Recombinant anti-polyamine antibodies: identification of a conserved binding site motif.

Polyamines are small linear polycations found ubiquitously in eukaryotic cells. They are involved in nucleic acid and protein synthesis and rises in cellular polyamine levels have been correlated with cell proliferation. Antibodies to these molecules have potential as prognostic indicators of disease conditions and indicators of treatment efficacy. Antipolyamine monoclonal antibodies of differing but defined specificities have been generated in our laboratory using polyamine ovalbumin conjugates as immunogens. These antibodies show small but significant cross reactivities with other polyamine species; IAG-1 cross reacts with spermidine (8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine (11%) and spermine (6%). We have rescued and sequenced the heavy and light chain variable regions of all three of these antibodies. While the light chains of two antibodies, IAG-1 and JSJ-1, were 93% homologous at the amino acid level, none of the heavy chains displayed any significant sequence homology. However, computer-generated models of all three antibody binding sites revealed a three-dimensionally conserved polyamine binding site motif. The polyamine appears to bind into a negatively charged cleft lined with acidic and polar residues. The cleft is partially or completely closed at one end and the specificity of the interaction is determined by placement of acidic residues in the cleft. Aromatic residues contribute to polyamine binding interacting with the carbon backbone. The polyamine-binding motif we have identified is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.

Humanization of the murine anti-human CD3 monoclonal antibody OKT3.

OKT3 is a murine monoclonal antibody which recognizes an epitope on the epsilon-subunit within the human CD3 complex. OKT3 possesses potent immunosuppressive properties in vivo and has been proven effective in the treatment of renal, heart and liver allograft rejection. Despite its efficacy, significant problems remain associated with OKT3 therapy, i.e. T-cell activation and the anti-murine antibody response. To address the problem of the anti-murine antibody response we have constructed humanized versions of OKT3. One of the humanized derivatives, gOKT3-7 incorporating the OKT3 complementarity determining regions plus a small number of alterations to the human framework, has an affinity of 1.4 x 10(9) M-1 compared with 1.2 x 10(9) M-1 for the murine OKT3. A humanized antibody (gOKT3-1) incorporating only the CDRs from OKT3 was found to be functionally inactive, confirming the requirement for nonCDR substitutions. gOKT3-7 retains the ability of mOKT3 to induce T cell proliferation in vitro and appears to be a good candidate for further development for in vivo therapy.

Colchicine induced polyploidy in chickpeas (Cicer arietinum L.).

The most successful induction of tetraploidy was obtained with 2 hour treatment by 0.25% aqueous colchicine solution of 18-hour watersoaked desi chickpeas material. However, kabuli types needed only 1 hour treatment under similar conditions. Gigantism accompanied induction of polyploidy in desi as well as kabuli types but yield and fertility were greatly reduced. The meiotic abnormalities accompanying polyploidy were multivalent association of chromosomes followed by unequal disjunction, chromosome bridges, laggards etc. The percentage of stainable pollen, however, was at par between diploids and tetraploids. Gene control of percentage seed setting was observed in both levels of ploidy. A striking feature of the studies was the high seed setting percentage in 4n F 1 material resulting from diverse crosses, viz., desi×kabuli. A probable reduction in multivalent association coupled with yield increases in segregants from the later generation of tetraploids indicates the possibility of selection for higher yield and fertility from polyploids, particularly from some hybrid material.