Alessandra Romanelli - Academia.edu (original) (raw)

Papers by Alessandra Romanelli

Research paper thumbnail of Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS

Journal of Mass Spectrometry, 2005

Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their abi... more Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.A powerful method currently available to study enzyme–inhibitor interactions is based on the use of the surface plasmon resonance (SPR) technique. When MMP interactions are studied, a procedure by which inhibitors are normally anchored on sensor chips and SPR technique is used in order to study their interaction with MMPs molecules is usually followed. This is because it is currently believed that MMPs cannot be anchored on the sensor-chip surface without losing their activity. However, this approach gives rise to problems, as the anchoring of low-molecular-weight inhibitors on gold surfaces easily affects their ability to interact with MMPs. For this reason, the anchoring of MMPs is highly desirable.A new experimental protocol that couples the Fourier transform-SPR (FT-SPR) technique with electrospray ionization-mass spectroscopy (ESI-MS) is described here for the evaluation of the activity of MMP-1 catalytic domain (cdMMP-1) anchored on gold surfaces. The cdMMP-1 surface coverage is calculated by using FT-SPR and the enzyme activity is estimated by ESI-MS. The proposed method is label-free. Copyright © 2005 John Wiley & Sons, Ltd.

Research paper thumbnail of The unusual helix stability of a VEGF mimetic peptide

Research paper thumbnail of Structural characterization of the helix stability of a de novo engineered VEGF mimicking peptide

Research paper thumbnail of Formulations for Natural and Peptide Nucleic Acids Based on Cationic

INTRODUCTION This article describes the production and characteriza- tion of cationic submicron p... more INTRODUCTION This article describes the production and characteriza- tion of cationic submicron particles constituted with Eudragit RS 100, plus different cationic surfactants, such as dioctadecyl-dimethyl-ammonium bromide (DDAB18) and diisobutylphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA), as a transport and delivery system for DNA/DNA and DNA/peptide nucleic acid (PNA) hybrids and PNA-DNA chimeras. Submicron particles could offer advantages over other delivery systems be- cause

Research paper thumbnail of Molecular interactions with nuclear factor κB (NF-κB) transcription factors of a PNA-DNA chimera mimicking NF-κB binding sites

European Journal of Biochemistry, 2001

The decoy approach against nuclear factor kB (NF-kB) is a useful tool to alter NF-kB dependent ge... more The decoy approach against nuclear factor kB (NF-kB) is a useful tool to alter NF-kB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF-kB specific cis-elements. Unfortunately, ODNs are not stable and need to be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N-(2-aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF-kB transcription factors to PNAs was either very low (binding to PNA-PNA hybrids) or exhibited low stability (binding to PNA-DNA hybrids). The main consideration of the present paper was to determine whether PNA-DNA chimeras mimicking NF-kB binding sites are capable of stable interactions with proteins belonging to the NF-kB family. Molecular modeling was employed for the design of PNA -DNA chimeras; prediction of molecular interactions between chimeras and NF-kB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA -DNA chimeras and NF-kB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF-kB PNA-DNA chimera and duplex NF-kB DNA-DNA. However, it was found that these differences do not prevent the duplex PNA -DNA chimera from binding to NF-kB transcription factors, being able to suppress the molecular interactions between HIV-1 LTR and p50, p52 and nuclear factors from B-lymphoid cells. Therefore, these results demonstrate that the designed NF-kB DNA-PNA chimeras could be used for a decoy approach in gene therapy.

Research paper thumbnail of Functional Binding Surface of a b-Hairpin VEGF Receptor Targeting Peptide Determined by NMR Spectroscopy in Living Cells

Research paper thumbnail of Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

Scientific reports, 2014

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their ... more Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

Research paper thumbnail of Structural Basis of a Temporin 1b Analogue Antimicrobial Activity against Gram Negative Bacteria Determined by CD and NMR Techniques in Cellular Environment

ACS Chemical Biology, 2015

We here report an original approach to elucidate mechanisms of action of antimicrobial peptides a... more We here report an original approach to elucidate mechanisms of action of antimicrobial peptides and derive crucial structural requirements for the design of novel therapeutic agents. The high resolution structure of TB_KKG6A, an antimicrobial peptide designed to amplify the spectrum of action of Temporin B, bound to E. coli is here determined by means of CD and NMR methodologies. We have also defined, through STD analysis, the residues in closer proximity to the bacterial membrane.

Research paper thumbnail of Functional Binding Surface of a β-Hairpin VEGF Receptor Targeting Peptide Determined by NMR Spectroscopy in Living Cells

Chemistry - A European Journal, 2014

In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growt... more In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast (15)N-edited NMR spectroscopic experiments. To this aim, (15)N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope-edited NMR spectroscopic experiments, including (15)N relaxation measurements, allowed a precise characterization of the in-cell HPLW epitope recognized by VEGFR2.

