin causes severe diarrhea and victims can die of shock. Death typically occurs within 3–5 days of the initial exposure. Abrin is a similar toxin, found in the highly ornamental rosary pea.

Deaths caused by ingestion of castor plant seeds are rare, partly because of the indigestible capsule, and partly because ricin can be digested (although it is resistant). The pulp from eight beans is considered toxic for an adult. A solution of saline and glucose has been used to treat ricin overdose. Rauber and Heard have written that close examination of early 20th century case reports indicates that public and professional perceptions of ricin toxicity "do not accurately reflect the capabilities of modern medical management".

Most acute poisoning episodes in humans are the result of oral ingestion of castor beans, 5-20 of which could prove fatal to an adult. Victims often manifest nausea, diarrhea, tachycardia, hypotension and seizures persisting for up to a week. Blood, plasma or urine ricin concentrations may be measured to confirm diagnosis.

Ricin is classified as a type 2 ribosome inactivating protein (RIP). Whereas Type 1 RIPs consist of a single enzymatic protein chain, Type 2 RIPs, also known as holotoxins, are heterodimeric glycoproteins. Type 2 RIPs consist of an A chain that is functionally equivalent to a Type 1 RIP, covalently connected by a single disulfide bond to a B chain that is catalytically inactive, but serves to mediate entry of the A-B protein complex into the cytosol. Both Type 1 and Type 2 RIPs are functionally active against ribosomes in vitro, however only Type 2 RIPs display cytoxicity due to the lectin properties of the B chain. In order to display its ribosome inactivating function, the ricin disulfide bond must be reductively cleaved.

The tertiary structure of ricin was shown to be a globular, glycosylated heterodimer of approximately 60-65 kDA. Ricin toxin A chain and ricin toxin B chain are of similar molecular weight, approximately 32 kDA and 34 kDA respectively.

1. Ricin A Chain (RTA) is an N-glycoside hydrolase composed of 267 amino acids. It has three structural domains with approximately 50% of the polypeptide arranged into alpha-helices andbeta-sheets. The three domains form a pronounced cleft that is the active site of RTA.

2. Ricin B Chain (RTB) is a lectin composed of 262 amino acids that is able to bind terminal galactose residues on cell surfaces. RTB form a bilobal, barbell-like structure lacking alpha-helicesor beta-sheets where individual lobes contain three subdomains. At least one of these three subdomains in each homologous lobe possesses a sugar-binding pocket that gives RTB its functional character.

Many plants such as barley have the A chain but not the B chain. People do not get sick from eating large amounts of such products, as ricin A is of extremely low toxicity as long as the B chain is not present.
Entry into the cytosol

The ability of ricin to enter the cytosol depends on hydrogen bonding interactions between RTB amino acid residues and complex carbohydrates on the surface of eukaryotic cells containing either terminal N-acetyl galactosamine or beta-1,4-linked galactose residues. Additionally, the mannose-type glycans of ricin are able to bind cells that express mannose receptors. Experimentally, RTB has been shown to bind to the cell surface on the order of 106-108 ricin molecules per cell surface.

The profuse binding of ricin to surface membranes allows internalization with all types of membrane invaginations. Experimental evidence points to ricin uptake in both clathrin-coated pits, as well as clathrin-independent pathways including caveolae and macropinocytosisVesicles shuttle ricin to endosomes that are delivered to the Golgi apparatus. The active acidification of endosomes are thought to have little effect on the functional properties of ricin. Because ricin is stable over a wide pH range, degradation in endosomes or lysosomes offer little or no protection against ricin. Ricin molecules are thought to follow retrograde transport via early endosomes, the trans-Golgi network, and the Golgi to enter the lumen of the endoplasmic reticulum (ER).

For ricin to function cytotoxically, RTA must be reductively cleaved from RTB in order to release a steric block of the RTA active site. This process is catalysed by the protein PDI (protein disulphide isomerase) that resides in the lumen of the ER. Free RTA in the ER lumen then partially unfolds and partially buries into the ER membrane, where it is thought to mimic a misfolded membrane-associated protein. Roles for the ER chaperones GRP94 and EDEM have been proposed prior to the 'dislocation' of RTA from the ER lumen to the cytosol in a manner that utilizes components of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. ERAD normally removes misfolded ER proteins to the cytosol for their destruction by cytosolic proteasomes. Dislocation of RTA requires ER membrane-integral E3 ubiquitin ligase complexes, but RTA avoids the ubiquitination that usually occurs with ERAD substrates because of its low content of lysine residues, which are the usual attachment sites for ubiquitin. Thus RTA avoids the usual fate of dislocated proteins (destruction that is mediated by targeting ubiquitinylated proteins to the cytosolic proteasomes). In the mammalian cell cytosol, RTA then undergoes triage by cytosolic molecular chaperones that results in its folding to a catalytic conformation that de-purinates ribosomes, thus halting protein synthesis.

