Characterization of a rare case of Ullrich congenital muscular dystrophy due to truncating mutations within the COL6A1 gene C-terminal domain: a case report.

BACKGROUND: Mutations within the C-terminal region of the COL6A1 gene are only detected in Ullrich/Bethlem patients on extremely rare occasions. CASE PRESENTATION: Herein we report two Brazilian brothers with a classic Ullrich phenotype and compound heterozygous for two truncating mutations in COL6A1 gene, expected to result in the loss of the α1(VI) chain C2 subdomain. Despite the reduction in COL6A1 RNA level due to nonsense RNA decay, three truncated alpha1 (VI) chains were produced as protein variants encoded by different out-of-frame transcripts. Collagen VI matrix was severely decreased and intracellular protein retention evident. CONCLUSION: The altered deposition of the fibronectin network highlighted abnormal interactions of the mutated collagen VI, lacking the α1(VI) C2 domain, within the extracellular matrix, focusing further studies on the possible role played by collagen VI in fibronectin deposition and organization.

Persistent dystrophin protein restoration 90 days after a course of intraperitoneally administered naked 2'OMePS AON and ZM2 NP-AON complexes in mdx mice.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2'-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

Exon skipping-mediated dystrophin reading frame restoration for small mutations.

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2'OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations.

Sodium azide induced neuronal damage in vitro: evidence for non-apoptotic cell death.

The features of neuronal damage induced by the mitochondrial toxin NaN(3) were investigated in rat primary cortical neuron cultures. Cell viability (MTT colorimetric determination) and transmembrane mitochondrial potential (J-C1 fluorescence) were concentration-dependently reduced 24 h after NaN(3); neither nuclear fragmentation by DAPI, nor Annexin V positivity by flow cytometry were detected, ruling out the occurrence of apoptosis. The loss in cell viability (to 54 +/- 2%) observed 24 h after a 10-min treatment with 3 mM NaN(3) was prevented by the NMDA glutamate receptor antagonist MK801 (1 microM), by the antioxidants trolox (100 microM) and acetyl-L-carnitine (1 mM) and by the nitric oxide synthase inhibitor, L-NAME (100 microM), but not by the guanylylcyclase inhibitor ODQ, 10 microM. The mitochondrial dysfunction induced by NaN(3) provides a common platform for investigating the mechanisms of both ischemic and degenerative neuronal injury, useful for screening potential protective agents against neuronal death.

Study of synthetic peptides derived from the PKI55 protein, a protein kinase C modulator, in human neutrophils stimulated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH.

Elucidation of the involvement of protein kinase C subtypes in several diseases is an important challenge for the future development of new drug targets. We previously identified the PKI55 protein, which acts as a protein kinase C modulator, establishing a feedback loop of inhibition. The PKI55 protein is able to penetrate the cell membrane of activated human T-lymphocytes and to inhibit the activity of alpha, beta(1) and beta(2) protein kinase C isoforms. The present study aimed to identify the minimal amino acid sequence of PKI55 that is able to inhibit the enzyme activity of protein kinase C. Peptides derived from both C- and N-terminal sequences were synthesized and initially assayed in rat brain protein kinase C to identify which part of the entire protein maintained the in vitro effects described for PKI55, and then the active peptides were tested on the isoforms alpha, beta(1), beta(2), gamma, delta, epsilon and zeta to identify their specific inhibition properties. Specific protein kinase C isoforms have been associated with the activation of specific signal transduction pathways involved in inflammatory responses. Thus, the potential therapeutic role of the selected peptides has been studied in polymorphonuclear leukocytes activated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH to evaluate their ability to modulate chemotaxis, superoxide anion production and lysozyme release. These studies have shown that only chemotactic function is significantly inhibited by these peptides, whereas superoxide anion production and lysozyme release remain unaffected. Western blotting experiments also demonstrated a selective reduction in the levels of the protein kinase C beta(1) isoform, which was previously demonstrated to be associated with the polymorphonuclear leukocyte chemotactic response.

