Distribution of ricin within the mammalian para-aortic lymph node. II. Comparison of the localization, after intramuscular dosage of colloidal gold-labelled ricin in vivo, with in vitro binding characteristics of the native toxin

Previous work has shown that, following an intramuscular injection of ricin, the toxin becomes localized within histiocytes in the sinuses of lymph nodes draining the 'wound' site. When ricin labelled with colloidal gold was similarly injected, it was found within the same lymphoid cells as seen with native ricin. Biologically inert Indian ink apparently follows a similar fate, as demonstrated by the appearance of carbon particles within sinus histiocytes, as soon as 1 h after intramuscular injection. When the binding in vitro of Indian ink or ricin toxin to sections of lymph node was examined, ricin was seen to bind to the surfaces of the same sinusoidal cells and also, with a much lower frequency, to follicular lymphocytes, whereas Indian ink failed to bind. This indicated an interaction between ricin and cell membrane components. Moreover, this binding was inhibited markedly by the galactose-containing disaccharide, lactose, a target sugar specified by the lectin binding site of ricin and to a much lesser extent by the monosaccharide mannose.     

Detection of residual toxin in tissues of ricin-poisoned mice by sandwich enzyme-linked immunosorbent assay and immunoprecipitation

This work aimed to evaluate a method to detect the residual ricin in animal tissues. Immunoprecipitation and sandwich enzyme-linked immunosorbent assay (ELISA) were used to detect ricin in the tissues of intoxicated mice. The monoclonal antibodies (Mabs) 4C13 and 3D74 were used to assay the whole ricin molecules via sandwich ELISA. Mab 4C13 was conjugated with Sepharose 4B to capture ricin or ricin A chain by immunoprecipitation. Mice injected intravenously with ricin at the dosage of 5 μg/mouse were killed at different time points after intoxication. The serum, liver, kidney, lung, and intestine were harvested. High levels of ricin were found in serum and liver samples at each poisoning time point by sandwich ELISA, suggesting the possibility of determining ricin intoxication by detecting residual ricin in the serum. However, this method turned out to be ineffective for examining ricin in the kidney, lung, and intestine of poisoned mice. Although the same tissue samples of intoxicated mice were analyzed by immunoprecipitation, positive bands were found. This indicated that some components in the kidney, lung, and intestine could bind with ricin and interfere in its binding activity with the coated antibody. Immunoprecipitation could be used to measure the existence of ricin in these samples.     

Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line.

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.     

In vivo potentiation of ricin toxicity by monensin delivered through liposomes.

Monensin, a carboxylic ionophore, which is known to raise intravesicular pH, was intercalated in liposomes and its effect on the toxicity of ricin in mice was studied. The toxicity of ricin in vivo was found to be significantly enhanced by the administration of monensin intercalated in liposomes (liposomal monensin). The observed enhancement of the toxicity of ricin by monensin was highly dose-dependent and was maximal when ricin was injected within 60 min of monensin injection. The survival time was found to be reduced in the range of 8-20 h, depending on the dose of ricin used, by liposomal monensin. Stability of liposomes containing monensin as inferred from the release of entrapped calcein or FITC-dextran under both in vivo and in vitro conditions was comparable to that observed for liposomes without monensin. Liposomal monensin remains in circulation for 2 h and was cleared from the blood stream after 4 h. In contrast, 15 min was required for the clearance of monensin when administered in free form. Studies on the distribution of liposomal monensin and 125I-ricin in various tissues have revealed that monensin is mainly localized in the liver and spleen which are also the major sites for ricin accumulation. Our observation on the substantial enhancement of ricin toxicity in vivo by liposomal monensin strongly supports the potential usefulness of the latter as a potentiating agent in the enhancement of the toxicity of immunotoxin or hormonotoxin for selective elimination of cancer cells.     

