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The interaction of plant toxin ricin and MLI binding subunits to liposomes containing monosialoganglioside (GM1), bearing a terminal galactose residue, has been examined as a possible receptor model. For the first time we demonstrate that ricin B-chain but not ricin provokes liposome aggregation at 10 M% GM1 concentration, whereas in the presence of either ricin A-chain or galactose the aggregation is inhibited. The B-subunit of plant toxin MLI from Viscum album has similar lectin specificity and activity but cannot aggregate GM1 liposomes. The ability of the B-chain to aggregate liposomes adds a new crucial step in the toxin transmembrane penetration mechanism. We demonstrate here possible ricin B-chain interactions with membranes proceeding via two sites, namely (a) a galactose-binding domain and (b) a hydrophobic interchain domain. In close contact with two phospholipid bilayers, ricin B-chain may determine the geometry of the fusion site. These events can provoke A-chain translocation which follows membrane fusion.

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Conjugates of anti-CD25 monoclonal antibodies against cell surface IL-2 receptor with MLIA and RTA were prepared and investigated. Both of the immunotoxins had high specific cytotoxic activity on target cells. The IC50 value of the anti-CD25/MLIA immunotoxin was 15-fold greater than that of the anti-CD25/RTA. Previous studies of the anti-CD5 immunotoxins with MLIA and RTA showed that the anti-CD5/MLIA IT was 80-fold more active than anti-CD5/RTA IT [Tonevitsky et al. (1991) Int. J. Immunopharmacol. 13, 1037-1041]. The surface hydrophobicity of the MLI A-chain was 4-fold higher than that of the ricin A-chain as estimated by binding with ANS. In model experiments with small unilamellar DMPC liposomes, MLIA but not RTA increased the turbidity of liposome suspensions at pH 4.5. Our results indicate that the greater cytotoxic activity of the MLI A-chain immunotoxin probably provided a higher surface hydrophobicity of the protein and the ability to interact with phospholipid membranes.

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The cytotoxic effect of mistletoe lectin I (MLI) on TA5 hybridoma cells which produce monoclonal antibodies (mAb) to MLI A-chain (MLA) was investigated. In vitro cytotoxic tests with colorimetric assay were carried out for LD50 determination. TA5 hybridoma cells were 100 times more resistant to MLI and 30 times to chimeric toxin consisting of MLA and ricin B-chain (MLA/RTB) than control cells. The TA5 mAb (IgG1) recognized MLI A-chain in Western blotting and bound 125I-labeled MLI with Ka of 0.43 x 10(8) M-1. The TA5 and control hybridomas had the same number of 125I-labeled MLI binding sites. Therefore cell-surface TA5 antibodies did not influence MLI binding with the cell. The cytotoxic effect and binding of MLI were completely blocked in the presence of 20 mM lactose. Thus, MLI cytotoxicity was mediated only by cell-surface galactosyl residues; intracellular mAb molecules block MLI toxicity. Our data suggest that MLA molecules mediating cytotoxicity pass through an anti-MLA antibody-containing vesicular compartment and that mAbs inhibit the translocation activity of MLI A-chain from intracellular vesicles into the cytosol.

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Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested.

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Escherichia coli cells transformed with plasmids containing ricin B-chain coding sequences are shown to express this heterologous protein in inclusion bodies. After denaturation and renaturation of the product in the presence of glutathione and lactose, the recombinant ricin B-chain is soluble, biologically active and stable. Cytotoxicity of heterodimer containing this protein and ricin A-chain is found to be only ten times lower, than that of native ricin. Recombinant B-chain alone was nontoxic to cells (ID50 > 10(-6) M). Our data suggest that ricin B-chain oligosaccharides are essential for stability preserving protein from proteolytic degradation in cells.