New chemotactic dimeric peptides show high affinity and potency at the human formylpeptide receptor.

A number of analogues of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe) have been studied in order to evaluate their ability to interact with formylpeptide receptors and to induce specific biological responses in human neutrophils. In vitro assays were carried out and receptor binding, chemotaxis, superoxide anion release and secretagogue activity were evaluated. The fMLP-OMe analogues synthesized, with the general formula for-Met-Leu-Phe-Xaa-Lys(OMe)-Phe-Leu-Met-for (Xaa=Gly, beta-Ala, gamma-aminobutyric acid, 5-aminovaleric acid, and 6-aminocaproic acid), were constituted by two fMLP units linked by a Lys residue, with an amino acid spacer between them. Competition binding experiments revealed that the new compounds have much more affinity for formylpeptide receptors than the reference ligand, with good correlation between receptor affinity and length of spacer. The EC(50) values for the killing mechanisms of each analogue were similar to each other, the affinity and potency, once again, being strictly dependent on the chain length. Furthermore the analogues proved to be more potent full agonists than the prototype fMLP-OMe in these functions, while chemotaxis was poorly induced. The dimeric fMLP-OMe analogues are one of the few examples of formylpeptides which exhibit a receptor affinity greater than the parent fMLP-OMe thereby rendering them suitable to be used as carriers for various drugs.

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices.

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (alpha, beta(1), beta(2), gamma, delta and epsilon) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10mM NaN(3), combined with 2mM 2-deoxyglucose (chemical ischemia); (iii) 1h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC beta(1), delta and epsilon isoforms total levels (cytosol+particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while alpha isoform was slightly reduced and gamma isoform was no longer detectable. After reperfusion, the changes displayed by alpha, beta(1), gamma, delta and epsilon were maintained and even potentiated, moreover, an increase in beta(2) (by 41+/-12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC alpha isoform, which following reperfusion was found only in the cytosol. PKC beta(1) and delta isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC beta(2) and epsilon isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1muM, prevented the translocation of beta(1) isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in beta(2) and epsilon isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.

Effects of chemical ischemia on cerebral cortex slices: focus on mitogen-activated protein kinase cascade.

A variety of harmful stimuli, among them energy depletion occurring during transient brain ischemia, are thought to unbalance protein kinase cascades, ultimately leading to neuronal damage. In superfused, electrically stimulated rat cerebral cortex slices, chemical ischemia (CI) was induced by a 5-min treatment with the mitochondrial toxin, sodium azide (10 mM), combined with the glycolysis blocker, 2-deoxyglucose (2 mM). Thereafter, 1 h reperfusion (REP) with normal medium followed. Western blot analysis of p21Ras, extracellular signal-regulated protein kinases (ERK)1/2 (p44/42), phospho-ERK1/2, mitogen-activated protein kinase (MAPK)-p38, phospho-p38, stress-activated protein kinases/c-Jun NH2-terminal protein kinases (SAPK/JNK), phospho-SAPK/JNK was carried out. The level of p21Ras was increased by 40% immediately after CI, and did not return to control values following REP. Both ERK1 and ERK2 levels were reduced by CI and recovered to control values following REP; no significant change in their phosphorylation degree (phosphorylated to total level ratio, about 50% in the controls) was observed. Neither p38 levels, nor phosphorylation degree were changed following CI/REP. The activation of SAPK/JNK was significantly reduced under CI, and did not recover following REP. All CI/REP-induced effects were prevented by the NMDA receptor antagonist MK-801, 10 microM, suggesting the involvement of glutamate. The present findings show that although CI stimulates the p21Ras protein, MAPK levels and/or phosphorylation are reduced, possibly because of acute energy depletion. Because the activation of SAPK/JNK has been related to both apoptosis and neuroprotection, the decrease observed under CI/REP conditions may instead be related to nonapoptotic neuronal death. These results could be of interest in developing preventive treatments for ischemia/REP-induced brain damage.

