Gianluca Damonte - Academia.edu (original) (raw)
Papers by Gianluca Damonte
Biochemical and Biophysical Research Communications, 1997
Peptide nucleic acid (PNA) molecules are very promising tools for antigene and antisense therapie... more Peptide nucleic acid (PNA) molecules are very promising tools for antigene and antisense therapies because of their remarkable refractoriness to degradation in biological fluids. However, very limited information is available on their uptake by potentially target cells and on their intracellular fate. A membrane-diffusable and fluorescence detectable PNA chimera, Phe-Leu-Phe-Leu-(Adenine)3-biotin, was obtained by solid phase peptide synthesis and characterized by
![Research paper thumbnail of Synthesis of 1 H -pyrazino[1,2- a ]pyrido[2,3- e ]pyrazine and 2 H -Pyrano[2,3- b ]pyrido[2,3- e ]pyrazine Derivatives](https://attachments.academia-assets.com/42598682/thumbnails/1.jpg)
](Journal of Heterocyclic Chemistry, 1996
Developmental Brain Research, 2004
The distribution of Pituitary adenylate cyclase-activating polypeptide (PACAP) was investigated i... more The distribution of Pituitary adenylate cyclase-activating polypeptide (PACAP) was investigated in the brain, pituitary and sensory organs of the zebrafish, Danio rerio, during development, in juvenile and adult specimens, using the immunofluorescence method. In 24 h post fertilization (hpf) embryos, PACAP immunoreactive cells appeared in the rostral telencephalon, dorsal diencephalon, caudal and medial rhombencephalon, spinal cord and retina. At 48
Journal of Biotechnology, 2015
Prolyl 4-hydroxylase (P4H) is a˛2ˇ2 tetramer catalyzing the post-translational hydroxylation of p... more Prolyl 4-hydroxylase (P4H) is a˛2ˇ2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens.
International journal of cancer. Journal international du cancer, Jan 20, 2015
MALDI-TOF MS was used to recognise serum peptidome profiles predictive of mortality in women affe... more MALDI-TOF MS was used to recognise serum peptidome profiles predictive of mortality in women affected by early BCa. Mortality was analysed based on signal profiling, and appropriate statistics were used. The results indicate that four signals were increased in deceased patients compared with living patients. Three of the four signals were individually associated with all-cause mortality, but only one having mass/charge ratio (m/z) 1,046.49 was associated with BCa-specific mortality and was the only peak to maintain an independent prognostic role after multivariate analysis. Two groups exhibiting different mortality probabilities were identified after clustering patients based on the expression of the four peptides, but m/z 1,046.49 was exclusively expressed in the cluster exhibiting the worst mortality outcome, thus confirming the crucial value of this peptide. The specific role of this peak was confirmed by competing risk analysis. MS findings were validated by ELISA analysis after...
European journal of medicinal chemistry, Jan 28, 2015
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in th... more The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the membrane of epithelial cells. Mutations affecting the CFTR gene cause cystic fibrosis (CF), a multi-organ severe disease. The most common CF mutation, F508del, impairs the processing and activity (gating) of CFTR protein. Other mutations, like G551D, only cause a gating defect. Processing and gating defects can be targeted by small molecules called generically correctors and potentiators, respectively. Aminoarylthiazoles (AATs) represent an interesting class of compounds that includes molecules with dual activity, as correctors and potentiators. With the aim to improve the activity profile of AATs, we have now designed and synthesized a library of novel compounds in order to establish an initial SAR that may provide indications about the chemical groups that are beneficial or detrimental for rescue activity. The new compounds were tested as correctors and potentiators in CFBE41o-expres...
Clinical Cancer Research, 2015
The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown t... more The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have anti-leukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current anti-leukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here we evaluated the anti-leukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n=6; B-CLL, n=19), and healthy leukocytes. Intracellular NAD+ and ATP levels, mitochondrial transmembrane potential (ΔΨm), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34+ progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD+ and ATP shortage, and induced ΔΨm dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including AML and B-CLL samples. Further evaluations of the combination of these agents in clinical setting should be considered.