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Monoclonal antibodies (mAb) reacting with native (TA5, TB12) and denatured (T33, T35) plant toxin mistletoe lectin I (MLI) from Viscum album have been obtained. The interaction between mAbs and native toxin with ML isoforms (MLII, MLIII) has been investigated. An immunological cross-reaction has been shown to take place for mAb TA5 (anti-A-chain of MLI) between MLII and MLIII isoforms of toxin. TA5 has not inhibited enzyme activity of the A-chain in a rabbit reticulocyte cell-free system. TB12 has been shown to react with the galactose-binding site of the B-chain. TA5 and TB12 have shown no cross-reaction with plant toxin ricin. The association constants for mAbs have been determined. The nature of heterogeneity of the lectins from Viscum album is discussed.

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A chimeric toxic protein was prepared from the mistletoe lectin I A-chain and ricin B-chain by using the disulfide exchange reaction. Ricin and chimeric protein were indistinguishable in binding to immobilized asialofetuin in ELISA. The chimeric protein was more toxic for Jurkat cells than native mistletoe lectin I, but not as effective as native ricin. In the presence of NH4Cl, which enhances the toxicity of some toxins and immunotoxins, but does not influence ricin toxicity, both ricin and chimeric toxin had equal cytotoxic activity. The possibility is discussed that the ricin B-chain protects the ricin A-chain from degradation during delivery from the cell surface to the place where it is translocated into the cytosol.

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Chimeric toxin protein was prepared from the mistletoe lectin I A-chain and ricin B-chain by using the disulfide exchange reaction. Ricin and chimeric protein were indistinguishable in binding to immobilized asialofetuin in ELISA. Chimeric protein was more toxic to Jurkat cells than native mistletoe lectin I, but not so effective as native ricin. In the presence of NH4Cl, which enhances the toxicity of some toxins and immunotoxins, but does not influence ricin toxicity, both ricin and chimeric toxin had equal cytotoxic activity. The possibility is discussed that the ricin B-chain protects the ricin A-chain (RTA) from degradation during delivering RTA from the cell surface to the place where RTA is translocated into the cytosol.

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In comparative experiments the conjugates of A-chain of a plant toxin ricin (RTA) and monoclonal antibody (MAb) HAE9 (IgM) directed against human erythroblast antigen (Ag-Eb) or Mab HAE3 against human glycophorin-A and immunotoxins (IT) directed to CD5 and CD7 antigens of human T-lymphocytes have been investigated. Proceeding from the number of receptors on a cell, we compared efficiency of the cytotoxic effects of the conjugates with different internalization rate. The efficiency of immunoconjugates against Ags with an extremely high internalization rate was only slightly higher than that of immunoconjugates against Ags with a lower internalization rate. The enhancing effect of ammonium chloride on immunoconjugate cytotoxicity was more pronounced in the case of immunotoxins with a high internalization rate.

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Hypoxia is a severe stress factor to which man and most other mammalian species are capable of adapting. However, the cellular mechanisms that enable cells to tolerate decreases in ambient oxygen tension are still unknown. We have previously shown that hypoxia induces the synthesis of unique proteins (molecular mass 38, 52, 74, 76 kDa) in human aortic endothelial cells and lymphocytes. In this study we investigated the specificity of hypoxia on the upregulation of these hypoxic stress proteins (HYP) in human peripheral blood lymphocytes and the role of calcium in this response. 35S-methionine pulse-labeling studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis and autoradiography demonstrated that normobaric hypoxia (4% O2-5% CO2-91% N2) enhanced synthesis of HYP, whereas heat-shock protein synthesis was not affected. Heat shock (42 degrees C) and cold stress (4 degrees C) did, however, induce synthesis of heat-shock protein but not HYP. The 38-kDa HYP is the major protein for which synthesis is upregulated by hypoxia. Its isoelectric point (pI) is 3.5-4.0, and it is localized in the cytosol. The 52-kDa HYP has a pI of greater than 6.5, and it is also localized in the cytosol. The 74- and 76-kDa HYPs appear to be membrane bound. In addition to hypoxia, an increase in calcium concentration in the culture media (25-50 mM) enhanced synthesis of HYP. An ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)/Ca2+ binding complex, when added to blood lymphocytes during exposure to hypoxia, significantly inhibited HYP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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