Nonspecific Cytotoxic Cells Express Cytosolic And Membrane Histone-Like Proteins   Donald L. Evans, Harjeet Kaur, Mustapha Oumouna and Liliana Jaso-Friedmann.   University of Georgia, Athens, GA   Many laboratories have previously described the antimicrobial activities of cellular lysates and tissue extracts obtained from many different teleost species.  Among these “antibiotics”, one class of endogenous type protein represents an especially novel approach to explaining mechanisms of innate anti-bacterial immunity.   In the present study, teleost nonspecific cytotoxic cells (NCC) were treated in vitro for 24h with synthetic oligodeoxynucleotides (ODNs) and whole cell lysates were examined for the presence of upregulated proteins by ligand blots using biotinylated ODNs.  An 18 kDa molecule was identified and subjected to tryptic digestion and analysis.  Amino acid sequencing of a 13-mer peptide revealed it was identical to human histone (H1).  In order to determine whether NCC expressed membrane receptors for ODNs, cells were activated with ODNs, labeled with NHS-sulfo-Biotin and cell lysates were prepared.  Surface membrane biotinylated lysates were next mixed with ODN-digoxigenin and these complexes precipitated with immobilized anti-digoxigenin.  Immunoprecipitates in the Ligand Blots were visualized with Avidin-Peroxidase.  Depending on the nucleotide sequence of the Dig-labeled ODN, 2-3 different molecular weight species of DNA binding receptors were identified.  Additional preliminary studies were done to determine (by flow cytometry) whether histone-like proteins (HLP) were expressed on NCC membranes.  Purified NCC were stained with commercially available polyclonal antibodies against a 23 kDa histone (H1) protein and a 15 kDa histone core protein.  Both polyclonals bound to NCC in relatively high proportions.  These data demonstrated that NCC express both cytosolic and membrane HLP.