Effects of Iodination on Cytoagglutination by and Toxicity of Ricinus communis Lectins

Iodinations of two Ricinus communis lectins, ricin D and hemagglutinin (CBH), with potassium iodide at pH 7.0 and 0°C led to inactivation of the cytoagglutinating activity on sarcoma 180 ascites tumor cells as well as the toxicity to HeLa cells of ricin D, whereas the cytoagglutinating activity of CBH was affected slightly. In the presence of lactose, which binds to ricin D, one tyrosyl residue in the B-chain of ricin D was protected from iodination and 40% of the cytoagglutinating activity was retained. This protection against iodination was not observed in the presence of glucose, which does not bind to ricin D. This suggested that the protected tyrosyl residue in the B-chain of ricin D may be situated at or near the saccharide binding site and directly involved in the binding to the saccharide moieties of the cellular receptors.

Adsorption of the iodinated ricin D to Sepharose 4B indicated that one of the two saccharide binding sites in ricin D is still intact and participates in the binding to saccharide: ricin D was altered from divalent to monovalent by the iodination.

We found from binding experiments with ¹²⁵I-labeled iodinated ricin D to HeLa cells, that the low toxicity of the iodinated ricin D may be attributed mainly to its decreased internalization into the cells and that the divalent binding of ricin D to the cellular receptors is important for this internalization.     

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Background

Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods

A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 10⁹ copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results

Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion

Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.     

Significance of the Tryptophan Residue at the Low Affinity Saccharide-binding Site of Ricin E

Chemical modification of tryptophan residues in ricin E was investigated with regard to saccharide-binding. Two out of ten tryptophan residues in ricin E were modified with N- bromosuccinimide at pH 4.5 in the absence of specific saccharide accompanied by a marked decrease in the cytoagglutinating activity. Such a loss of the cytoagglutinating activity was found to be principally due to the oxidation of one tryptophan residue per B-chain. In the presence of lactose, one tryptophan residue/mol was protected from the modification with retention of a fairly high cytoagglutinating activity. However, G a IN Ac did not show such a protective effect. The binding of lactose to ricin E altered the environment of the tryptophan residue at the low affinity binding site of ricin E, leading to a blue shift of the fluorescence spectrum and an UV-difference spectrum with a maximum at 290 nm and a trough at 300 nm. The ability to generate such spectroscopic changes induced by lactose was retained in the derivative in which one tryptophan residue/mol was oxidized in the presence of lactose, but not in the derivative in which two tryptophan residues/mol were oxidized in the absence of lactose. Based on these results, it is suggested that one of the two surface-localized tryptophan residues is responsible for saccharide binding at the low affinity binding site of ricin E, which can bind lactose but lacks the ability to bind GalNAc.     

Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices

Background

In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits.

Methodology/Findings

This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index–time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material.

Conclusions/Significance

The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Demonstration of ricin within the mammalian para-aortic lymph node I. Comparison of the localization, after intramuscular injection, with three immunocytochemical methods

Summary Following a supralethal injection of ricin into thigh muscle of the adult rat, the toxin was demonstrated post-mortem in the para-aortic lymph node, ipsilateral to the side of injection. The relative merits of two immunoenzyme methods, peroxidase anti-peroxidase (PAP) and avidin—biotin—peroxidase complex (ABC) and a silver-enhanced immunogold method (IGSS) were assessed in the detection of ricin in the lymph node tissue. The toxin was clearly seen to be located in association with histiocytes found both within and lining the sinuses of the nodes and also, in some cases, in the subcapsular sinus of the node; the toxin was not demonstrable within lymphoid follicles by light microscopy. However, using electron microscopy and the IGSS technique, cells carrying discrete particles of gold could be visualized within follicular areas. The IGSS and ABC-peroxidase methods were both found to give excellent results without background staining at the light microscopy level. However, when these techniques were used prior to embedding and viewing by electron microscopy, the IGSS technique proved to be far superior.     