Ribosome Inactivation
Study of the N-glycosidase activity of ricin was pioneered by Endo and Tsurugi who showed that RTA cleaves a glycosidic bond within the large rRNA of the 60S subunit of eukaryotic ribosomes. They subsequently showed RTA specifically and irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 (A4324) within the 28S rRNA, but leaves the phosphodiester backbone of the RNA intact. The ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes. The sequence, 5’-AGUACGAGAGGA-3’, termed the sarcin-ricin loop, is important in binding elongation factors during protein synthesis. The depurination event rapidly and completely inactivates the ribosome, resulting in toxicity from inhibited protein synthesis. A single RTA molecule in the cytosol is capable of depurinating approximately 1500 ribosomes per minute.

Depurination Reaction
Within the active site of RTA, there exist several invariant amino acid residues involved in the depurination of ribosomal RNA. Although the exact mechanism of the event is unknown, key amino acid residues identified include tyrosine at positions 80 and 123, glutamic acid at position 177, and arginine at position 180. In particular, Arg180 and Glu177 have been shown to be involved in thecatalytic mechanism, and not substrate binding, with enzyme kinetic studies involving RTA mutants.

The model proposed by Mozingo and Robertus, based x-ray structures, is as follows:

1. Sarcin-ricin loop substrate binds RTA active site with target adenine stacking against tyr80 and tyr123.
2. Arg180 is positioned such that it can protonate N-3 of adenine and break the bond between N-9 of the adenine ring and C-1’ of the ribose.
3. Bond cleavage results in an oxycarbonium ion on the ribose, stabilized by Glu177.
4. N-3 protonation of adenine by Arg180 allows deprotonation of a nearby water molecule.
5. Resulting hydroxyl attacks ribose carbonium ion.
6. Depurination of adenine results in a neutral ribose on an intact phosphodiester RNA backbone.

Ricin is easily purified from castor oil manufacturing waste. The aqueous phase left over from the oil extraction process is called waste mash. It would contain about 5–10% ricin by weight, but heating during the oil extraction process denatures the protein, making the resultant seed cake safe for use as animal feed. From fresh seed, separation requires chromatographic techniques similar to other plant proteins.

Patented Extraction Process
A process for extracting ricin has been described in a patent. The described extraction method is very similar to that used for the preparation of soy protein isolates.

The patent was removed from the United States Patent and Trademark Office (USPTO) database sometime in 2004. Modern theories of protein chemistry cast doubt on the effectiveness of the methods disclosed in the patent.

Potential Medicinal Use
Some researchers have speculated about using ricins in the treatment of cancer, as a so-called "magic bullet" to destroy targeted cells. Because ricin is a protein, it can be genetically linked to a monoclonal antibody to target malignant cells recognized by the antibody. The major problem with ricin is that its native internalization sequences are distributed throughout the protein. If any of these native internalization sequences are present in a therapeutic, then the drug will be internalized by, and kill, untargeted epithelial cells as well as targeted cancer cells.

Some researchers hope that modifying ricin will sufficiently lessen the likelihood that the ricin component of these immunotoxins will cause the wrong cells to internalize it, while still retaining its cell-killing activity when it is internalized by the targeted cells. Generally, however, ricin has been superseded for medical purposes by more practical fragments of bacterial toxins, such as diphtheria toxin, which is used in denileukin diftitox, an FDA-approved treatment for leukemia and lymphoma. No approved therapeutics contain ricin.

A promising approach is also to use the non-toxic B subunit as a vehicle for delivering antigens into cells thus greatly increasing their immunogenicity. Use of ricin as an adjuvant has potential implications for developing mucosal vaccines.

