DTT was then removed by gel filtration. Some samples were additioned with mM Fig. The reaction mixture ml contained 70 mU TrxR. After B Time-course of ricin 1. Reaction compound auranofin. C Reduction of ricin by microsomal membranes was stopped after 60 min by addition of non-reducing sample buffer. Moreover, pre-incubation of minute amounts of RTA. Taken together, these results of each cell extract with 30 nM gold I compound aur- further support the hypothesis that TrxR associated with anofin totally suppressed the reductive cleavage of ricin by microsomes is necessary to sustain disulfide reductase cell extracts Fig.
As expected, immu- activity of PDI. This band also asses the role of the various enzymes. TrxR was found in similar of RTA and unknown protein s. This aspect warrants amounts in extracts of all cell lines, whereas Trx and PDI further investigation.
Unlike what observed in microsomes showed higher levels of expression in the U cell line. Inhibition of ricin and of pre-reduced ricin cytotoxicity by auranofin Cleavage of disulfide bonds is a critical event in the activation of endocytosed toxins and ITs in the cell intox- ication phenomena brought about by these molecules. In the case of ricin this step appears to be rate limiting [8] and may be linked to the process of translocon-assisted trans- membrane passage [9].
Once in the cytosol RTA will inhibit protein synthesis thereby killing the cell. Because results of our in vitro experiments suggested that reductive activation of ricin cytotoxicity was crucially dependent on TrxR, we decided to evaluate the role of TrxR in intact cells by inhibiting its activity with auranofin. B in treated and untreated cells. As summarized in Fig. Lane 1: control untreated RTA; lane 3: control untreated ricin; lanes 4 and 5: U percentage of 14 C-leucine incorporation was significantly cell extracts; lanes 6 and 7: Molt-3 cell extracts; lanes 8 and 9: Jurkat cell higher, and therefore ricin cytotoxicity lower, in cells extracts; lanes 10 and DU cell extracts.
Reduction of 1. To auranofin; lane control untreated ricin; lanes 13 and U cell rule out that auranofin could interfere with other steps of extraxcts; lanes 15 and Molt-3 cell extracts; lanes 17 and Jurkat cell extracts; lanes 19 and DU cell exctracts. C Reduction of 1 mM ricin-mediated intoxication, cells were also exposed in ricin by U cell extracts 30 mg total protein immunosubtracted of Trx short-time assays to high doses of pre-reduced ricin lane 2 , PDI lane 4 , and TrxR lane 6 or Trx and PDI lane 8.
Lane 1: Fig. Discussion The reductive cleavage of disulfide bonds is an event of crucial importance to activate many bacterial and plant toxins in the cell. Indeed, the replacement of the intermolecular disulfide by a non-cleavable thioether link- age renders almost ineffective the toxicity of RTA-based ITs [43]. Reducing substrates such as GSH can facilitate thiol—disulfide exchange in the absence of enzymes but the presence of oxidoreductases increases the reaction rate by Fig.
Inhibition of ricin cytotoxicity by gold I compound auranofin. U- several thousand-fold [44]. The involvement of oxidore- circle , Molt-3 triangle , Jurkat square , and DU diamonds ductases is, therefore, likely to facilitate the intracellular cells were pre-incubated in the absence empty symbols or in the presence activation of ricin and RTA-ITs and the subsequent cell of 30 nM auranofin for 6 h filled symbols and were then exposed to ricin for 4 h.
Protein synthesis was then measured by evaluating 14 C-leucine intoxication phenomena. Results are expressed as percent 14 C-leucine incorporation Considering its intracellular localization, PDI appeared with respect to mock-treated control cultures. The direction of concentrations where significant differences between cells treated with or the reaction catalyzed by protein disulfide oxidoreductases without auranofin were observed. However, when PDI or Trx catalyze the reduction ricin was not significantly different in cells pre-incubated of disulfide bridges they become oxidized and therefore with auranofin with respect to controls.
Therefore, aura- must be enzymatically regenerated by a reductase in order nofin preferentially blocks toxicity of intact ricin but not to maintain their catalytic activity as disulfide reductases. Noteworthy, 30 nM gold I can This is particularly true for PDI, which acts in the unfavor- totally inhibit reductive activation of ricin by microsomes able redox oxidizing environment of the ER [41,45].
