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Maurizio Simmaco

Publications and source records attributed to Maurizio Simmaco.

13 recordsLinked to original sources
PubMed2006

Cardiac resynchronization therapy increases plasma levels of the endogenous inotrope apelin.

BACKGROUND: Cardiac resynchronization therapy (CRT) has been introduced to treat drug refractory chronic heart failure (CHF). Apelin, the endogenous ligand of the APJ receptor, is under evaluation for its potential role in human CHF pathophysiology. This study aims to assess whether biventricular pacing affects plasma apelin levels in patients with severe CHF. METHODS AND RESULTS: Fourteen patients (9 men, 5 women, mean age 68+/-13 years) undergoing biventricular pace-maker/ICD implantation were studied. Patients underwent baseline clinical and echocardiographic evaluation, and assessment of plasma apelin and NT-proBNP levels. The evaluation was repeated 48 h and 9+/-2 months after device implantation to assess the acute and chronic effects of CRT on apelin and NT-proBNP levels. Eight healthy age- and sex-matched subjects served as controls. In CHF patients, baseline apelin levels were reduced and NT-proBNP increased compared to control subjects (apelin: 0.47+/-0.2 vs. 0.97+/-0.3 ng/mL, p<0.001; NT-proBNP: 2007+/-114 vs. 229+/-72 pmol/L, p<0.001). Short-term evaluation did not reveal any effect of CRT on apelin or NT-proBNP levels. By contrast, at 9+/-2 months follow-up, CRT responders showed left ventricular reverse remodelling and an increase in ejection fraction, together with a significant increase in plasma apelin levels (0.99+/-0.1 vs. 0.47+/-0.2 ng/mL, p<0.001) and decrease in NT-proBNP (938+/-591 vs. 2007+/-114 pmol/L, p<0.05). CONCLUSIONS: Long-term CRT increases plasma levels of the endogenous inotrope apelin in patients with CHF.

Aged↗
PubMed2006

A synergism between temporins toward Gram-negative bacteria overcomes resistance imposed by the lipopolysaccharide protective layer.

Temporins are short and homologous antimicrobial peptides (AMPs) isolated from the frog skin of Rana genus. To date, very little is known about the biological significance of the presence of closely related AMPs in single living organisms. Here we addressed this question using temporins A, B, and L isolated from Rana temporaria. We found that temporins A and B are only weakly active toward Gram-negative bacteria. However, a marked synergism occurs when each is mixed with temporin L. To shed light on the underlying mechanisms involved in these activities, we used various experimental strategies to investigate: (i) the effect of the peptides' interaction on both the viability and membrane permeability of intact bacteria and spheroplasts; (ii) their interaction with lipopolysaccharides (LPS) and the effect of LPS on the oligomeric state of temporins, alone or combining one with another; (iii) their structure in solution and when bound to LPS, by using circular dichroism and ATR-FTIR spectroscopies. Our data reveal that temporin L synergizes with A and B by preventing their oligomerization in LPS. This should promote their translocation across the outer membrane into the cytoplasmic membrane. To the best of our knowledge, this is the first study that explains how a combination of native AMPs from the same species can overcome bacterial resistance imposed by the LPS leaflet.

Amphibian Proteins↗
PubMed2006

Effect of natural L- to D-amino acid conversion on the organization, membrane binding, and biological function of the antimicrobial peptides bombinins H.

Antimicrobial peptides (AMPs) are evolutionarily old components of innate immunity found in all living pluricellular organisms. Interestingly, some organisms express families of AMPs with only a slight variation among their members, possibly to increase their spectrum of activity. Despite the growing body of knowledge about their biological activity and mode of action on bacteria, only a few of them have been tested on Leishmania, a worldwide spread protozoan pathogen, and the parameters contributing to this activity are yet to be determined. We report on the anti-Leishmania activity and mode of action of bombinins H2 and H4 isolated from the skin secretion of the frog Bombina variegata. H4, the most active, is the first natural AMP of animal origin with a single L- to D-amino acid isomerization. Membrane depolarization and membrane permeation assays, as well as electron microscopy, suggest that the lethal mechanism involves plasma membrane permeation and/or disruption. To better understand the enhanced activity of H4, we determined the peptide's structure in membranes mimicking those of mammals, bacteria, and Leishmania by using ATR-FTIR and CD spectroscopies and assessed their membrane binding by using surface plasmon resonance. The data reveal that (i) H2 but not H4 partially aggregates in membranes mimicking those of Leishmania, (ii) H2 is slightly more helical than H4 in all membranes, and (iii) H4 binds the Leishmania model membrane approximately 5-fold better than H2. This study highlights the importance of a single alpha-amino acid epimerization as a tool used by nature to modulate the activity of AMPs. In addition, our findings suggest bombinins H as potential templates for the development of new drugs with a new mode of action against Leishmania.