Environmental Microbiology, 2015
The interactions of Vibrio aestuarianus 01/032 with haemolymph of the bivalves Mytilus galloprovi... more The interactions of Vibrio aestuarianus 01/032 with haemolymph of the bivalves Mytilus galloprovincialis and Crassostrea gigas were investigated to understand if haemolymph components (haemocytes and soluble factors) could be involved in the higher resistance to microbial infection shown by mussels in comparison with oysters. Although 01/032 bacteria adhered to haemocytes of both bivalves, they were sensitive to the bactericidal activity of whole haemolymph from mussel, but not from oyster; in addition, adhesion to mussel (but not oyster) haemocytes was affected by D-mannose. Mussel serum opsonins directed towards D-mannose-binding bacterial ligands were purified by affinity chromatography and were shown to mediate 01/032 interactions with M. galloprovincialis haemocytes. Nano-High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI-MS/MS) analysis showed that the purified opsonin matched the protein precursor of Mytilus edulis extrapallial protein (EP). In the presence of M. galloprovincialis EP protein (MgEP), C. gigas haemocytes killed V. aestuarianus 01/032 almost as efficiently as mussel phagocytes. These findings suggest that the different sensitivity of 01/032 strain to the antibacterial activity of oyster and mussel haemolymph might partly depend on the fact that C. gigas serum lacks MgEP-like opsonins. These results represent the basis for understanding the different sensitivity to microbial infections shown by the two bivalve species.
BMC Biotechnology, 2006
BACKGROUND: Taxol is an effective antineoplastic agent, originally extracted from the bark of Tax... more BACKGROUND: Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce
N-terminally truncated amyloid-b (Ab) peptides are present in early and diffuse plaques of indivi... more N-terminally truncated amyloid-b (Ab) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbN3(pE))40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Ab accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Ab1-40, Ab1-42, AbN3(pE))40 and AbN3(pE))42. Our data show that fibre morphology of Ab peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbN3(pE))40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbN3(pE))40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while fulllength peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance b-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.
Thrombosis and Haemostasis, 2006
Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, an... more Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, and standard chromatographic or capillary electrophoretic procedures are readily available. However, in the analyses of fibrinopeptide mixtures derived from the action of thrombin on human fibrinogen, a few unidentified peaks are usually present. The composition of these peaks was studied by reverse-phase HPLC/MS, revealing a single major anomalous peptide having a molecular mass of 1384.4. A further MS/MS analysis allowed the identification of this form, as a Nterminally truncated fibrinopeptide B (fpB) lacking the first two residues (pyroglutamic acid and glycine). This previously unidentified, relatively low-abundance form ( approximately 7%) has been found consistently in our fibrinopeptides preparations, and analysis of the parent Bbeta-chain suggest that it is likely present in circulating fibrinogen. In addition, deamidated forms of all fpB species (including desArgB), resulting from the conversion of asparagine to aspartic acid, were also identified. Overall, these previously unreported forms constitute a substantial amount of fpB (up to approximately 17% of the total), and should be taken into account for a reliable quantitative analysis of fpB release.
Journal of Proteome Research, 2009
Biosilica is an amazing example of natural order and complexity. Siliceous sponge spicules, in pa... more Biosilica is an amazing example of natural order and complexity. Siliceous sponge spicules, in particular, are characterized by a large variety of dimensions and shapes, with an ultrastructure based on silica nanoparticles strictly packaged around an axial filament constituted by a family of proteins called silicateins. These peculiar proteins have a high sequence homology with cathepsins and they play a double role of enzyme and template in the control of biosilica precipitation. However, their natural structural organization inside the spicules is far from being understood in details. In this work, axial filaments extracted from spicules of Petrosia ficiformis have been extensively analyzed by mass spectrometry, exploiting MALDI and ESI analysis of both the intact protein and the peptides coming from digestion of the axial filament with different proteases. Results demonstrate that P. ficiformis spicules contain almost only silicatein beta. Several post-translational modifications, like methylations at the N-terminal region, three phosphorylation sites, and the oxidation of a histidine and of a cysteine to cysteic acid, are described.
Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed t... more Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA transla- tion. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a
Escherichia coli is a robust, economic and rapid expression system for the production of recombin... more Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284:26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.