Research paper thumbnail of Synthetic Strategies for Obtaining PNA and PNA Conjugates

Research paper thumbnail of Reactions of Pd(PPh3)4 with 3′,5′-Di-O- acetylthymidine: Oxidative addition of Pd(PPh3)4 on thymidine N3 and C4 atoms

Organometallics, 2005

Oxidative addition reactions of Pd(PPh 3 ) 4 on the pyrimidine nucleosides 3′,5′-di-Oacetylthymid... more Oxidative addition reactions of Pd(PPh 3 ) 4 on the pyrimidine nucleosides 3′,5′-di-Oacetylthymidine and 3′,5′-di-O-acetyl-4-chlorothymidine and on the nucleobase 1-methylthymine have been investigated. N3 and C4 metal coordinated complexes 3 and 7 were isolated and characterized by spectroscopic techniques. Moreover, the crystal structure of the trans-[PdCl(1-methyl thymine)(PPh 3 ) 2 ]‚H 2 O (4) is reported.

Research paper thumbnail of Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS

Journal of Mass Spectrometry, 2005

Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their abi... more Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.

Research paper thumbnail of Targeting pre-miRNA by Peptide Nucleic Acids

Artificial DNA: PNA & XNA, 2012

PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with... more PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs.

Research paper thumbnail of Crystallization and preliminary X-ray diffraction studies of a D-lysine-based chiral PNA–DNA duplex

Acta Crystallographica Section D-biological Crystallography - ACTA CRYSTALLOGR D-BIOL CRYST, 2002

Research paper thumbnail of Development of an efficient and low-cost protocol for the manual PNA synthesis by Fmoc chemistry

Tetrahedron Letters, 2010

An efficient and low-cost protocol for the manual synthesis of Peptide Nucleic Acids is reported ... more An efficient and low-cost protocol for the manual synthesis of Peptide Nucleic Acids is reported here. The protocol relies on coupling reactions carried out with 2.5 equiv of PNA monomers activated with HOBT/ HBTU, in the presence of pyridine/NMM. The protocol has been tested on four PNA oligomers with a length ranging from 9 to 12 bases and a purine content up to 67%.

Research paper thumbnail of A new ferrocenemethyl-thymidine nucleoside: Synthesis, incorporation into oligonucleotides and optical spectroscopic studies on the resulting single strand, duplex and triplex structures

Tetrahedron, 1999

A new thymidine analogue, bearing a ferrocenemethyl residue at the N-3 position of the base, was ... more A new thymidine analogue, bearing a ferrocenemethyl residue at the N-3 position of the base, was synthesized in high yields via Mitsunobu reaction of ferrocenemethanol with sugar protected thymidine, converted into the corresponding 3'-phosphoramidite and incorporated into oligonucleotides. Duplex and triplex formation experiments, evaluated by UV and CD spectroscopy, showed a dramatic decrease of the affinity towards complementary single strands, while for triplexes, the introduction of a ferrocene residue in the third strand resulted in higher melting temperatures, associated with a reduced content of triplex structure.

Research paper thumbnail of Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction

Proceedings of the National Academy of Sciences, 2004

Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, t... more Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The 1 JNC coupling constant for the (؊1) scissile peptide bond at the N-extein-intein junction was found to be Ϸ12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (؊1) scissile bond in its unusual conformation. Overall, these studies support the ''ground-state destabilization'' model as part of the mechanism of catalysis.

Research paper thumbnail of Synergistic Antibacterial and Anti-Inflammatory Activity of Temporin A and Modified Temporin B In Vivo

PLoS ONE, 2009

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Ra... more Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10-14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and antiinflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3-6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.

Research paper thumbnail of Semisynthesis of Dimeric Proteins by Expressed Protein Ligation

Organic Letters, 2008

A one-pot synthesis of homodimeric proteins is described. The synthetic strategy is based on a do... more A one-pot synthesis of homodimeric proteins is described. The synthetic strategy is based on a double expressed protein ligation reaction between thioester peptides and a new bis-cysteinyl linker. The protocol was also applied to the synthesis of heterodimers.

Research paper thumbnail of Peptides from Royal Jelly: studies on the antimicrobial activity of jelleins, jelleins analogs and synergy with temporins

Journal of Peptide Science, 2011

Peptides isolated from natural fonts are the object of several studies aimed at finding new molec... more Peptides isolated from natural fonts are the object of several studies aimed at finding new molecules possessing antibacterial activity. We focused our studies on peptides originally isolated from the Royal Jelly, the jelleins and on some analogs having a UV reporter at the N-or C-terminus. We found that jelleins are mainly active against gram-positive bacteria; interestingly, they act in synergy with peptides belonging to the family of temporins such as temporin A and temporin B against Staphylococcus aureus A170 and Listeria monocytogenes.