Ricinine has some insecticidal effects on three insect pests as well as a hepatoprotective activity. Ricinine, when administered to mice at low doses has memory-improving effects. The signs of intoxication caused by ricinine can be used as chemical model of epilepsy in the screening of anticonvulsant drugs.

Incidents Involving Ricin: Main article: Incidents involving ricin

Ricin has been involved in a number of incidents, including the high-profile assassination of Georgi Markov using a weapon disguised as an umbrella.

The ingestion of Ricinus communis cake is responsible for fatal ricin poisoning in animals.

Use as a Chemical/Biological Warfare Agent
The United States investigated ricin for its military potential during the First World War. At that time it was being considered for use either as a toxic dust or as a coating for bullets and shrapnel. The dust cloud concept could not be adequately developed, and the coated bullet/shrapnel concept would violate the Hague Convention of 1899 (adopted in U.S. law at 32 Stat. 1903), specifically Annex § 2, Ch.1, Article 23, stating "...it is especially prohibited...[t]o employ poison or poisoned arms". The First World War ended before the U.S. weaponized ricin.

During the Second World War the United States and Canada undertook studying ricin in cluster bombs. Though there were plans for mass production and several field trials with different bomblet concepts, the end conclusion was that it was no more economical than using phosgene. This conclusion was based on comparison of the final weapons rather than ricin's toxicity (LCt50 ~40 mg·min/m3). Ricin was given the military symbol W or later WA. Interest in it continued for a short period after the Second World War, but soon subsided when the U.S. Army Chemical Corps began a program to weaponize sarin.

The Soviet Union also possessed weaponized ricin. There were speculations that the KGB even used it outside of the Soviet bloc; however, this was never proven. In 1978, the Bulgarian dissident Georgi Markov was assassinated by Bulgariansecret police who surreptitiously 'shot' him on a London street with a modified umbrella using compressed gas to fire a tiny pellet contaminated with ricin into his leg. He died in a hospital a few days later; his body was passed to a special poison branch of the British Ministry of Defence (MOD) that discovered the pellet during an autopsy. The prime suspects were the Bulgarian secret police: Georgi Markov had defected from Bulgaria some years previously and had subsequently written books and made radio broadcasts which were highly critical of the Bulgarian communist regime. However, it was believed at the time that Bulgaria would not have been able to produce the pellet, and it was also believed that the KGB had supplied it. The KGB denied any involvement although high-profile KGB defectors Oleg Kalugin and Oleg Gordievsky have since confirmed the KGB's involvement. Earlier, Soviet dissident Aleksandr Solzhenitsyn also suffered (but survived) ricin-like symptoms after a 1971 encounter with KGB agents.

Despite ricin's extreme toxicity and utility as an agent of chemical/biological warfare, it is extremely difficult to limit the production of the toxin. The castor bean plant from which ricin is derived is a common ornamental and can be grown at home without any special care, and the major reason ricin is a public health threat is that it is easy to obtain.

Under both the 1972 Biological Weapons Convention and the 1997 Chemical Weapons Convention, ricin is listed as a schedule 1 controlled substance. Despite this, more than 1 million tonnes of castor beans are processed each year, and approximately 5% of the total is rendered into a waste containing negligible concentrations of undenatured ricin toxin.

Ricin is several orders of magnitude less toxic than botulinum or tetanus toxin, but the latter are harder to come by. Compared to botulinum or anthrax as biological weapons or chemical weapons, the quantity of ricin required to achieve LD50 over a large geographic area is significantly more than an agent such as anthrax (tons of ricin vs. only kilogram quantities of anthrax). Ricin is easy to produce, but is not as practical nor likely to cause as many casualties as other agents. Ricin is inactivated (the protein changes structure and becomes less dangerous) much more readily than anthrax spores, which may remain lethal for decades. Jan van Aken, a Dutch expert on biological weapons, explained in a report for The Sunshine Project that Al Qaeda's experiments with ricin suggest their inability to produce botulinum or anthrax.

Ian Davison, a British white supremacist and neo-Nazi, was arrested in 2009 for planning terrorist attacks involving ricin.

In 2011 the United States government discovered information that terrorist groups were attempting to obtain large amounts of castor beans for weaponized ricin use.

On November 1, 2011 the FBI arrested 4 North Georgia men and charged them in plots to purchase explosives, a silencer and to manufacture the biological toxin ricin from castor beans
 (Wikipedia, 2012).