[Mutations in beta'-subunit of the Escherichia coli RNA-polymerase influence interaction with downstream duplex DNA in the elongation complex]. / Mutatsii v beta'-sub''edinitse RNK-polimerazy Escherichia coli, vliiaiushchie na vzaimodeistvie s perednim dupleksom DNK v élongatsionnom komplekse.

RNA polymerase (RNAP) exhibits absolute processivity being capable of synthesizing RNA 10(3)-10(5) nucleotides in length without breaking contact with the DNA template. Stability of the elongation complex is thought to depend, in particular, on the RNAP-DNA interactions downstream along the run of transcription. We studied the effects of several deletions and insertions in the RNAP beta'-subunit N-terminal region, which presumably interacts with the downstream duplex DNA in the elongation complex. Most of the mutations obtained led to gross defects in RNAP assembly and disturbed catalytic activity of the enzyme. The mutations reduced stability of both promoter and elongation complexes, probably because they altered the contacts between RNAP and the downstream duplex DNA.

[Chimeric toxin of the A-subunit of viscumin and the B-subunit of ricin]. / Khimernyi toksin A-sub''edinitsy viskumina i B-sub''edinitsy ritsina.

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.

Comparison of the cytotoxic activity of the immunotoxins with different internalization rate.

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.

Cytotoxic effect of ricin A-chain conjugates containing monoclonal antibodies against human erythroid cells.

Selective elimination of human erythroblastoid cells by the conjugate of the A-chain of a plant toxin ricin (RA) and monoclonal antibody (MAb) HAE9 (IgM) directed against human erythroblast antigen (Ag-Eb) has been demonstrated. In comparative experiments, MAb HAE3 (IgM) against human glycophorin-A was used. On average, the conjugates obtained contained two A-chain molecules and one antibody molecule. Efficiency of cytotoxic action of native ricin and conjugates was compared both with the amount of binding sites on the surface of K562 cells and the internalization rate of these proteins. The association constants of the proteins proved to be almost the same (ka = 10(8) M-1). The ID50 values were 1.1 x 10(-11), 3.2 x 10(-10) and 3.1 x 10(-9) M for ricin, HAE9/RA and HAE3/RA, respectively. Ammonium chloride at a concentration of 10 mM increases the cytotoxic effect of the HAE9/RA conjugate approximately 10 times and does not change the activity of the HAE3/RA conjugate.

[Cytotoxic properties of a recombinant hybrid of the A protein of Staphylococcus aureus with a fragment of exotoxin A of Pseudomonas aeruginosa]. / Tsitotoksicheskie svoistva rekombinantnogo gibrida A-belka Staphylococcus aureus s fragmentom ékzotoksina A Pseudomonas aeruginosa.

By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and . Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.

Immunotoxin with mistletoe lectin I A-chain and ricin A-chain directed against CD5 antigen of human T-lymphocytes; comparison of efficiency and specificity.

Monoclonal anti-CD5 antibody was coupled to the enzymatically active subunit of plant toxin [either mistletoe lectin I (ML) or ricin]. The obtained conjugates proved to be selectively toxic to CD5-bearing target cells. The immunotoxin prepared from ML A-chain (MLA) was as toxic as native ML and approximately 80-fold more active than the corresponding conjugate with ricin A-chain (RTA). The comparative studies of the structural properties of isolated MLA and RTA were carried out using intrinsic fluorescence spectroscopy. The results showed similar properties for both proteins. No antigenic cross-reactivity against both toxins was detected when using polyclonal antibodies. The results suggest that MLA-antibody conjugates may be potential candidates for therapeutical use.