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Query: UNIPROT:P06889 (Mol)
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A monoclonal antibody specific for human EGF receptors was cross-linked to subunit A of toxic ricin. Using this conjugate, we isolated a variant of A431 cells, designated C1-B7, with approximately 40 times less EGF binding capacity. Unlike the parental cells, the C1-B7 variant was resistant to EGF-induced suppression of cell growth. The EGF receptors retained in this variant were of high-affinity type and susceptible to EGF-induced autophosphorylation. Membrane prepared from C1-B7 cells was highly phosphorylated in the presence of 2 microM [gamma-32P]-ATP, primarily on the lipid components shown as phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. This same level of lipid phosphorylation was observed on A431 membrane only in the presence of higher ATP concentrations. After addition of EGF to A431 membrane, phosphatidylinositol phosphorylation was significantly decreased with a concomitant increase in EGF-dependent protein phosphorylation. Thus, the EGF-dependent receptor-mediated protein phosphorylation precedes phosphatidylinositol phosphorylation. These observations support the idea that the growth inhibitory effect of EGF on A431 cells is caused by high ATP consumption due to the EGF-induced protein phosphorylation and reduction of phosphatidylinositol turnover.
Somat Cell Mol Genet 1985 Nov
PMID:Variant of A431 cells isolated by ricin A-conjugated monoclonal antibody directed to EGF receptor: phosphorylation of EGF receptor and phosphatidylinositol. 300 1

The A-chain of a plant toxin ricin has been coupled to poly- and monoclonal antibodies specific to the L-chains of human IgG. The inhibitory effect of the conjugates has been compared with the ability of the antibodies to bind to target cells. Cytotoxicity of the conjugates has been monitored following incorporation of 14C-leucine radioactivity into Burkitt lymphoma cells with surface Ig. The 50% inhibition of protein synthesis is observed 18 h after treatment of cells with immunotoxins, when the concentration of the conjugates with poly- and monoclonal antibodies is 1.2.10(-9) M and 0.7.10(-9) M, respectively. The data take into account that only part of the polyclonal antibodies molecules is able to react with target cells. The control conjugates containing either monoclonal antibodies that do not react with the lymphoma cells surface L-chains or nonimmune serum IgG proved to have no effect on target cells even at the level of 10(-7) M. The immunotoxins with poly- and monoclonal antibodies produce almost the same kinetics of protein synthesis inhibition, when incubated with lymphoma cells for 60 min. However, a 30 min treatment reveals a considerably higher cytotoxicity of the conjugate with monoclonal antibodies.
Mol Biol (Mosk)
PMID:[Selective cytotoxicity of antibody-ricin-A-chain conjugate for human tumor B cells. II. Comparison of activity of immunotoxins conjugated with poly- and monoclonal antibodies]. 314 80

We describe here the properties of a variant cell line, termed AF192, selected by exposing mouse LMTK- cells to a cytotoxic form of transferrin prepared by conjugating transferrin to diphtheria toxin. AF192 cells were mildly resistant to the transferrin-diphtheria toxin conjugate and were cross-resistant to the protein toxins modeccin, abrin, ricin, and Pseudomonas aeruginosa exotoxin A. AF192 cells had an aberrant transferrin cycle characterized by an approximately 50% reduction in the rate of iron uptake from diferric transferrin, an approximately 25% reduction in the number of surface transferrin receptors, and a time course for transferrin recycling that resolved into two apparent first-order rate processes. The aberrant transferrin cycle was not the result of a failure of endocytosed transferrin to discharge iron; rather, part, but not all, of the transferrin taken up by AF192 cells was diverted to an intracellular site from which it was recycled very slowly.
Somat Cell Mol Genet 1988 Sep
PMID:Two pathways of transferrin recycling evident in a variant of mouse LMTK- cells. 317 65

A variant cell line of Leishmania donovani (named R2D2) has been selected for resistance to the cytotoxic lectin ricin agglutinin and shown to be defective in the synthesis of its major glycoconjugate lipophosphoglycan. Compared to the parental line, R2D2 cells showed a marked resistance to the toxic effects of ricin and an increased sensitivity toward concanavalin A. The synthesis of lipophosphoglycan by R2D2 cells was judged to be less than 1% relative to that of wildtype cells as determined by incorporation of radioactive mannose and galactose and by electrophoretic and chromatographic analyses. Although lacking lipophosphoglycan, R2D2 parasites were capable of infecting cultured U937 macrophages.
Mol Biochem Parasitol 1988 Apr
PMID:A ricin agglutinin-resistant clone of Leishmania donovani deficient in lipophosphoglycan. 338 85

Protein A of Staphylococcus aureus was covalently bound to reduced ricin A chain toxin by N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate consisting mainly of one molecule of protein A bound to two molecules of A chains (Mr 107,000) was purified by tandem affinity chromatography on ConA-Sepharose 4B and IgG-Sepharose 4B. The purified protein A-A chain conjugate was able to bind and kill human lymphoma cells coated either with monoclonal mouse IgG2a anti-kappa antibody or with polyclonal rabbit anti-kappa antibody. The cytotoxic activity of protein A-A chain conjugate in conjunction with either mouse or rabbit anti-kappa antibodies was 10 times higher than that of rabbit IgG anti-mouse IgG coupled with A chain on Daudi cells coated with mouse anti-kappa antibody and 100 times higher than that of rabbit anti-kappa antibody coupled with A chain on non-coated Daudi cells. The cytotoxic effect of protein A-A chain conjugate on antibody-coated Daudi cells (9 x 10(-12) M) was comparable with that of ricin toxin on non-coated Daudi cells (2 x 10(-12) M). The results recommend the use of protein A-ricin A chain toxin conjugate as a unique specific toxin for the "in vitro" killing of antibody-coated target cells.
Mol Immunol 1988 May
PMID:Protein A vectorized toxins--II. Preparation and "in vitro" cytotoxic effect of protein A-ricin A chain conjugate on antibody coated human tumour cells. 341 31