The see above; Fig. This may suggest that additional mechanism s the active site of PDI in vitro or indirectly by means of Trx leads to ricin activation in intact cells. Results of experiments reported herein indicated that neither ricin nor ST. Our results clearly indicate that Trx and PDI can catalyze the reductive activation of both the toxin and the IT in a time-scale of seconds to minutes whereas the presence of TrxR appears to be essential to promote and sustain the disulfide reduc- tase activity of the two oxidoreductases.
Indeed, the reductive activation of ricin and of ST. Comparison of cytotoxic effect of intact ricin empty symbols and an irreversible alkylating agent of selenium, which is pre-reduced ricin filled symbols in U circle , Molt-3 triangle , Jurkat square , and DU diamonds cells in the absence or in the necessary for the reductase activity of TrxR.
To the best presence of 30 nM gold I compound auranofin. Protein before. Results are expressed as percent 14 C-leucine incorporation with respect to The mechanism by which TrxR activates PDI and mock-treated control cultures. Nevertheless, comparable expressed in microsomal membranes. However, it must be GSSG ratio allowed the time-dependent reduction considered that levels of protein expression and enzyme of ricin and ST.
Moreover, to explain inhibited by pre-incubating microsomes with auranofin. In the comparable reductive cleavage of ricin obtained in cell addition, results of immunosubtraction experiments con- extracts from the four different cell lines we must also take firmed that PDI and TrxR are indeed involved in the into account that PDI can supply disulfide reductase activ- reduction of ricin in microsomes.
Immunosubtraction experiments carried out with cell The possibility that auranofin could block other crucial extracts indicated that PDI can supply disulfide reductase steps in the cell intoxication mechanism by ricin was ruled activity on ricin also when Trx is absent.
Consistent with out by results obtained in control experiments with pre- the central role of TrxR in promoting the disulfide reduc- reduced ricin. In fact, as already described by Lewis and tase activity of both Trx and PDI, incubation of cell extracts Youle [36] we also observed comparable cytotoxicity in with auranofin inhibited the reductive cleavage of ricin cells treated with high concentrations of intact or pre- Fig.
However, incubation with the selective TrxR Similar ricin reduction patterns were observed in micro- inhibitor auranofin resulted in decreased cytotoxicity only somes deprived of PDI Fig. In both cases, an effective reduction RTB subunits is needed within the cell. Of course, differences in sensitivity to ricin e.
With pre- the reductive activation of ricin under the control of TrxR. However, ricin tionally replace TrxR [47]; alternatively reduction could be cytotoxicity is not completely prevented by incubating catalyzed or by other selenium-dependent TrxRs e.
Inhibition of ricin reduction by non-immunodepleted the cytosol of living cells. In fact auranofin can also induce U cell extracts pre-incubated with auranofin Fig. However reductive activation of ricin in takes place in compartmentalized environments it is likely intact cells is a strictly compartmentalized event and it that the cytosolic Trx and glutaredoxin systems participate might, therefore, take place in the ER or in ER-related only negligibly to this cell intoxication step.
Instead, due to organelles. Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Physiological functions of thioredoxin and related intracellular compartments not shown.
Eur J Biochem ;—9. In conclusion, our finding that TrxR exerts a fundamen- [16] Holmgren A. Antioxid Redox Signal ;— Thioredoxin expression and localization in human cell lines: of these reagents, both in terms of negative e. Exp Cell Res ; ricin intoxication due to accidental or criminal occur- — J Biol Chem ; — Cloning and characterization of the nucleoredoxin gene that encodes Acknowledgments a novel nuclear protein related to thioredoxin. Genomics ; —9. Ann NYAcad Sci ;— Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.
J Histochem Cytochem ; References — The Florey Lecture, Crystal structure of cells. A new selenoprotein from human lung Biol ;— The crystal structure of diphtheria toxin.
Three- — Crystallization of exotoxin A from pseudo- cations for mechanism and evolution of a selenocysteine-dependent monas aeruginosa.
Although ricin has been prepared in crystalline condition in the laboratory in small quantities, it becomes necessary, for purposes of toxicological warfare, to prepare relatively large quantities in a high state of purity. This necessitates that as much as possible of the non-toxic material present be removed in the process.
In preparing the protein material, the castor beans are first ground and pressed to remove most of the oil. In the event that the expressing step is supplemented by solvent extraction, it is important to prevent detoxification of the protein during the solvent removal step. If residual solvent is removed from the ground beans by blowing with steam, considerable detoxification results. Blowing with nitrogen efiectively prevents detoxification but is expensive when carried out on a large scale.