Research paper thumbnail of Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS

Journal of Mass Spectrometry, 2005

Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their abi... more Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.A powerful method currently available to study enzyme–inhibitor interactions is based on the use of the surface plasmon resonance (SPR) technique. When MMP interactions are studied, a procedure by which inhibitors are normally anchored on sensor chips and SPR technique is used in order to study their interaction with MMPs molecules is usually followed. This is because it is currently believed that MMPs cannot be anchored on the sensor-chip surface without losing their activity. However, this approach gives rise to problems, as the anchoring of low-molecular-weight inhibitors on gold surfaces easily affects their ability to interact with MMPs. For this reason, the anchoring of MMPs is highly desirable.A new experimental protocol that couples the Fourier transform-SPR (FT-SPR) technique with electrospray ionization-mass spectroscopy (ESI-MS) is described here for the evaluation of the activity of MMP-1 catalytic domain (cdMMP-1) anchored on gold surfaces. The cdMMP-1 surface coverage is calculated by using FT-SPR and the enzyme activity is estimated by ESI-MS. The proposed method is label-free. Copyright © 2005 John Wiley & Sons, Ltd.

Research paper thumbnail of The unusual helix stability of a VEGF mimetic peptide

Research paper thumbnail of Structural characterization of the helix stability of a de novo engineered VEGF mimicking peptide

Research paper thumbnail of Formulations for Natural and Peptide Nucleic Acids Based on Cationic

INTRODUCTION This article describes the production and characteriza- tion of cationic submicron p... more INTRODUCTION This article describes the production and characteriza- tion of cationic submicron particles constituted with Eudragit RS 100, plus different cationic surfactants, such as dioctadecyl-dimethyl-ammonium bromide (DDAB18) and diisobutylphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA), as a transport and delivery system for DNA/DNA and DNA/peptide nucleic acid (PNA) hybrids and PNA-DNA chimeras. Submicron particles could offer advantages over other delivery systems be- cause

Research paper thumbnail of Molecular interactions with nuclear factor κB (NF-κB) transcription factors of a PNA-DNA chimera mimicking NF-κB binding sites

European Journal of Biochemistry, 2001

The decoy approach against nuclear factor kB (NF-kB) is a useful tool to alter NF-kB dependent ge... more The decoy approach against nuclear factor kB (NF-kB) is a useful tool to alter NF-kB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF-kB specific cis-elements. Unfortunately, ODNs are not stable and need to be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N-(2-aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF-kB transcription factors to PNAs was either very low (binding to PNA-PNA hybrids) or exhibited low stability (binding to PNA-DNA hybrids). The main consideration of the present paper was to determine whether PNA-DNA chimeras mimicking NF-kB binding sites are capable of stable interactions with proteins belonging to the NF-kB family. Molecular modeling was employed for the design of PNA -DNA chimeras; prediction of molecular interactions between chimeras and NF-kB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA -DNA chimeras and NF-kB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF-kB PNA-DNA chimera and duplex NF-kB DNA-DNA. However, it was found that these differences do not prevent the duplex PNA -DNA chimera from binding to NF-kB transcription factors, being able to suppress the molecular interactions between HIV-1 LTR and p50, p52 and nuclear factors from B-lymphoid cells. Therefore, these results demonstrate that the designed NF-kB DNA-PNA chimeras could be used for a decoy approach in gene therapy.

Research paper thumbnail of Functional Binding Surface of a b-Hairpin VEGF Receptor Targeting Peptide Determined by NMR Spectroscopy in Living Cells

Research paper thumbnail of Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

Scientific reports, 2014

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their ... more Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

Research paper thumbnail of Structural Basis of a Temporin 1b Analogue Antimicrobial Activity against Gram Negative Bacteria Determined by CD and NMR Techniques in Cellular Environment

ACS Chemical Biology, 2015

We here report an original approach to elucidate mechanisms of action of antimicrobial peptides a... more We here report an original approach to elucidate mechanisms of action of antimicrobial peptides and derive crucial structural requirements for the design of novel therapeutic agents. The high resolution structure of TB_KKG6A, an antimicrobial peptide designed to amplify the spectrum of action of Temporin B, bound to E. coli is here determined by means of CD and NMR methodologies. We have also defined, through STD analysis, the residues in closer proximity to the bacterial membrane.

Research paper thumbnail of Functional Binding Surface of a β-Hairpin VEGF Receptor Targeting Peptide Determined by NMR Spectroscopy in Living Cells

Chemistry - A European Journal, 2014

In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growt... more In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast (15)N-edited NMR spectroscopic experiments. To this aim, (15)N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope-edited NMR spectroscopic experiments, including (15)N relaxation measurements, allowed a precise characterization of the in-cell HPLW epitope recognized by VEGFR2.

