Modification of the carbohydrate in ricin with metaperiodate and cyanoborohydride mixtures: effect on binding, uptake and toxicity to parenchymal and non-parenchymal cells of rat liver

The carbohydrate in the toxic glycoprotein ricin was chemically modified by simultaneous treatment with sodium metaperiodate and sodium cyanoborohydride. This treatment causes oxidative cleavage of the sugar residues and reduction of the aldehyde groups which are formed to primary alcohols. The modification markedly decreased the rapid removal of ricin from the blood by hepatic non-parenchymal cells with only a relatively small increase in accumulation of the toxin by parenchymal cells. Binding, uptake and toxicity of the modified ricin in primary monolayer cultures of hepatic non-parenchymal cells were all decreased to a much greater extent than in parenchymal cells. The results indicate that native ricin binds to non-parenchymal cells by a dual recognition process which involves both interaction of cell receptors with the mannose-containing oligosaccharides of the toxin and binding of ricin to galactose-containing glycoproteins and glycolipids on the cells. However, uptake and toxicity of native ricin in non-parenchymal cells appears to result principally from entry of the toxin through the mannose recognition pathway. By contrast, uptake and toxicity of the expressed essentially through the galactose-recognition route.     

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.     

The preparation of deglycosylated ricin by recombination of glycosidase-treated A- and B-chains: effects of deglycosylation on toxicity and in vivo distribution

Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target.     

Modification of vesicle surfaces with amphiphilic sterols. Effect on permeability and in vivo tissue distribution

In this paper, we describe the permeability of vesicles prepared with various synthetic cholesterol derivatives. Cholesterol derivatives with side-chains ending in hydroxyl groups reduced the permeability of unilamellar vesicles. However, addition of cholesterol derivatives with terminal amino groups makes the vesicles more permeable. Vesicles prepared with a short-chain amino-cholesterol derivative were found to be less permeable in phosphate-buffered saline, but not in bovine serum, while long-chain amino-cholesterol-containing vesicles were very permeable in both media. Studies in vivo indicate a rapid clearance rate for intravenously administered amino-cholesterol-containing vesicles with a concomitant increase in liver uptake. However, no difference was found in either the clearance or tissue distribution of control vesicles and the less permeable hydroxyl-cholesterol-containing vesicles.     

The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity

The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the sarcin-ricin-loop (SRL) on the 60S ribosomal subunit is a major source of its toxicity. The model proposes that SRL depurination leads to protein synthesis inhibition, evoking ribotoxic stress with concomitant induction of numerous metabolic pathways, which lead to cell death. However, the direct relationship between the depurination and its impact on the translational machinery in vivo has never been satisfactorily explained. In this work, we approached a long-standing question about the influence of SRL depurination on the functioning of the translational machinery in vivo. We have shown that an already low level of depurinated ribosomes exert an effect on cell metabolism, indicating that minute modification within the ribosomal pool is sufficient to elicit a toxic effect. Importantly, depurination does not affect notably any particular step of translation, and translational slowdown caused by ricin is not a direct consequence of depurination and cannot be considered as the sole source of cell death. Instead, SRL depurination in a small fraction of ribosomes blocks cell cycle progression with no effect on cell viability. In this work, we have provided a comprehensive picture of the impact of SRL depurination on the translational apparatus in vivo. We propose that ribosomes with depurinated SRL represent a small imprinted ribosomal pool, which generates a specific signal for the cell to halt the cell cycle.     

Potentiation of TNF-alpha toxicity by conjugation with ricin A-chain

Hybrid toxins containing a cytokine moiety have been used effectively to selectively kill cells expressing the complementary cytokine receptor both in vivo and in vitro. To date all cytokines incorporated into hybrid toxins, e.g. interleukin 2 are biologically active as monomers, so attachment of a toxin group causes minimal interference with the cytokine structure. By contrast, the pro-inflammatory and anti-cancer cytokine tumour necrosis factor alpha (TNF-alpha) is biologically active as a homotrimer in which the grooves created between the hydrophobically associated monomers form the receptor binding region, so maintenance of this structure is crucial for activity. In this report the authors show that TNF-alpha can be modified by reaction with a crosslinking agent and by subsequent attachment of the toxin ricin A-chain without loss of TNF-alpha cytotoxic activity in the WEHI assay. Structural association of the hybrid toxin composed of TNF-alpha and ricin A-chain was confirmed by Western blot analysis. The hybrid toxin was toxic to HeLa cells (IC50=4 pM) not sensitive to native TNF-alpha, and sensitive WEHI cells with substantially increased lethality (LD50=0.01 fM). This increased TNF-alpha cytotoxic activity suggests that hybrid toxins containing TNF-alpha may have therapeutic applications in the treatment of cancer.     

