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Abrin Sentezi | Gel Electrophoresis | Proteins

The rate of inhibition of protein synthesis and ribosome-dependent GTPase activity by abrin and ricin was determined

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04/01/2018 · Eur

It is suggested that the intact ribosomes thus trapped are inaccessible to the toxins and that the isolation of polysomes results in release of functionally intact ribosomes capable of supporting poly(U)‐directed polymerization of phenylalanine.AB - The rate of protein synthesis in HeLa cells was measured at various periods of time after addition of abrin and ricin to the medium and compared with the concurrent ability of the isolated ribosomes to support poly(U)‐stimulated synthesis of polyphenylalanine in a cell‐free system.

Abrin works by penetrating the cells of the body and inhibiting cell protein synthesis

Ribosome-inactivating proteins (RIPs) are a family of protein toxins that bring about the inhibition of protein synthesis either directly by inactivating the ribosomes or indirectly by modifying factors involved in translation (). They are distributed widely in nature and are found in bacteria, fungi, and plants (). One of the most potent members of the RIP family is abrin produced by the subtropical climber Abrus precatorius (). Abrin is an AB type toxin with a 30-kDa A chain, an RNA N-glycosidase that irreversibly inactivates the 28S rRNA of the mammalian 60S ribosomal subunit. Once in the cytosol, the A chain depurinates the adenine of the alpha-sarcin-ricin loop and thereby arrests host cell protein synthesis (, ). The B chain is a galactose-specific lectin and hence binds to cell surface glycosylated receptors, which allows the toxin entry (, ). Apart from inhibiting protein synthesis, RIPs induce apoptosis (). Abrin shows significant similarities to ricin at the sequence level as well as the structural level, but abrin is several times more potent than ricin (). There is much interest in understanding the bioactivities of these proteins, owing to their extreme toxicity (the 50% lethal dose [LD50] of abrin for mice is 0.04 μg/kg of body weight, and the LD50 of ricin is 3 μg/kg) (), stability, and easy availability. Both proteins cause pulmonary edema, with acute destructive alveolitis and apoptosis and necrosis in the lower respiratory tract epithelium (, ). RIPs from different plants are potent elicitors of sensitization and immunoglobulin E (IgE) production (). The use of these proteins as bioterrorist weapons is also of considerable concern (, , ). The passive administration of antibodies has proven to be a specific and effective mode of defense against poisoning by various biological toxins (). Although both anti-A chain and anti-B chain antibodies are able to neutralize toxins in vitro and in vivo, antibodies against the A chain of ricin have better protective efficacy than anti-B chain antibodies (, ).

ABRIN : Biotoxin - Centers for Disease Control and Prevention

Kinetics of inhibition of protein synthesis by F1G4-IT is slower than that of abrin

Toward the objective of establishing antibodies that would neutralize abrin, a panel of MAbs against the rABA was established and characterized extensively. The MAbs recognized specifically the rABA as well as the abrin A chain under native as well as denaturing conditions. There is a divergence of more than 75% between plant and bacterial RIPs in primary structures, even though they have identical mechanisms of action. This large amount of sequence variability may result in the plant and bacterial RIPs' having distinct neutralizing epitopes (). In agreement with the earlier predictions by other groups (), in spite of the high level of sequence similarity observed among different RIPs (abrin, ricin, R. communis agglutinin I, and A. precatorius agglutinin I), mostly unique epitopes were recognized by the anti-rABA antibodies. The inhibition of protein synthesis and apoptosis are the two major effects of RIPs on cells (, ). MAb D6F10 but none of the other MAbs rescued MCF-7 cells from abrin-mediated inhibition of protein synthesis (Fig. ). We also observed that, in the presence of this antibody, there was a significant reduction in apoptosis induced by abrin among Jurkat cells (Fig. ) and that a 200-fold molar excess of antibody was required to inhibit the abrin toxicity in OVCAR-3 cells (Fig. ). It is pertinent to mention here that all the toxin binding and neutralization assays were carried out with all three cell lines, Jurkat, MCF-7, and OVCAR-3, and we obtained very comparable data.