Signal transduction pathways triggered by selective formylpeptide analogues in human neutrophils.

Human neutrophils are highly specialised for their primary function, i.e. phagocytosis and destruction of microorganisms. Leukocyte recruitment to sites of inflammation and infection is dependent upon the presence of a gradient of locally produced chemotactic factors. The bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was one of the first of these to be identified and is a highly potent leukocyte chemoattractant. It interacts with its receptor on the neutrophil membrane, activating these cells through a G-protein-coupled pathway. Two functional fMLP receptors have thus far been cloned and characterized, namely FPR (formyl peptide receptor) and FPRL1 (FPR like-1), with high and low affinities for fMLP, respectively. FMLP is known to activate phospholipase C (PLC), PLD, PLA2 and phosphatidylinositol-3-kinase (PI3K), and it also activates tyrosine phosphorylation. The second messengers resulting from the fMLP receptor interaction act on various intracellular kinases, including protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs). The activation of these signal transduction pathways is known to be responsible for various biochemical responses which contribute to physiological defence against bacterial infection and cell disruption. This review will consider the ability of selective analogues (ligands able to discriminate between different biological responses) to activate a single spectrum of signal transduction pathways capable of producing a unique set of cellular responses, hypothesising that a distinctive imprint of signal protein activation may exist. Through more complete understanding of intracellular signaling, new drugs could be developed for the selective inflammatory blockade.

A 'pure' chemoattractant formylpeptide analogue triggers a specific signalling pathway in human neutrophil chemotaxis.

As it has not yet been established whether the second messengers involved in the neutrophil response have identical or specific signalling requirements for each physiological function, protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAPKs) were studied in human chemotaxis triggered by the full agonist for-Met-Leu-Phe-OMe (fMLP-OMe) and the 'pure' chemoattractant for-Thp-Leu-Ain-OMe [Thp1,Ain3] analogue. Experiments were performed in the presence or absence of extracellular Ca2+, known to be an important modulator of second messengers. Our data demonstrate that specific PKC beta1 translocation and p38 MAPK phosphorylation are strongly associated with the chemotactic response of the neutrophils triggered by both peptides, while Ca2+ is not necessary for chemotaxis to occur. PKC and MAPK inhibitors were used in Western blotting assays and in cell locomotion experiments to investigate if the MAPK signalling pathway was controlled by PKC activation. The most important finding emerging from this study is that PKC and MAPK activate the chemotactic function of human neutrophils by two independent pathways.

Biological activity of for-Met-Leu-Phe-OMe analogs: relevant substitutions specifically trigger killing mechanisms in human neutrophils.

Two analogs of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe), for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2), carrying unusual hydrophilic residues, were synthesized in order to investigate whether they provoked specific biological responses, as well as intracellular calcium mobilization, in human neutrophils. Whereas neither compound stimulates chemotaxis, both are able to elicit lysosomal enzyme production. However compound 1 is able to trigger copious superoxide anion production while compound 2 only elicits minor superoxide anion production. In binding experiments on formylpeptide receptors, the newly synthesized compounds for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2) showed affinity values in the micromolar range. These derivatives demonstrate inability to find a positive contribute from single substitutions. A very important result of this research is the evidence of the ability of the formyl group alone to trigger the primary target of the human neutrophil activity, i.e. killing mechanisms, by activating the specific receptor conformation.

Formylpeptides trigger selective molecular pathways that are required in the physiological functions of human neutrophils.