Effects of carbohydrate headgroups on the stability of induced cubic phases in binary mixtures of glycolipids

This paper is part in a series of papers, investigating the influence of carbohydrate headgroups on the mesogenic properties of glycolipids. While previous papers focussed on the synthesis and mesogenic properties of the pure compounds, we will discuss here our results obtained with binary mixtures. Mixtures of compounds, one forming a lamellar phase and the other one a columnar phase in their pure state, displayed always an induced cubic phase. The stability of this induced cubic phase depends significantly on the structure of the carbohydrate headgroup of both components. Thus it was possible to derive structure–property relationships by comparison of the phase diagrams that have been obtained, if the carbohydrate headgroup of one component was changed systematically. We observed an interesting effect of galactose headgroups which might be of great biological importance. Furthermore, the observed kind of kinetic of the S_A→cub transition might also be of great biological relevance.     

Effects of electrical stimulation in vivo of M. pectoralis on carbohydrate metabolism with reference to certain liver and blood values in the pigeon

The pectoralis muscles of two groups of anaesthetized pigeons were exercised in vivo by electrical stimulation for periods of 1 h and 5 h respectively. There was no significant change from controls in the level of blood glucose in both groups. Blood lactate level was significantly higher in the exercised groups but was relatively lower in the 5-h control group in comparison with its 1-h counter part. Blood lactate dehydrogenase (LDH) activity was significantly higher in the 1-h stimulated pigeons as was also the case with liver LDH in the same group but markedly lower in the 5-h ones. No significant change was seen in liver glycogen content in the stimulated pigeons. Liver phosphorylase activity was markedly low in the 5-h stimulated pigeons as was also the case with liver LDH activity. Circulating level of corticosterone was significantly higher in both the stimulated groups. Blood thyroxine (T4) as well as triiodothyronine (T3) levels were considerably reduced in both stimulated groups. The T3/T4 ratio was higher in the 5-h stimulated pigeons. It was concluded that, while initially carbohydrate was used as fuel for exercise, in prolonged exercise, lipid became the chief fuel as was shown in earlier studies. While fat continued to be used as the main fuel, carbohydrate was spared and also gluconeogenesis was enhanced. It was also concluded that the r?le of the thyroid hormones in promoting oxidative metabolism was enhanced by markedly increasing peripheral deiodination of T4 to T3 in prolonged exercise.     

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.     

The effects of cyclophosphamide on the toxicity and immunogenicity of ricin A chain immunotoxin in rats

We conducted a study to determine if treatment with cyclophosphamide (CY) could suppress the formation of anti-murine and anti-ricin A chain antibodies in rats treated with a murine monoclonal antibody-ricin A chain immunotoxin (IT). Female Sprague-Dawley rats received intravenous doses of IT at a dose of 5 mg/kg body weight alone or in combination with CY at a dose level of either 10 or 20 mg/kg body weight. The IT was given as one or two courses consisting of five consecutive daily intravenous injections (days 0 to 4, or days 0 to 4 and days 21 to 25 of the study). Cyclophosphamide was given on days 2, 4, 6, 13, and 17 of the study to the group receiving a single course of IT; additional doses of CY were administered on days 23, 25, and 27 to the group receiving two courses of IT. On days 4, 14, 21, 28, and 35, animals from each group were evaluated for antibodies to murine IgG and ricin A chain, and for clinical laboratory parameters and histopathology. Animals receiving IT alone developed significant titers of both anti-murine and anti-ricin A chain antibodies. Compared with the response in the animals receiving single-course IT, the response to both of the components of the IT was significantly increased on days 28 and 35 in the animals receiving a second course of IT. The groups receiving a combination of either one or two courses of CY and IT demonstrated a significantly decreased antibody response to both the murine IgG and the ricin A chain compared with the group receiving IT alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sialoglycoprotein and carbohydrate complexes in chromium toxicity

Chromium(VI) compounds are amongst the most widely encountered industrial carcinogens and are of increasing concern with respect to environmental exposure. Sialoglycoproteins and carbohydrates play a crucial role in stabilizing oxoCr(V) intermediates, which are produced by extracellular and intracellular reduction of chromium(VI). Recent research has addressed the molecular characterization of oxoCr(V)-sialoglycoprotein and -carbohydrate complexes and the roles that these species may play in Cr(VI) metabolism and carcinogenesis. Particular highlights include the role of oxoCr(V) complexes of extracellular sialoglycoproteins, intracellular D-glucose, and related species and their potential roles in Cr(VI)-induced genotoxicity.     

