PR-39, a proline/arginine-rich peptide antibiotic, has been purified from pig intestine and later shown to originate in the bone marrow. Intending to isolate a clone for a human counterpart to PR-39, we synthesized a PCR probe derived from the PR-39 gene. However, when this probe was used to screen a human bone marrow cDNA library, eight clones were obtained with information for another putative human peptide antibiotic, designated FALL-39 after the first four residues. FALL-39 is a 39-residue peptide lacking cysteine and tryptophan. All human peptide antibiotics previ-

A 78 residue antimicrobial, basic peptide, NK‐lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK‐lysin. The clone was used to show that stimulation with human interleukin‐2 induced synthesis of NK‐lysin‐specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin‐2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK‐lysin was present in cells positive for CD8, CD2 and CD4. NK‐lysin showed high anti‐bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK‐sensitive mouse tumour cell line, YAC‐1, but it did not lyse red blood cells. The amino acid sequence of NK‐lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK‐lysin is a new effector molecule of cytotoxic T and NK cells.

Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1 % trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three) : reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1 -11,l-15 and 1 -16, intact thymosin p-4, defensins HNPl, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contttin factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.

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Human 5-lipoxygenase (EC 1.13.11.34), the key enzyme involved in the transformation of arachidonic acid to the potent biologically active leukotrienes, has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus (3B6) carrying the human 5-lipoxygenase coding sequence downstream of the strong polyhedrin protein promoter was isolated. Approximately 48 hr after infection of Spodoptera frugiperda cells with the recombinant baculovirus, maximal intracellular enzyme activity and protein levels were dptected. The recombinant 5-lipoxygenase in 10,000 x g supernatant fractions was able to synthesize large amounts of 5-hydroperoxy-6,8,10,14-icosatetraenoic acid, together with smaller amounts of the nonenzymatic hydrolysis products of leukotriene A4, and exhibited a dependence on Ca2l and ATP for maximal activity. Immunoblot analysis of supernatant proteins from human leukocytes and recombinant virus-infected cells indicated the presence of indistinguishable '80-kDa bands. However, 5-lipoxygenase protein in recombinant-infected cells was found to be present in amounts 50-200 times that present in leukocytes on a per-cell basis. Histidine-362 and histidine-372, potential iron-atom ligands within a putative iron-binding domain, were changed to serine residues. Recombinant baculoviruses carrying the mutations were isolated and used to infect insect cells. Although infected cells were able to express mutant 5-lipoxygenase protein, enzyme activity was not substantially altered, suggesting the nonessential nature of these histidines in binding iron at the putative ferric catalytic site.