For-Met-Delta(z)Leu-Phe-OMe ([Delta(z)Leu(2)]) is a conformationally restricted for-Met-Leu-Phe-OMe (fMLP-OMe) analogue able to discriminate between different responses of human neutrophils. In contrast, [Delta(z)Leu(2)] significantly activates the transduction pathways-involving Ca(2+), inositol phosphate, and cyclic AMP (cAMP) enhancement, as is the case with the full agonist fMLP-OMe. Here, we have studied the specific involvement of protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAPKs) in the presence or absence of extracellular Ca(2+), being the cation clearly involved in the activation of neutrophils by fMLP. A strong correlation has been found between PKC isoforms, MAPKs and the selective physiological functions by [Delta(z)Leu(2)]-activated neutrophils. In a calcium-free condition, our data suggest that the failure of PKC beta1 translocation and of p38 MAPK phosphorylation by the analogue refers to its inability to induce chemotaxis, and that the failure by both fMLP-OMe and [Delta(z)Leu(2)] to evoke extracellular response kinase 1 and 2 (ERK1/2) phosphorylation would suggest a reduction in superoxide anion production.

Hydrophilic residues at position 3 highlight unforeseen features of the fMLP receptor pocket.

The peptides for-Met-Leu-Tyr-OMe, for-Met-Leu-Glu-OMe, for-Met-Leu-Asp-OMe and for-Met-Leu-Ser-OMe were synthesized to investigate the importance of a hydrophilic side chain of the residue at position 3 on biological activities of human neutrophils. A number of in vitro essays were carried out, including: chemotaxis, superoxide anion production, lysozyme release and receptor binding. Our results highlight that for-Met-Leu-Asp-OMe acts as a full agonist with a higher efficacy than formyl-Met-Leu-Phe-OMe, the tripeptide normally used as a model chemoattractant for the study of cell functions. The other analogs show efficacies that are in the same range or a little less than the prototype. The main point emerging from this study is that the role of Phe substitution needs to be re-hypothesised.

Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.

Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches.

Structural characterization of promoter sequences of the gene coding human PKI55 protein, a protein kinase C inhibitor.

The PKI55 protein was identified in our laboratory as specific protein kinase C inhibitor. We previously demonstrated that PKI55 is poorly translated in vivo and acts promoting PKC degradation and establishing a feedback loop of inhibition. However, our understanding of mechanisms by which the expression of PKI55 is regulated, is limited. In the present work we investigated the mRNA expression of PKI55 in human tissues by Northern blotting and RT-PCR, demonstrating that it is highly expressed in brain tissue. Moreover, since the computational analysis of the gene promoter region showed two sites (Box 1 and Box 2) similar to consensus sequences for AP1 and GAGA factors, we investigated their ability to bind to these proteins. Electrophoretic mobility shift assays showed that GAGA factors preferentially interacted with Box 2, while AP1 elements linked preferentially Box 1 sequence. We suggest that the interaction of these transcription factors with Box 1 and Box 2 could regulate the transcription of the PKI55 gene and, consequently, the expression of PKC.

Oligomeric formylpeptide activity on human neutrophils.

A series of oligomeric formylpeptides were synthesized by cross-linking the prototype fMLP using a Lys residue. They were then investigated for their ability to stimulate chemotaxis, superoxide anion production, and lytic enzyme release in human neutrophils. Although active in stimulating the different receptor isoforms, leading to the different biological responses, these analogues showed a lesser potency and affinity than the standard peptide. On the basis of the results reported here, we can hypothesise that: (i) the increased bulk of these molecules seems to hinder their correct positioning into the receptor pocket, thereby hindering favourable receptor interaction; and that: (ii) fMLP space positions do not seem to allow the ligand to increase biological responses.

Effects of PKI55 protein, an endogenous protein kinase C modulator, on specific PKC isoforms activity and on human T cells proliferation.

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.

Structure-activity relationship of for-L-Met L-Leu-L-Phe-OMe analogues in human neutrophils.

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.

The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca(2+)-dependent pathway(s).

Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca2+ levels and activation of PKC alpha, thereby upregulating D1 and D3 cyclin, downregulating p21waf1 and p27kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca2+ increase and PKC alpha activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKC alpha or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation.

Properties of a novel chemotactic esapeptide, an analogue of the prototypical N-formylmethionyl peptide.

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.