The abilities of the MAbs to neutralize abrin toxicity were tested by a protein synthesis assay. The 50% inhibitory concentrations (IC50s) for the inhibition of protein synthesis by abrin on MCF-7 cells (Fig. ) and OVCAR-3 cells (data not shown) were estimated to be ~0.4 to 0.8 ng/ml. Abrin at concentrations >10-fold higher than the IC50, 10 ng/ml, was mixed with 25 μg of each of the purified MAbs/ml, and the mixtures were added to MCF-7 cells grown in cell culture plates. Only the MAb D6F10 showed complete rescue of the cells from abrin-mediated inhibition of protein synthesis, while none of the other MAbs showed any detectable neutralization activity (Fig. ). In agreement with the observation that the MAb D6F10 did not bind RIPs other than abrin under native conditions (Fig. ), we found no neutralizing activity of MAb D6F10 toward ricin (50 ng/ml) and A. precatorius agglutinin I (1 μg/ml) (Fig. ). MAb D6F10 at 2.5 μg was able to neutralize 100% of the toxicity induced by 12.5 ng of abrin/ml (Fig. ). As abrin also induces apoptosis (), cells cultured with abrin (10 ng/ml) in the presence of MAb D6F10 (25 μg/ml) were analyzed for apoptosis. There was complete inhibition of abrin-induced apoptosis of Jurkat cells (Fig. ). The apoptotic population was also quantified by using FACScan (Fig. ).

Abrin - an overview | ScienceDirect Topics

At the cellular level, abrin inhibits protein synthesis, thereby causing cell death.

The abilities of the MAbs to protect against the cytotoxic effect of abrin were determined by a protein synthesis assay. As described earlier (), cells (0.2 × 106/well) were cultured for 8 h either in the absence or in the presence of different concentrations of abrin or abrin preincubated for 30 min with the purified antibodies. After leucine starvation for 2 h, 20 μCi of [3H]leucine was added and the cells were incubated for 1 h. The total cell protein was precipitated with 5% trichloroacetic acid, washed twice with 20% ice-cold ethanol, dried, and dissolved in 200 μl of 1% sodium dodecyl sulfate in 0.1 N NaOH. The incorporated radioactivity was measured in a scintillation counter (Beckman Coulter).

Plant ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that inhibit protein synthesis in cells. Abrin, a type II RIP, is an AB type toxin, which is one of the most lethal types of toxin known. The B chain facilitates the entry of the molecule into the cell, whereas the A chain exerts the toxic effect. We have generated hybridomas secreting antibodies of the immunoglobulin G class specific to the recombinant A chain of abrin. One monoclonal antibody, namely, D6F10, rescued cells from abrin toxicity. Importantly, the antibody also protected mice from lethal doses of the toxin. The neutralizing effect of the antibody was shown to be due to interference with abrin attachment to the cell surface.

01/03/2014 · The sequestration by BASP1 renders cells increasingly resistant to the inhibition of protein synthesis by abrin and the ..
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  • What is Abrin? (with pictures) - wiseGEEK

    Abrin obtained from the plant Abrus precatorius inhibits protein synthesis and also triggers apoptosis in cells

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Abrin and Immunoneutralization: A Review - Springer