Effects of ricin on the ribosomal sites involved in the interaction of the elongation factors.

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell-free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)-directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible. Ricin specifically inhibits elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes but has no effect on the non-enzymic binding of aminoacyl-tRNA. Ricin also inhibits formation of the complex elongation-factor-2 - ribosome - nucleotide with GTP, GDP or GMP-P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.     

Modification of the action of misonidazole. I. Effect of TAN on toxicity

The toxicity of misonidazole, an electron affinic radiosensitizer, is greatly reduced by TAN, a free radical radiosensitizer. The production of single-strand breaks in DNA of mammalian cells incubated in dilute suspension with misonidazole (15 mM) under hypoxic conditions is greatly decreased by the presence of TAN (10 mM). The survival of such cells is also greatly enhanced if TAN is present at a concentration of 10mM even less. The implications and possible mechanisms of this finding are discussed.     

Effects of carbohydrate supplementation on performance and carbohydrate oxidation after intensified cycling training.

To study the effects of carbohydrate (CHO) supplementation on performance changes and symptoms of overreaching, six male endurance cyclists completed 1 wk of normal (N), 8 days of intensified (ITP), and 2 wk of recovery training (R) on two occasions in a randomized crossover design. Subjects completed one trial with a 6% CHO solution provided before and during training and a 20% solution in the 1 h postexercise (H-CHO trial). On the other occasion, subjects consumed a 2% CHO solution at the same time points (L-CHO). A significant decline in time to fatigue at approximately 63% maximal power output (H-CHO: 17 +/- 3%; L-CHO: 26 +/- 7%) and a significant increase in mood disturbance occurred in both trials after ITP. The decline in performance was significantly greater in the L-CHO trial. After ITP, a significant decrease in estimated muscle glycogen oxidation (H-CHO: N 49.3 +/- 2.9 kcal/30 min, ITP 32.6 +/- 3.4 kcal/30 min; L-CHO: N 49.1 +/- 30 kcal/30 min, ITP 39.0 +/- 5.6 kcal/30 min) and increase in fat oxidation (H-CHO: N 16.3 +/- 2.4 kcal/30 min, ITP 27.8 +/- 2.3 kcal/30 min; L-CHO: N 16.9 +/- 2.6 kcal/30 min, ITP: 25.4 +/- 3.5 kcal/30 min) occurred alongside significant increases in glycerol and free fatty acids and decreases in free triglycerides in both trials. An interaction effect was observed for submaximal plasma concentrations of cortisol and epinephrine, with significantly greater reductions in these stress hormones in L-CHO compared with H-CHO after ITP. These findings suggest that CHO supplementation can reduce the symptoms of overreaching but cannot prevent its development. Decreased endocrine responsiveness to exercise may be implicated in the decreased performance and increased mood disturbance characteristic of overreaching.     

The toxicity to fish of mixtures of poisons: II. Copper-ammonia and zinc-phenol mixtures

Tests with rainbow trout in mixtures of ammonium chloride with copper sulphate and of phenol with zinc sulphate have shown that the threshold of toxic concentration for a 50% mortality occurs in solutions for which a value of I is obtained by summing the concentrations of the individual poisons expressed as fractions of their individual threshold concentrations. With ammonia-copper mixtures this method of predicting the threshold concentration becomes progressively less adequate as lower percentage mortalities are considered.     

Effect of prostaglandins in vivo on enzymes involved in thyroid carbohydrate metabolism.

Treatment of adult guinea pigs with prostaglandins produces changes in the levels of enzymes involved in carbohydrate metabolism of the thyroid gland. A decrease in glucose-6-phosphate dehydrogenase activity is observed with a concomitant increase in 6-phosphogluconic dehydrogenase; the glycolytic enzymes appear unaffected by the same treatment. The results indicate that prostaglandins do not have the biochemical effects obtained with thyrotropin and cAMP administration, showing that these compounds play an antagonistic role in comparison with the above mentioned stimulating agents.