In the perspective of therapeutic approaches the monoclonal antibody, MBrl, with a quite restricted spectrum of reactivity for human breast carcinoma, was coupled to restrictocin (Res), a ribosome inactivating protein produced by Aspergillus restrictus. In a cell-free system this toxin was found to have an activity comparable to that of other plant toxins, but its in vitro toxicity was shown to be low on different cell lines. Three batches of MBr1-Res conjugate were prepared and their specificity, efficiency, and maximum level of cytotoxicity were analyzed on the cell line MCF-7 expressing the relevant antigen, on several irrelevant tumor cell lines, and on normal cells. Conjugates were from 600 to 1500 times more efficient than the uncoupled derivatized Res towards MCF-7 cells and were completely ineffective on the other target cells. The antigen-driven cytotoxicity was confirmed by the nontoxicity of an irrelevant conjugate on MCF-7 cells. The cytotoxic efficiency of MBr1-Res was low when compared to the binding level of MBr1 at the same concentration and a portion of treated cells (from 10% to 30%) survived the treatment. The heterogeneity of expression of the relevant antigen, together with its only partial internalization, could account for these limitations. The lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin were tested as potentiating agents but in both cases the cytotoxicity remained unmodified. A neutralization assay performed on a xenogenic model indicated that the MBr1-Res conjugate was capable of reducing the tumor take. These data indicate the possibility of using the Res to prepare a reproducible and highly selective breast cancer conjugate. However, there are still a number of problems which must first be solved before we can consider its clinical application.

The protein toxin restrictocin, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of M , 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restrictocin toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later.After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBrl, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBrl, MLuCl and MOvl7 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBrl, MLuCl and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites internalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.

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When Escherichia coli grows in a casamino acid-supplemented medium, the removal of amino acids results in the inability to synthesize tryptophanase even in the presence of the specific inducer. However the transcription of the tryptophanase gene is still operative, since readdition of amino acids, even in the presence of an inhibitor of RNA synthesis, elicits enzyme production. Therefore, under these conditions tryptophanase is synthesized in two distinct steps, (a) accumulation of untranslated mRNA during amino acid deprivation, and (b) translation of the previously accumulated mRNA on readdition of amino acids plus an inhibitor of RNA synthesis. The first step is stimulated by cyclic AMP and inhibited by compounds which cause transient repression, while the second step is unaltered by either type of effector. I n consequence it is concluded that cyclic AMP controls tryptophanase synthesis at the transcriptional level.The differential rate of synthesis of some inducible bacterial enzymes changes under different growth conditions [l]. Recent evidence indicates that the synthesis of these enzymes requires appropiate cellular levels of cyclic AMP and that the phenomena known as catabolite and transient repression result from a lowering of the levels of the cyclic nucleotide [2]. The requirement for cyclic AMP has been studied in detail in the case of B-galactosidase, using whole cells and cell-free systems from Escherichia coli [3 -51. The results obtained have demonstrated that cyclic AMP exerts a positive control on the initiation of lac mRNA synthesis. However, it has not been possible to investigate whether cyclic AMP affects also a transcriptional step of the synthesis of tryptophanase [ 6 ] , another cyclic AMP-dependent enzyme. In contrast, a stimulatory effect of cyclic AMP on tryptophanase translation was observed [7], and it has been proposed that B-galactosidase and tryptophanase are controlled by cyclic AMP through different mechanisms [2]. The experimental results reported here show that cyclic AMP stimulates tryptophanase synthesis a t the level of transcription and that transient repression inhibits tryptophanase production at the same level. These facts suggest that the control of the synthesis of tryptophanase by the cyclic nucleotide may be similar to that operating in the lac and gal operons [5,8].Unusual Abbreviations. Cyclic AMP, adenosine-3'-5'-monophosphate; mRNA, messenger ribonucleic acid.Enzymes. ,9-Galactosidase (EC 3.2.1.23) ; tryptophanase or tryptophan hydrolase (L-tryptophan --f indole + pyruvate + ammonia). ____ MATERIALS AND METHODS Biological MaterialsThe experiments were carried out with the strain RSl of E.coli. This strain did not grow on agar plates containing 5 pg/ml rifampicin and was selected from the strain 9483 of the National Collection of Industrial Bacteria (Aberdeen, Scotland).I n all the experiments the bacteria were grown aerobically at 37 "C in a mineral base [9] supplemented with 30mM glycerol, 0.6 pM thiamine hydrochloride and I0 mg/ml casamino acids. Cultures in...

Sheep immunoglobulin IgG1 and immunoglobulin IgG2 heavy chains were treated with cyanogen bromide. The fractions from the C-terminal end of the heavy chains were isolated and purified, and the amino acid sequences gamma1 and gamma2 heavy chains had the identical sequence: Met-His-Glx-Ala-Leu-His-Asx-His-Tyr-Thr-Glx-Lys-Ser-Ile-Ser-Lys-Pro-Pro-Gly. Comparison with the C-terminal peptides of other species, reported in the literature, suggests that the subclasses are the results of recent evolutionary processes. Residues at position 4 from the C-terminus may be phylogenetically related.