Animals↗
PubMed2004

Temporins, small antimicrobial peptides with leishmanicidal activity.

Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action.

Adenosine Triphosphate↗
PubMed2004

Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli.

Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4',6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.

Antimicrobial Cationic Peptides↗
PubMed2003

Ranacyclins, a new family of short cyclic antimicrobial peptides: biological function, mode of action, and parameters involved in target specificity.

We report on two new cyclic 17-residue peptides that we named ranacyclins E and T, the first isolated from Rana esculenta frog skin secretions and the second discovered by screening a cDNA library from Rana temporaria. Ranacyclins have a loop region that is homologous with that of an 18-mer peptide, pLR, isolated from the skin of the Northern Leopard frog, Rana pipiens, with no reported antimicrobial activity. Here we show that ranacyclins and pLR have antimicrobial and antifungal activity. However, despite the high structural similarity, they differ in their spectrum of activity. The data reveal that ranacyclins and pLR have several properties that differentiate them from most known antimicrobial peptides. These include the following: (i) they adopt a significant portion of random coil structure in the membrane as revealed by ATR-FTIR and CD spectroscopy (50% for ranacyclin T and 70% for both ranacyclin E and pLR); (ii) they bind similarly to both zwitterionic and negatively charged membranes as revealed by using tryptophan fluorescence and surface plasmon resonance (SPR; BIAcore biosensor); (iii) they insert into the hydrophobic core of the membrane and presumably form transmembrane pores without damage to the bacterial wall, as revealed by SPR, ATR-FTIR, and transmission electron microscopy (TEM); and (iv) despite being highly and equally active in permeating bacterial spheroplasts and negatively charged membranes, they differ significantly in their potencies against target cells. Furthermore, a significant fraction of a given secondary structure is not prerequisite for membrane permeation and antimicrobial activity. However, increasing the fraction of a secondary structure and reducing peptide assembly in the membrane make it easier for the peptide to diffuse through the cell wall, which is different for each microorganism, into the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed2003

Stereochemistry of the reactions of glutamate-1-semialdehyde aminomutase with 4,5-diaminovalerate.

Conversion of glutamate 1-semialdehyde to the tetrapyrrole precursor, 5-aminolevulinate, takes place in an aminomutase-catalyzed reaction involving transformations at both the non-chiral C5 and the chiral C4 of the intermediate 4,5-diaminovalerate. Presented with racemic diaminovalerate and an excess of succinic semialdehyde, the enzyme catalyzes a transamination in which only the l-enantiomer is consumed. Simultaneously, equimolar 4-aminobutyrate and aminolevulinate are formed. The enzyme is also shown to transaminate aminolevulinate and 4-aminohexenoate to l-diaminovalerate as the exclusive amino product. The interaction of the enzyme with pure d- and l-enantiomers of diaminovalerate prepared by these reactions is described. Transamination of l-diaminovalerate yielded aminolevulinate quantitatively showing that reaction at the C5 amine does not occur significantly. A much slower transamination reaction was catalyzed with d-diaminovalerate as substrate. One product of this reaction, 4-aminobutyrate, was formed in the amount equal to that of the diaminovalerate consumed. Glutamate semialdehyde was deduced to be the other primary product and was also measured in significant amounts when a high concentration of the enzyme in its pyridoxal form was reacted with d-diaminovalerate in a single turnover. Single turnover reactions showed that both enantiomers of diaminovalerate converted the enzyme from its 420-nm absorbing pyridoxaldimine form to the 330-nm absorbing pyridoxamine via rapidly formed intermediates with different absorption spectra. The intermediate formed with l-DAVA (lambdamax = 420 nm) was deduced to be the protonated external aldimine with the 4-amino group. The intermediate formed with d-DAVA (lambdamax = 390 nm) was deduced to be the unprotonated external aldimine with the 5-amino group.

Aldehydes↗
PubMed2003

An amphibian antimicrobial peptide variant expressed in Nicotiana tabacum confers resistance to phytopathogens.

