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PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M. Ferrati, C. Piubello, F.C. Marincola, E. Gandini, J. Mol. Evol. 57 (2003) 131-139] is synthesized following protein kinase C (PKC) activation and acts as a PKC modulator, establishing a feedback loop of inhibition. In this work, PKI55 was found to inhibit recombinant alpha, beta(1), beta(2), gamma, delta, zeta and eta PKC isoforms; the effect on conventional PKC was lost in the absence of calcium. Confocal immunofluorescence experiments showed that PKI55 can penetrate into peripheral blood mononuclear cells (PBMC), following a coordinated movement of calcium ions. The addition of PKI55 protein down-regulated the PKC enzyme activity in phytohaemagglutinin-activated PBMC, decreasing the activity of alpha, beta(1) and beta(2) PKC isoforms. Moreover, inhibition in PBMC proliferation was observed. Similar effects were detected in Jurkat T cells transfected with a plasmid containing the coding sequence of PKI55. The PKI55 protein functional role could be to control the pathological over-expression of specific PKC isoforms and to regulate proliferation.

Neutrophils constitute the first line of defence against bacterial invasion. They migrate to infected tissues along a concentration gradient of chemoattractant molecules, the most important of which is for-Met-Leu-Phe-OH (fMLP). Different responses arise from formylpeptides binding to different isoforms of the specific receptor. The aim of the studies reported herein was to clarify (i) the role of fMLP-OMe amide bonds in receptor-ligand cross-linking, (ii) the nature of the group occupying the N- and C-terminal positions, (iii) the features peculiar to the Met, Leu, and Phe receptor pockets, and (iv) the features which determine the specific neutrophil response.

A series of new beta-peptido sulfonamides, related to the chemotactic tripeptide fMLF-OMe, has been synthesized. The examined 1a,b-7a,b models contain the achiral -HN-(CH(2))(2)-SO(2)- taurine (Tau) residue or the chiral -HN-CH(nBu)-CH(2)-SO(2)- and -HN-CH(iBu)-CH(2)-SO(2)- residues, corresponding to the beta-aminocarboxylic acid counterparts beta(3)-HNle and beta(3)-HLeu, respectively. The biological activity of the new analogues has been determined on human neutrophils and compared with that of the reference ligand as well as that of the previously studied related models. The results are analyzed in terms of structure-activity relationships. The conformational preferences of the new tripeptides 1b and 2b, containing a central chiral beta-amino-ethanesulfonic acid residue, have also been discussed.

New synthetic analogues of the chemotactic N-formyltripeptide HCO-Met-Leu-Phe-OMe have been synthesized. The reported new models, namely Boc-Met-beta-Ala-Phe-OMe (1), HCO-Met-beta-Ala-Phe-OMe (2), Boc-Met-Tau-Phe-OMe (3), HCO-Met-Tau-Phe-OMe (4) and HCl.Met-Tau-Phe-OMe (5), are characterized by the presence at the central position of a residue of beta-alanine or 2-aminoethanesulfonic acid (taurine) replacing the native L-leucine. Whereas tripeptides 1 and 2 have been found quite inactive as chemoattractants, all the three models containing the Tau residue exhibit a remarkable activity. Superoxide anion production and lysozyme release have been also evaluated and the biological results are discussed together with the conformational preferences of the examined models.

The new disulphur-bridged peptide, for-Met-Leu-Cys(OMe)-Cys(OMe)-Leu-Met-for, has been synthesized and its biological properties resulting from its binding to the formyl-peptide receptor of human neutrophils characterized. Three activities resulting from this interaction were measured: directed cell migration (i.e., chemotaxis); superoxide anion production; and lysozyme enzyme release. The properties were compared with those observed for the prototypical peptide, for-Met-Leu-Phe-OMe. Chemotaxis is strongly triggered while both superoxide anion production and lysosomal enzyme release are elicited only at high concentrations and never reach the response peak observed for the prototype peptide at physiologically relevant concentrations. The derivative appears to bind with a good affinity to the formyl-peptide receptors. These results provide new information regarding the structure-activity relationship of the formyl-peptide receptor.

We report here the synthesis and activity of new analogs of the N-formyl and N-tert-butyloxycarbonyl (Boc) derivatives of the tripeptide Met-Leu-Phe-OMe containing an achiral omega-amino acid residue replacing the hydrophobic central leucine. The tripeptides HCO-Met-NH-(CH2)n-CO-Phe-OMe and Boc-Met-NH-(CH2)n-CO-Phe-OMe (n = 3-5) containing the central homomorphic residue of 5-aminopentanoic acid (delta-aminovaleric acid; delta-Ava; n = 4) and the two non-homomorphic residues of 4-aminobutanoic acid (gamma-aminobutyric acid; gamma-Abu; n = 3) and 6-aminohexanoic acid (epsilon-aminocaproic acid; epsilon-Aca; n = 5) have been examined. The activity as agonists and antagonists in chemotaxis, lysozyme release, and superoxide anion production of the new analogs has been determined. The N-Boc derivatives 2a and 2b, incorporating the gamma-Abu and the delta-Ava residues, show good and selective antagonist activity on superoxide anion production.

