Biomedical subjects

Sofia Falzarano

Publications and source records attributed to Sofia Falzarano.

9 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

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.

Chemotactic Factors↗

PubMed2006

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.

Anti-Inflammatory Agents↗

PubMed2005

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.

Binding, Competitive↗

PubMed2005

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.

Blotting, Western↗

PubMed2004

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.

Blood Proteins↗

PubMed2003

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.

Dose-Response Relationship, Drug↗

PubMed2003

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.

Chemotaxis↗

PubMed2003

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.

Calcium↗