Esculentin-1 is a 46-residue antimicrobial peptide present in skin secretions of Rana esculenta. It is effective against a wide variety of micro-organisms, including plant pathogens with negligible effects on eukaryotic cells. As a possible approach to enhance plant resistance, a DNA coding for esculentin-1, with the substitution Met-28Leu, was fused at the C-terminal end of the leader sequence of endopolygalacturonase-inhibiting protein, under the control of the cauliflower mosaic virus 35S promoter region, and introduced into Nicotiana tabacum. The antimicrobial peptide was isolated from the intercellular fluids of healthy leaves of transgenic plants, suggesting that it was properly processed, secreted outside cells and accumulated in the intercellular spaces. The morphology of transgenic plants was unaffected. Challenging these plants with bacterial or fungal phytopathogens demonstrated enhanced resistance up to the second generation. Moreover, transgenic plants displayed insecticidal properties.

Amino Acid Sequence↗
PubMed2003

A peptidylprolyl cis/trans isomerase from Xenopus laevis skin: cloning, biochemical characterization and putative role in the secretion.

In amphibian skin secretions, a peptidylprolyl cis/trans isomerase activity was detected. A Xenopus laevis skin cDNA coding for this protein was cloned, sequenced and over-expressed in Escherichia coli. The primary structure of the protein shows extensive similarity with members of the cyclophilin A family. Catalytic parameters of the recombinant protein are similar to those of the human enzyme. The enzymatic activity is inhibited by cyclosporin A. Data suggesting that peptidylprolyl isomerization influences the biological activity of antibacterial peptides of amphibian origin are presented, and its putative role in the defence mechanism discussed.

Amino Acid Sequence↗
PubMed2003

Functional characterisation of the 1-18 fragment of esculentin-1b, an antimicrobial peptide from Rana esculenta.

Esculentin-1 is a 46-amino acid residue peptide isolated from skin secretions of Rana esculenta, displaying the most potent antimicrobial activity among the bioactive molecules found in the secretion, with negligible effects on eukaryotic cell membranes. From skin secretions, the 19-46 fragment of esculentin-1, devoid of antibacterial activity, was also isolated. We studied in detail the activity of the N-terminal fragment (1-18) of esculentin-1 using a synthetic amidated analogue. The results show that this fragment is highly active against most bacterial and fungal species, although at a lower extent than the full-length peptide, being four-fold more potent against Phytophthora nicotianae. It has a reduced activity against human erythrocytes with respect to the full-length peptide. The killing curves in liquid medium are similar for the two molecules and the shorter peptide is able to increase the bacterial outer and inner membrane permeability. Overall these data indicate that the antimicrobial properties of esculentin-1 are exerted by its N-terminal 1-18 region and that the positively charged residue distribution as well as peptide length represent important determinants for cell selectivity.

Amphibian Proteins↗
PubMed2002

Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles.

The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.

Animals↗
PubMed2002

Interactions of the antimicrobial peptides temporins with model biomembranes. Comparison of temporins B and L.

Temporins are short (10-13 amino acids) and linear antimicrobial peptides first isolated from the skin of the European red frog, Rana temporaria, and are effective against Gram-positive bacteria and Candida albicans. To get insight into their mechanism(s) of action, we compared the effects on model membranes exerted by two members of this family, viz., temporin B (LLPIVGNLLKSLL-NH(2)) and temporin L (FVQWFSKFLGRIL-NH(2)). More specifically, we measured their insertion into lipid monolayers as well as their effects on the structural dynamics of liposomal bilayers as revealed by diphenylhexatriene (DPH)- and pyrene-labeled phospholipids. We also observed the impact of these peptides on the topology of giant vesicles. Both temporins readily penetrate into lipid monolayers, their intercalation being enhanced in the presence of the common bacterial negatively charged phospholipid phosphatidylglycerol. Instead, the eukaryotic lipid cholesterol did to some extent counteract their penetration into the lipid films. Both temporin B and temporin L caused an enrichment of phospholipids in the bilayers, and in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), these peptides increased acyl chain order. Temporin B had practically no effect on giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), whereas rapid vesiculation was observed when POPG was present. In contrast, temporin L induced vesiculation of both SOPC and SOPC/POPG giant vesicles while the presence of cholesterol in SOPC giant vesicles attenuated this effect.

Amino Acid Sequence↗