A murine homologue of the Drosophila brainiac gene shows homology to glycosyltransferases and is required for preimplantation development of the mouse.

The neurogenic gene brainiac was first isolated in Drosophila melanogaster, where it interacts genetically with members of the Notch signaling cascade. We have isolated a murine homologue of the Drosophila brainiac gene and delineated its highly specific expression pattern during development and adult life. We find particularly strong expression in the developing central nervous system, in the developing retina, and in the adult hippocampus. Targeted deletion of mouse Brainiac 1 expression leads to embryonic lethality prior to implantation. Null embryos can be recovered as blastocysts but do not appear to implant, indicating that mouse Brainiac 1, likely a glycosyltransferase, is crucial for very early development of the mouse embryo.

CD3X anti-nitrophenyl bispecific diabodies: universal immunotherapeutic tools for retargeting T cells to tumors.

We developed a universal recombinant bispecific molecule (BiMol) that is capable of redirecting cytotoxic T cells to tumor cells via tagged anti-tumor ligands such as antibody fragments or cytokines. A recombinant bispecific diabody with binding specificities for the CD3 molecule on T cells as well as for the hapten nitrophenyl (NIP) was produced. This bispecific molecule is capable of redirecting cytotoxic T cells to kill a series of malignant cells, including B cell lymphoma, Hodgkin's lymphoma, and colon carcinoma via NIP-conjugated ligands to tumor-associated antigens. Cytotoxic activity of the diabody was found to be comparable to tetradoma-derived bispecific antibodies with similar specificities. Our findings demonstrate that universal CD3xanti-NIP diabodies could be used for T cell based cellular immunotherapy in a variety of human malignancies. Additionally, these bispecific molecules allow fast and economic testing of tumor-associated antigens on malignant cells for their potential use as immunotherapeutic target structures if corresponding hapten-conjugated antibodies or ligands are available.

Sequence analysis of immunoglobulin heavy-chain variable region genes from the synovium of a rheumatoid arthritis patient shows little evidence of mutation but diverse CDR3.

To gain insight into the nature of B-lymphocyte responses in the synovium of rheumatoid arthritis (RA) patients, we amplified and sequenced immunoglobulin heavy-chain variable region genes expressed in seven IgM and three IgG-secreting synovial-derived hybridomas established from one patient. Each hybridoma V-region was encoded by unique VH-D-JH combination demonstrating that none of these hybridomas derived from clonally related B-lymphocytes in vivo. The expressed VH genes closely resembled (95.6%-100% homology) known germline VH genes in most hybridomas, including VH genes frequently used to encode autoantibodies. The antibodies produced by these hybridomas, with the exception of one IgM rheumatoid factor, did not bind to any of a large panel of autoantigens in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluoresence, suggesting that frequent expression of 'autoantibody-associated' VH genes does not correlate with detectable autoreactivity in this patient. Hybridoma CDR3 DNA was diverse in length and gene composition. Conserved heavy-chain cross-reactive idiotypes were expressed on 4/7 IgM- and 2/3 IgG-secreting hybridomas. The close similarity of expressed VH genes to germline counterparts of these hybridomas suggests that polyclonal activation is a prominent mechanism in B-lymphocyte activation in the synovium of this rheumatoid arthritis patient.

Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display.

Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. "Phage display" enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.

Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones.

Temporal and spacial distribution of mannopine synthase (mas) promoter activity was determined throughout the development of transgenic tobacco plants using bacterial luciferase luxA and luxB as reporter genes. Luciferase activity was determined by luminometry in vitro and visualized by computer-enhanced single-photon video imaging in vivo. The activity of the mas dual promoters increased basipetally in developing plants and was wound-inducible in leaf and stem tissue. Hormone bioassays with isolated plant tissues and tumors deficient in the transferred DNA (T-DNA)-encoded genes iaaM, iaaH, and ipt indicated that activity of the mas dual promoters is regulated by auxin and enhanced by cytokinin in both differentiated and tumorous plant cells.