Protein A of Staphylococcus aureus (mol. wt 43,000) was covalently bound to ricin toxin (mol. wt 60,000) by N-succinimidyl 3-(2-pyridyldithio) propionate. The conjugate consisting of one molecule of protein A bound to one molecule of ricin toxin (mol. wt 100,000) was purified by successive affinity chromatographies on IgG-Sepharose 4B and ConA-Sepharose 4B. The purified protein A-ricin toxin conjugate was able to bind and kill IgG antibody coated leukemia EL4 cells leaving unaffected EL4 cells not coated with antibody. The cytotoxic efficacy of the conjugate was comparable to that of nonconjugated ricin toxin. The results recommend the use of protein A-ricin toxin conjugate as a "universal" specific toxin for the "in vitro" killing of various antibody-coated target cells.
Mol Immunol 1986 Dec
PMID:Protein A vectorized toxins--I. Preparation and properties of protein A-ricin toxin conjugates. 349 27

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.
Mol Biol (Mosk)
PMID:[Effect of pH on the conformation and stability of the plant toxin ricin]. 360 Jun 23

Sodium butyrate (Btr) (3 mM) causes a 10-fold increase in production of the glycoprotein hormone alpha-subunit in HeLa cells. The following report demonstrates that this response could be inhibited about 95% by 5 mM 2-deoxy-D-glucose (dGlc), whereas alpha-subunit production in uninduced cells was affected little or not at all. Addition of D-mannose restored the Btr induction of Hela-alpha in cultures that had been treated with dGlc. When the alpha-subunits secreted by cells cultured in Btr plus dGlc or in Btr alone were compared by gel filtration (Sephadex G-75) and lectin affinity (concanavalin A and ricin) chromatography, differences were noted that probably reflect changes in their carbohydrate moieties. Immunoprecipitation of [35S]methionine-labeled HeLa-alpha and incubation with endoglycosidase H indicated that the subunit secreted from cells in the presence of dGlc contained oligosaccharide side chains that were not processed to the complex type. Cells that were simultaneously treated with Btr plus dGlc showed no increase in alpha-subunit production over cells receiving Btr only; in contrast, cells that were preincubated with Btr for either 16 or 36 h before dGlc was added exhibited high levels of subunit synthesis. Measurement of alpha-mRNA levels at various times after Btr and dGlc were added to cultures indicated that Btr brought about a dramatic increase in alpha-specific mRNA about 24 h after being added to cultures. This increase could be prevented by dGlc when added simultaneously with Btr but not when added after a 24-h preincubation. Although dGlc prevented the induction of alpha-subunit and alpha-mRNA in response to Btr, it had no effect on histone hyperacetylation, suggesting that if this chromatin modification is necessary for the induction process, it is not in itself sufficient. Together, the data demonstrate that dGlc inhibits the accumulation of alpha-subunit mRNA normally produced in response to Btr and that the subunit produced contains altered oligosaccharide constituents.
Mol Cell Biol 1987 May
PMID:Dual effects of 2-deoxyglucose on synthesis of the glycoprotein hormone common alpha-subunit in butyrate-treated HeLa cells. 360 Jun 39

Based on a galactose oxidase/NaB[3H]4 technique of radiolabeling macromolecules, the major glycoconjugate (lipophosphoglycan) of Leishmania donovani promastigotes was determined to be located on the cell surface. Incorporated radioactivity was analyzed by gel filtration on Sephadex G-100, chromatography on ricin agglutinin-coupled agarose, and lability to mild acid hydrolysis. Lipophosphoglycan was present throughout the various phases of growth of promastigotes, but was preferentially expressed during the latter part of logarithmic phase and in the stationary phase. In addition, metabolically labeled lipophosphoglycan was released into the culture medium. Expression of this unusual glycoconjugate on the cell surface of L. donovani suggests that it may play a major role in host cell-parasite interactions.
Mol Biochem Parasitol 1987 May
PMID:Cell surface lipophosphoglycan of Leishmania donovani. 361 72

The binding of the plant toxin ricin to various life cycle stages of Schistosoma mansoni has been studied. Fluorescein isothiocyanate (FITC)-ricin exhibited binding to schistosomula and adult worms, but not to cercariae or to freshly transformed schistosomula. Binding was specifically inhibited by the presence of galactose in the medium. Analysis of protein synthesis in treated parasites illustrated that ricin failed to intoxicate schistosomula or adult worms. Indeed, fluorescence quenching studies suggested that the fluorescent toxin was confined to the outer bilayer and was not internalised.
Mol Biochem Parasitol 1987 May
PMID:Interaction of the plant toxin ricin with different life cycle stages of Schistosoma mansoni. 361 73


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