After the oil has been removed, the pressed cake or pomace is extracted by agitating with water at a pH of 3. The extraction process is operative within a pH range of about 3 to 4. A careful pH control is essential in order that as much non-toxic protein as possible may be eliminated and also that the filtration rate may be held at a satisfactory value.
Either HCl or H 80 may be used to get the desired pH for the extraction water, but H 50 is preferred due to its lower corrosion rate and ease of handling in concentrated form.
The acid should be used in reasonably dilute form to prevent undue local concentrations during its addition. Following the extraction, the slurry is filtered using either a conventional recessed plate filter or a continuous string discharge vacuum filter. The filtrate from the water extraction step, which contains the ricin, was treated with a This amount and concentration of salt solution was about optimum considering the factors of cost and toxin recovery.
Somewhat higher concentrations and larger amounts of solution can be used, however. The precipitation process is not limited to the use of Na SO since a saturated solution of NaCl can be used successfully, but Na SO solution gives better nitrogen fractionation, more rapid precipitation, and can be operated under wider pH limits. It is desirable to raise the pH to about before precipitation as this gives better ecovery and greater non-toxic nitrogen removal.
The base used was quite dilute in order to prevent detoxification due to high local concentrations in the solution. Washing the filter cake with Na SO solution removed additional non-toxic nitrogen which is desirable. In this washing step a After filtration the filter cake, which contains the ricin in combination with the Na SO may be dried and slurried with CCL, to separate the ricin by flotation.
Separation of the ricin after a single precipitation and washing step is possible, but it is preferred to carry the process through an additional extraction and precipitation step. This is accomplished by slurrying the filter cake in three times its weight of water and the pH of the slurry is again brought to 3. In filtering out the precipitate, no filter aid was used and the filter cake was washed with Na SO solution on the filter whereby an additional amount of nontoxic nitrogen was removed from the cake.
This washing was effective only the first time and repeated washings had little effect in removing further non-toxic nitrogen. The ricin-Na SO precipitate was dried at about 50 to 60 C. Need an account? Click here to sign up.
Download Free PDF. Chemical Warfare and Terrorism Medicine, Allister Vale. A short summary of this paper. Download Download PDF. Translate PDF. VX is non-volatile Sulphur mustard was used in the Iran—Iraq War. This is a measure of how quickly nerve agents evaporate and is More recently, the release of sarin by terrorists in Japan in increased by rises in ambient temperature. For example, the vapour and , and the discovery of ricin in London and Paris in pressure of sarin is 0.
Nerve agents, ricin and sulphur mustard are Vapour density discussed in this contribution. Nerve agents Solubility in water The solubility of tabun is 9. Two classes of nerve Stability agent are recognized — G and V.
GE and GF were synthesized Persistence subsequently. The V agents were introduced later and are exem- Non-persistent agents disperse rapidly after release and are an plified by VX, synthesized in the s. The G agents are both immediate, short-duration hazard. In contrast, aerosolized, are contact poisons.
VX continue to be a contact hazard and may vaporize over time to produce an inhalation hazard. Factors influencing the impact of nerve agents Toxicity An understanding of the impact of the physicochemical proper- LC50 by inhalation minute exposure in the mouse is 15 mg. This results in accumulation of acetylcholine, which in self-contained breathing apparatus should be used in contam- turn leads to enhancement and prolongation of cholinergic effects inated areas.
Casualties should be moved to hospital as soon as and depolarization blockade. The rate of spontaneous reactivation possible. This is of particular importance in poisoning with soman, because of the very rapid aging of the soman—enzyme Aging: with soman in particular, an additional reaction occurs complex.
This involves monodealkylation of the dialkyl- rhoea should be administered atropine as a matter of urgency, phosphonyl enzyme, which is then resistant to spontaneous and whichever oxime is available. This can be achieved most hydrolysis and reactivation by oximes e.
Mono- conveniently by the use of an autoinjector such as the ComboPen. Very relation to the treatment of soman poisoning, in which it is a severely intoxicated casualties may require intramuscular admin- serious problem. Cas- soman, sarin and tabun are 1. Aging occurs more slowly and reactivation relatively rapidly every 5—10 minutes until secretions are minimal and the patient with nerve agents other than soman, but early oxime administra- is atropinized dry skin and sinus tachycardia.
An oxime e. In cases of GF or soman vapour exposure. Features follow most rapidly after inhalation. The duration of oxime treatment depends on for several days, occurs rapidly following exposure to nerve agent the presence of features, the clinical response and the RBC AChE vapour and is a sensitive index of exposure.
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