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Abstract: In order to explore the properties of chemotactic N-formylpeptides containing isopeptide bonds within their backbones, a group of lysine-containing analogs of the prototypical chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO-Met or Boc-Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N[sup α] or N[sup ε] bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO-Met-Lys(Z)-Phe-OMe (2a, b), RCO-Met-Lys(Z-Phe)-OMe (3a, b), Z-Lys(RCO-Met)-Phe-OMe (4a, b), Z-Phe-Lys(RCO-Met)-OMe (5a, b), RCO-Met-Phe-Lys(Z)-OMe (6a, b) and Z-Lys(RCO-Met-Phe)-OMe (7a, b), with R=OC(CH[sub 3])[sub 3] and R=H for compounds a and b, respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b) and antagonists (compounds a). All N-formyl derivatives 2b-7b are less potent than fMLF-OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a-7a do not show antagonistic activity towards fMLF induced chemotaxis and O[sub 2][sup -] production, however, all these compounds except 4a antagonize lysozyme release by 60%. [ABSTRACT FROM AUTHOR]

Abstract: We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II β-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an α-helix within VRRSL and a type II β-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No β-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the β-turn formation in the REGD sequence. [ABSTRACT FROM AUTHOR]

Human neutrophils can damage the non-immunized K562 cell line when stimulated by phorbol myristate acetate (PMA). The combination of the activation of protein kinase C by phorbol and the increase of free intracellular calcium by ionophore potentiates the lytic reaction. The OKT9- immunized target cells are not able to induce by themselves the lytic response in neutrophils. hut by combining the two signals, the antigenic stimulus and PMA. a high level of cytolytic response is attained. The addition of EGTA does not affect neutrophil cytotoxicity against antibody-coated targets. while it markedly reduced the lytic reaction against non-immunized targets: in contrast, the addition of ECTA together with the ionophore ionomycin completely suppresses the lysis of immunized and non-immunized targets. The treatment of neutrophils with the protein kinase C inhibitor H-7 causes a dose-related inhibition of the lytic functions that is greater on unsensitized K562. Thus the interaction of Fc receptors with immunized targets is required for reaching the maximal cytolysis. The enhanced lytic activity that occurs in the presence of immunized targets is mediated by calcium flux. as detected by using the monoclonal antibody AB8.28 which hinds to FcIII receptors (FcRIII), thus supporting that both signals are involved. [ABSTRACT FROM AUTHOR]

The human erythroid myeloid leukaemia cell line K562 was used as target for human neutrophil cytotoxicity. Neutrophils demonstrated cytotoxicity against K562 only in the presence of a second stimulus, tetradecanoil phorbol acetate (TPA), a result consistent with previous observations.We now demonstrate that antibody-coated K562 (using OKT9 and 345 monoclonal antibodies) are similarly only sensitive to neutrophils when TPA is added. The presence of both antibody and TPA in the cytotoxic assay resulted in significantly higher levels of cytotoxicity than in the absence of antibody; the result being consistent with a synergistic action between protein kinase C activation and Fe receptor perturbation in the neutrophil. The cytotoxicity against non-coated and antibody-coated targets was markedly inhibited, particularly against the former, by the protein kinase C inhibitor, l-(5-isoquinollne-sulfonyl)-2-methyl piperazine (H-7). There were marked differences in the extra-cellular calcium dependency of the two types of cytotoxicity reactions. TPA-activated respiratory burst was unaffected by the presence of non-coated and OKT9-coated targets, whereas TPA-induced lysosomal enzyme release was significantly increased by non-coated targets and a further increase occurred in the presence of OKT9-coated K562. [ABSTRACT FROM AUTHOR]