A molecular modeling study on binding of drugs to calmodulin.

Computer-graphical methods have been used to study the interaction between a series of drugs and calmodulin. Based on the X-ray crystallographic coordinates of the alpha-C atoms of calmodulin, a molecular model of the helical sequences was built. The model has been used to derive two possible binding sites for phenothiazines and one binding site for penfluridol. The principal binding forces occur through contacts between acidic amino acids of calmodulin and the protonated side-chain nitrogen of the drugs as well as between a basic amino acid and the electronegative substituents of the aromatic rings. Calculated interaction energies show a good correlation with experimental inhibition data.

Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation.

Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity.

Role of alphabeta and gammadelta T cells in the host response to Salmonella infection as demonstrated in T-cell-receptor-deficient mice of defined Ity genotypes.

Salmonella spp. are facultative intracellular bacteria which enter the body through the intestinal tract. We studied the roles of T cells expressing either the alpha and beta chains or the gamma and delta chains of the T-cell receptor (alphabeta T cells or gammadelta T cells, respectively) in the host defense against Salmonella using mice genetically deficient in either alphabeta T cells, gammadelta T cells, or both T-cell subsets. These mutant strains of mice were infected orally or intraperitoneally with Salmonella dublin, and the progression of the disease was monitored by determining bacterial numbers in the feces, gut wall, Peyer's patches, mesenteric lymph nodes, spleen, and liver. Since susceptibility to Salmonella infection in mice is strongly affected by the alleles at the Ity locus, T-cell-mutant mice with either the Ity-sensitive or Ity-resistant phenotype were tested for resistance to S. dublin infection. We found that even though large numbers of intraepithelial and mucosal alphabeta and gammadelta T cells populate the normal intestine, they have no role in controlling the invasion of S. dublin into the intestine or the subsequent bacterial replication in the Peyer's patches or gut wall. Furthermore, systemic infections were equally severe for the first 6 days in normal, alphabeta T-cell-deficient, and gammadelta T-cell-deficient mice, and alphabeta but not gammadelta T cells were required for clearance of S. dublin, regardless of the Ity phenotype. However, mice that lacked both T-cell subsets had higher bacterial counts in their livers 15 to 18 days after infection than did alphabeta T-cell-deficient mice, suggesting that gammadelta T cells can contribute to acquired immunity to S. dublin.

Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells.

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria-mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.