The octapeptide D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH(2) ([D-Ala(1)]TNH(2)), an analog of peptide T (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH) associated with CD(4)/T(4) receptors involved in human immunodeficiency virus infection, was combined with the chelating polyazamacrocycle 1,4,8,11-tetraazacyclotetradecane (cyclam) to afford the bifunctional ligand cyc-[D-Ala(1)]TNH(2). This was then reacted with [(99m)TcO(4)](-) and Sn(2+) to yield the monocationic complex [(99m)Tc(O)(2)(cyc-[D-Ala(1)]TNH(2))](+). Biological activity of both the cyclam-peptide conjugate and the resulting Tc-99m complex were evaluated by measuring their chemotactic indexes. Results showed that N-cyclam acylation and subsequent labeling with Tc-99m of [D-Ala(1)]TNH(2) were tolerated, and both cyc-[D-Ala(1)]TNH(2) and [(99m)Tc(O)(2)(cyc-[D-Ala(1)]TNH(2))](+) retained the high chemotactic capacity of the original octapeptide. Biodistribution of the Tc-99m complex was carried out in rats. Fast blood clearance and no accumulation in organs of interest were observed.

N-formyl- and N-acetyl-peptides were synthesized and compared in order to understand which features can best elicit biological responses. The behavior of N-formyl-peptides confirms the previously found sequential obligations in the residues, while acetyl-derivatives do not seem suitable for an efficacious stimulation of human neutrophils. Copyright 2000 Academic Press.

Based on the sequence of the prototypical chemotactic tripeptide HCO-Met-Leu-Phe-OH (fMLF) and by taking into account the versatility shown by its N-terminal carbamate analogues, the new biscarbamates MeOCO-Met-Leu-gPhe-COOMe (2) and Boc-Met-Leu-gPhe-COOMe (4) were synthesized. These two new ligands are characterized by the presence of a gem-diamino residue (gPhe) replacing the C-terminal Phe and a carbamate functionality positioned at both the ends of the molecule. The activity of the two new compounds has been determined on human neutrophils and compared to that shown by the corresponding N-terminal monocarbamates MeOCO-Met-Leu-Phe-OMe (1) and Boc-Met-Leu-Phe-OMe (3).

The formyl tripeptides containing 2-azetidinecarboxylic acid 2, 2-piperidinecarboxylic acid 3 and norvaline 4 in position 2 were synthesized and their biological activity was evaluated. The conformation of peptides was studied by CD and FT-IR techniques. While 2 and 3 do not show either chemotactic activity or superoxide production, 4 retains both activities.

For-Met-delta ZLeu-delta ZPhe-OMe (3) has been synthesized as a new analogue of the prototypical chemotactic agent For-Met-Leu-Phe-OMe (fMLP-OMe). Compound 3 is characterized by presence of two consecutive alpha,beta-didehydro amino acid residues [delta ZLeu = (Z)-alpha,beta-didehydroleucine; delta ZPhe = (Z)-alpha,beta- didehydrophenylalanine] located at the central and C-terminal position, respectively. When tested on human neutrophils the N-formyltripeptide 3, although less active than the parent, is able to induce chemotaxis, superoxide anion production and lysozyme release. The activity of 3 has been compared to that of related fMLP-OMe analogues containing a single delta ZPhe residue located at the C-terminal position.

In our research there was spontaneous adhesion between resting fibroblasts and neutrophils in vitro which could be increased by stimulating either the coculture of cells or each cell type separately with various stimulants. Interferon-gamma, interleukin-1, and interleukin-6 significantly increased adhesion; however, the highest adhesive response was obtained when cocultures were treated with phorbol myristate acetate (PMA). As PMA-stimulated fibroblasts show the highest adhesion to resting neutrophils, it was suggested that adhesion was primarily due to an effect on fibroblasts. Without Mg2+ PMA did not stimulate fibroblast adhesion, whereas in the absence of Ca2+ the response was only partially reduced. Spontaneous adhesion was independent of both neutrophil integrins and fibroblast ICAM-1, whereas cytokine-stimulated adhesion was blocked by mAbs against ICAM-1; PMA-stimulated adhesion was not affected by mAbs anti-ICAM-1, but was partially inhibited by mAbs anti-beta 2 integrins. These results suggested the presence of mechanisms able to modulate the adhesive fibroblast-neutrophil interaction in inflammatory and wound healing processes.

The peptide H-Lys-Val-Leu-Asp-OH (KVLD), derived from antiflammins, and its analog H-Lys-Val-Asp-Leu-OH (KVDL) were prepared by solution synthesis. KVLD does not inhibit platelet aggregation in contrast to the results recently published.

Human neutrophils stimulated with phorbol myristate acetate were able to damage human erythroleukemic K562 cells, in the absence of specific antibody, as assessed by a two hour 51Cr release assay. Neutrophils treated with formyl-peptide fMet-Leu-Phe did not display tumoricidal response, but the addition of diacylglycerol kinase inhibitor R59022 together with formyl-peptide induced the cytotoxic capacity against tumor target cells. Phorbol ester is a potent activator of certain functions of neutrophils because of its ability to directly and irreversibly stimulate protein kinase C; formyl-peptide, on the contrary, activates protein kinase C by inducing a rapid and transient production of diacylglycerol, that is quickly metabolized. The addition of an inhibitor of diacylglycerol kinase, R59022, however potentiated the action of formyl-peptide. These results indicate that protein kinase C is involved in the tumoricidal activity of neutrophils against K562 cells, and that maximal activation of the enzyme is required to achieve the cytotoxic response.

Myelin basic protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation. Pre-treatment of human neutrophils with myelin basic protein selectively inhibits the formyl-peptide-induced chemotaxis, without affecting chemotaxis evoked by casein and activated serum. Furthermore, both the degranulation and superoxide anion production stimulated by the chemotactic peptide are not modified by the prior treatment of the neutrophils with myelin basic protein.