Binding of Lactose and Galactose to Native and Iodinated Ricin D

The binding of lactose and galactose to native and iodinated ricin D was investigated by equilibrium dialysis and ultraviolet difference spectroscopy. The results provided direct evidence that native ricin D has two independent saccharide binding sites with different affinities, of which the high-affinity (HA-) binding site is able to bind with both lactose and galactose while the low-affinity (LA-) binding site binds only with lactose. In contrast, the iodinated ricin D possesses only one binding site both for lactose and galactose with high affinity.

By UV-difference spectroscopic analysis we found that there is one tyrosyl residue at or near the HA-binding site in ricin D which may be involvled in binding with saccharide. This tyrosyl residue was not iodinated in the presence of lactose but was iodinated in the absence of lactose and was perturbed by an addition of lactose even after iodination.

From these results, it was inferred that the binding site abolished by the iodination is the LA-binding site and this may be due to the conformational alteration of the LA-binding site caused by the iodination of the tyrosyl residue(s) present near the LA-binding site.     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

Absorption of Iron-Dextrin (Fe59) and Iron-Sorbitol (Fe59) from an I. M. Injection in Piglets

Absorption of iron has been followed after the intramuscular injection of 12 piglets with iron-dextrin and iron-sorbitol, labelled with Fe59. Disappearance from the site of injection was followed by a method developed for external radioactivity measurements. Blood levels of Fe59 were measured at intervals during a period of 51 days. The tissue distribution pattern of Fe59 was examined in six of the piglets after 30 days. Both iron-dextrin and iron-sorbitol were rapidly absorbed, iron-sorbitol more rapidly. After 20 minutes, 50 per cent of the iron-sorbitol had been absorbed and less than one per cent remained at the site of injection after 48 hours. The amount of Fe59 remaining at the site of injection of iron-dextrin had decreased by half by 60 minutes but some ten per cent had still not been absorbed after ten days. When given as iron-dextrin (94 mg Fe per piglet), the level of Fe59 in the blood remained fairly constant for 14 days. On piglets given iron-sorbitol, (100 mg Fe each) the blood level declined steadily. The amount of Fe59 in the liver, spleen and bone marrow was much greater 30 days after the injection of iron-dextrin than after iron-sorbitol. The relation was reversed in the kidneys, presumably because of the greater excretion of iron-sorbitol in the urine.     

Ricin A-Chain and Ricin A-Chain Immunotoxins Rapidly Damage Human Endothelial Cells: Implications for Vascular Leak Syndrome

The results of Phase I/II clinical trials indicate that ricin A-chain-containing immunotoxins cause vascular leak syndrome, characterized by hypoalbuminemia with resultant weight gain and edema. Vascular leak syndrome may be a dose-limiting factor during treatment with ricin A-chain-containing immunotoxins. In this report, we determined the effect of ricin A-chain and ricin A-chain-containing immunotoxins on human umbilical vein endothelial cells with the aim of developing an in vitro model to study vascular leak syndrome. The major findings of our study are: (1) Human umbilical vein endothelial cells undergo rapid and dramatic changes in morphology after treatment with ricin A-chain and ricin A-chain-containing immunotoxins. These changes include rounding of the cells and, eventually, the formation of gaps between them. (2) The permeability of human umbilical vein endothelial cell monolayers to passage of molecules increases after exposure to ricin A-chain or ricin A-chain-containing immunotoxins and this is consistent with the morphologic changes. (3) Human umbilical vein endothelial cells bind ¹²⁵ I-rRTA in a dose-dependent manner but binding is not specific. (4) Human umbilical vein endothelial cells are moderately more sensitive to ricin A-chain-induced inhibition of protein synthesis and proliferation than simian virus-transformed mouse endothelial cells. (5) The morphologic changes are observed 1 h after exposure to the toxins, whereas inhibition of protein synthesis is not detectable until 4 h after a similar exposure. The in vitro model represents a first step in dissecting the complex events which occur in cancer patients who develop vascular leak syndrome after treatment with ricin A-chain-containing immunotoxins.     

The uptake and redistribution of 241pu within the gonads.