N2 - Abrin-a consists of A-chain with N-glycosidase activity, which inhibits protein synthesis, and lectin-like B-chain responsible for binding with cell-surface receptors and penetrating of abrin-a molecule into the cells. As a lectin component, the B-chain can also participate in cell signal transduction. It has been reported that abrin induces apoptosis, but the molecular mechanism(s) of this induction have been obscure and several alternative variants have been discussed. The present study demonstrates that abrin-a induces apoptosis in human cultured cell lines, derived from acute lymphoblastic leukemia (ALL) (Jurkat, CCRF-CEM, MOLT-4, HPB-ALL). The apoptosis was estimated by: phosphatidylserine (PSer) exposure at the cell surface, activation of caspase cascade, and DNA fragmentation. The penetrating of abrin-a into the cells was detected by fluorescent confocal microscopy, using fluorescein isothiocyanate (FITC) as a fluorescent marker. It was established that the effect of abrin-a on the apoptosis induction in leukemic cells was dose- and time-dependent. The process was initiated 1 h after abrin-a application (before its penetrating into the cells) and was characterized with PSer translocation from the inner to the outer monolayer of plasma membrane, caspase activation on the first to second hour after beginning of treatment, with maximum on the third to fourth hour, and DNA fragmentation on the fourth to sixth hour, depending of the cell line. The exposure of PSer on the cell surface was detected in Jurkat, CCRF-CEM, and MOLT-4 cells. In HPB-ALL, no significant changes in PSer exposure on the cell surface was observed. Activation of caspase-3, -8, and -9 was detected in Jurkat, MOLT-4, and HPB-ALL. Surprisingly, the activity of caspase-3 increased on the first hour after beginning of treatment, while the activity of caspase-8 and -9 began to increase on the second hour. In CCRF-CEM, activation of caspases was not measured, but the apoptosis progressed to DNA fragmentation in a dose- and time-dependent manner. DNA fragmentation was also detected in Jurkat, but not in MOLT-4 and HPB-ALL cells. It seems that the mechanisms of abrin-a-induced apoptosis are different and the progress of apoptosis depends of the cell line. There was a very good positive correlation between the agglutinating activity of abrin-a and development of apoptosis to DNA fragmentation. The time-dependent effects of abrin-a on apoptosis as well as its time-dependent penetration into the cells suggest that the B-chain probably triggers the apoptosis, while the A-chain and breakage of the disulfide bond are responsible for its progress.

01/04/2014 · Introduction to Abrin

Abrin-a consists of A-chain with N-glycosidase activity, which inhibits protein synthesis, and lectin-like B-chain responsible for binding with cell-surface receptors and penetrating of abrin-a molecule into the cells. As a lectin component, the B-chain can also participate in cell signal transduction. It has been reported that abrin induces apoptosis, but the molecular mechanism(s) of this induction have been obscure and several alternative variants have been discussed. The present study demonstrates that abrin-a induces apoptosis in human cultured cell lines, derived from acute lymphoblastic leukemia (ALL) (Jurkat, CCRF-CEM, MOLT-4, HPB-ALL). The apoptosis was estimated by: phosphatidylserine (PSer) exposure at the cell surface, activation of caspase cascade, and DNA fragmentation. The penetrating of abrin-a into the cells was detected by fluorescent confocal microscopy, using fluorescein isothiocyanate (FITC) as a fluorescent marker. It was established that the effect of abrin-a on the apoptosis induction in leukemic cells was dose- and time-dependent. The process was initiated 1 h after abrin-a application (before its penetrating into the cells) and was characterized with PSer translocation from the inner to the outer monolayer of plasma membrane, caspase activation on the first to second hour after beginning of treatment, with maximum on the third to fourth hour, and DNA fragmentation on the fourth to sixth hour, depending of the cell line. The exposure of PSer on the cell surface was detected in Jurkat, CCRF-CEM, and MOLT-4 cells. In HPB-ALL, no significant changes in PSer exposure on the cell surface was observed. Activation of caspase-3, -8, and -9 was detected in Jurkat, MOLT-4, and HPB-ALL. Surprisingly, the activity of caspase-3 increased on the first hour after beginning of treatment, while the activity of caspase-8 and -9 began to increase on the second hour. In CCRF-CEM, activation of caspases was not measured, but the apoptosis progressed to DNA fragmentation in a dose- and time-dependent manner. DNA fragmentation was also detected in Jurkat, but not in MOLT-4 and HPB-ALL cells. It seems that the mechanisms of abrin-a-induced apoptosis are different and the progress of apoptosis depends of the cell line. There was a very good positive correlation between the agglutinating activity of abrin-a and development of apoptosis to DNA fragmentation. The time-dependent effects of abrin-a on apoptosis as well as its time-dependent penetration into the cells suggest that the B-chain probably triggers the apoptosis, while the A-chain and breakage of the disulfide bond are responsible for its progress.

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