Research paper thumbnail of Synthetic Strategies for Obtaining PNA and PNA Conjugates

Research paper thumbnail of Reactions of Pd(PPh3)4 with 3′,5′-Di-O- acetylthymidine: Oxidative addition of Pd(PPh3)4 on thymidine N3 and C4 atoms

Organometallics, 2005

Oxidative addition reactions of Pd(PPh 3 ) 4 on the pyrimidine nucleosides 3′,5′-di-Oacetylthymid... more Oxidative addition reactions of Pd(PPh 3 ) 4 on the pyrimidine nucleosides 3′,5′-di-Oacetylthymidine and 3′,5′-di-O-acetyl-4-chlorothymidine and on the nucleobase 1-methylthymine have been investigated. N3 and C4 metal coordinated complexes 3 and 7 were isolated and characterized by spectroscopic techniques. Moreover, the crystal structure of the trans-[PdCl(1-methyl thymine)(PPh 3 ) 2 ]‚H 2 O (4) is reported.

Research paper thumbnail of Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS

Journal of Mass Spectrometry, 2005

Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their abi... more Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.

Research paper thumbnail of Targeting pre-miRNA by Peptide Nucleic Acids

Artificial DNA: PNA & XNA, 2012

PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with... more PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs.

Research paper thumbnail of Crystallization and preliminary X-ray diffraction studies of a D-lysine-based chiral PNA–DNA duplex

Acta Crystallographica Section D-biological Crystallography - ACTA CRYSTALLOGR D-BIOL CRYST, 2002

Research paper thumbnail of Development of an efficient and low-cost protocol for the manual PNA synthesis by Fmoc chemistry

Tetrahedron Letters, 2010

An efficient and low-cost protocol for the manual synthesis of Peptide Nucleic Acids is reported ... more An efficient and low-cost protocol for the manual synthesis of Peptide Nucleic Acids is reported here. The protocol relies on coupling reactions carried out with 2.5 equiv of PNA monomers activated with HOBT/ HBTU, in the presence of pyridine/NMM. The protocol has been tested on four PNA oligomers with a length ranging from 9 to 12 bases and a purine content up to 67%.

Research paper thumbnail of A new ferrocenemethyl-thymidine nucleoside: Synthesis, incorporation into oligonucleotides and optical spectroscopic studies on the resulting single strand, duplex and triplex structures

Tetrahedron, 1999

A new thymidine analogue, bearing a ferrocenemethyl residue at the N-3 position of the base, was ... more A new thymidine analogue, bearing a ferrocenemethyl residue at the N-3 position of the base, was synthesized in high yields via Mitsunobu reaction of ferrocenemethanol with sugar protected thymidine, converted into the corresponding 3'-phosphoramidite and incorporated into oligonucleotides. Duplex and triplex formation experiments, evaluated by UV and CD spectroscopy, showed a dramatic decrease of the affinity towards complementary single strands, while for triplexes, the introduction of a ferrocene residue in the third strand resulted in higher melting temperatures, associated with a reduced content of triplex structure.

Research paper thumbnail of Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction

Proceedings of the National Academy of Sciences, 2004

Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, t... more Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The 1 JNC coupling constant for the (؊1) scissile peptide bond at the N-extein-intein junction was found to be Ϸ12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (؊1) scissile bond in its unusual conformation. Overall, these studies support the ''ground-state destabilization'' model as part of the mechanism of catalysis.

Research paper thumbnail of Synergistic Antibacterial and Anti-Inflammatory Activity of Temporin A and Modified Temporin B In Vivo

PLoS ONE, 2009

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Ra... more Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10-14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and antiinflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3-6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.

Research paper thumbnail of Semisynthesis of Dimeric Proteins by Expressed Protein Ligation

Organic Letters, 2008

A one-pot synthesis of homodimeric proteins is described. The synthetic strategy is based on a do... more A one-pot synthesis of homodimeric proteins is described. The synthetic strategy is based on a double expressed protein ligation reaction between thioester peptides and a new bis-cysteinyl linker. The protocol was also applied to the synthesis of heterodimers.

Research paper thumbnail of Peptides from Royal Jelly: studies on the antimicrobial activity of jelleins, jelleins analogs and synergy with temporins

Journal of Peptide Science, 2011

Peptides isolated from natural fonts are the object of several studies aimed at finding new molec... more Peptides isolated from natural fonts are the object of several studies aimed at finding new molecules possessing antibacterial activity. We focused our studies on peptides originally isolated from the Royal Jelly, the jelleins and on some analogs having a UV reporter at the N-or C-terminus. We found that jelleins are mainly active against gram-positive bacteria; interestingly, they act in synergy with peptides belonging to the family of temporins such as temporin A and temporin B against Staphylococcus aureus A170 and Listeria monocytogenes.