Male and female hamsters and a female rabbit were injected with 241Pu citrate. The hamsters were killed serially at 15 min, 2 hours, 1 day and 10 days after injection, and the rabbit 1 week after injection. The gonads were examined for 241Pu by tissue-section autoradiography. Soon after injection the plutonium was concentrated by the contents of atretic Graafian follicles and by thecal rings in the ovary, but was found to be dispersed throughout the testes. It is suggested that the disperse distribution in the testes which is only seen soon after injection may be an artefact of tissue processing. One day after injection, plutonium was accumulated by macrophages in both the follicles of the ovary and in the interstitial tissue of the testes. Macrophages containing plutonium later migrated away from the aretic ovarian follicles towards the ovarian medulla. This pattern of distribution and redistribution in the ovary is regarded as likely to lower the effective dose from a-emitting plutonium isotopes to the viable oocytes. No migration of macrophages was seen in the testes. Histochemical staining methods revealed the presence of acid protoglycans, including chondroitin sulphate, and glycoproteins at the sites of plutonium concentration in the ovary. These molecules are regarded as likely receptor sites for plutonium. In the testes no acidic carbohydrates were found, and it is suggested that the initial binding site for plutonium may be a compound lipid. This was deduced from the apparent inability of the interstitial tissue of the testes to bind plutonium in situ.     

A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.

We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples.     

Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition.

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.     

Protection Induced by Protein-Lipopolysaccharide Complexes in Tobacco Leaves: Role of Protected Tissue Free-space Solutes*

Hypersensitive confluent necrosis in tobacco leaves, caused by Pseudomonas syringae pv. aptata, is prevented by an intercellular injection of protein-lipopolysaccharide (pr-LPS) complexes 48 h earlier. An increase (48 %) in the peroxidase activity and a new low molecular weight protein (ε 17.5 kD) were found in the intercellular fluid of protected tissue (treated fluid) 48 h after pr-LPS infiltration. Up to 24 h after pr-LPS infiltration the treated fluid did not inhibit the in vitro growth of heterologous bacteria. Injected into tobacco leaves 30 min before the bacteria, the treated fluid retarded the rate of intercellular bacterial growth between 8 and 12 h after infiltration of the bacteria compared with the control fluid. Up to 4 h after injection, the attachment of live and dead bacteria to the cell walls occurred in both the protected tissue and in the control; 4 h after injection, however, the dead bacteria were only weakly attached in the protected tissue. Within 20 min after the intercellular infiltration, there was a decrease in the heterologous bacterial number in the protected tissue, 33 % less than that in the control tissue. Protected tissue free-space solutes did not have a direct antibacterial effect but activated an antibacterial response in the leaf tissue. Attachment of bacteria to the cell walls did not in itself trigger hypersensitive confluent necrosis. The protected tissue was initially more favourable for the survival of heterologous bacteria.     

A cytotoxic, photolabile cross-linking derivative of ricin : Action on various cells and application to the study of ricin toxicity

Ricin has been coupled to the cleavable, photolabile, hetero-bifunctional cross-linking reagent N-[4-(p-azidophenylazo)benzoyl] 3-aminopropyl-N¹-oxysuccinimide ester. Approx. 1.3–1.9 moles/mole of ricin are present in the conjugate. The conjugate is fully toxic when tested in the dark on intact cells. Cells, incubated with low amounts of the conjugate at 2 °C for 45 min and shifted to 37 °C for 3 h, become markedly protected against the effect of low concentrations of the toxin if irradiated after 2.5–3 h, when the toxic effect on protein synthesis in non-irradiated cells is maximal. Irradiation at intermediate times produces no or only partial protection. Less protection is obtained with high concentrations of the conjugate or in the presence of methylamine, a reagent that enhances uptake of ricin into cells.Subcellular fractionation of cells exposed in the dark to the photolabile ricin derivative for various times reveals that the conjugate is cross-linked to plasma membrane-derived fractions at early times. After 45 min at 37 °C the ricin is associated with subcellular fractions enriched in lysosomes and Golgi-derived elements and after prolonged times at 37 °C with subcellular fractions supporting RNA synthesis.