Experimental Parasitology, 2005
The Sec61α protein is defined as a highly conserved essential integral component of the endoplasm... more The Sec61α protein is defined as a highly conserved essential integral component of the endoplasmic reticulum in eukaryotic cells. We report a detailed immunolocalization of the Entamoeba histolytica homologue of the Sec61α subunit (EhSec61α), which shows an irregular pattern throughout the cell and is also found on the cell surface, its effective down-regulation by means of antisense peptide nucleic acids
Proceedings of The National Academy of Sciences, 2005
ADP-ribosyl cyclases are ubiquitous enzymes responsible for synthesis from NAD+ of the intracellu... more ADP-ribosyl cyclases are ubiquitous enzymes responsible for synthesis from NAD+ of the intracellular calcium-releasing signal molecules cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP+). Here, we show that cyclases from lower and higher Metazoa also synthesize three adenylic dinucleotides from cADPR and adenine: diadenosine diphosphate and two isomers thereof. These dinucleotides are present and metabolized in mammalian cells
Bioorganic & Medicinal Chemistry, 2015
The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants... more The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds.
Journal of Biological Chemistry, 2014
Background: Much evidence indicates that giant viruses, including Megavirus chilensis, possibly e... more Background: Much evidence indicates that giant viruses, including Megavirus chilensis, possibly encode autonomous glycosylation systems. Results: The Megavirus genome encodes proteins involved in the synthesis of 2-acetamido-2,6-dideoxy hexoses. Conclusion: N-Acetyl-rhamnosamine that is found in the virion glycans is produced by a novel viral biosynthetic pathway. Significance: These results will help in understanding the origin and function of the virally encoded glycosylation machineries.
Tetrahedron Letters, 2003
A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) ... more A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.
Biochemical and Biophysical Research Communications, 1997
Peptide nucleic acid (PNA) molecules are very promising tools for antigene and antisense therapie... more Peptide nucleic acid (PNA) molecules are very promising tools for antigene and antisense therapies because of their remarkable refractoriness to degradation in biological fluids. However, very limited information is available on their uptake by potentially target cells and on their intracellular fate. A membrane-diffusable and fluorescence detectable PNA chimera, Phe-Leu-Phe-Leu-(Adenine)3-biotin, was obtained by solid phase peptide synthesis and characterized by
Journal of Heterocyclic Chemistry, 1996
Developmental Brain Research, 2004
The distribution of Pituitary adenylate cyclase-activating polypeptide (PACAP) was investigated i... more The distribution of Pituitary adenylate cyclase-activating polypeptide (PACAP) was investigated in the brain, pituitary and sensory organs of the zebrafish, Danio rerio, during development, in juvenile and adult specimens, using the immunofluorescence method. In 24 h post fertilization (hpf) embryos, PACAP immunoreactive cells appeared in the rostral telencephalon, dorsal diencephalon, caudal and medial rhombencephalon, spinal cord and retina. At 48
Journal of Biotechnology, 2015
Prolyl 4-hydroxylase (P4H) is a˛2ˇ2 tetramer catalyzing the post-translational hydroxylation of p... more Prolyl 4-hydroxylase (P4H) is a˛2ˇ2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens.
International journal of cancer. Journal international du cancer, Jan 20, 2015
MALDI-TOF MS was used to recognise serum peptidome profiles predictive of mortality in women affe... more MALDI-TOF MS was used to recognise serum peptidome profiles predictive of mortality in women affected by early BCa. Mortality was analysed based on signal profiling, and appropriate statistics were used. The results indicate that four signals were increased in deceased patients compared with living patients. Three of the four signals were individually associated with all-cause mortality, but only one having mass/charge ratio (m/z) 1,046.49 was associated with BCa-specific mortality and was the only peak to maintain an independent prognostic role after multivariate analysis. Two groups exhibiting different mortality probabilities were identified after clustering patients based on the expression of the four peptides, but m/z 1,046.49 was exclusively expressed in the cluster exhibiting the worst mortality outcome, thus confirming the crucial value of this peptide. The specific role of this peak was confirmed by competing risk analysis. MS findings were validated by ELISA analysis after...
European journal of medicinal chemistry, Jan 28, 2015
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in th... more The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the membrane of epithelial cells. Mutations affecting the CFTR gene cause cystic fibrosis (CF), a multi-organ severe disease. The most common CF mutation, F508del, impairs the processing and activity (gating) of CFTR protein. Other mutations, like G551D, only cause a gating defect. Processing and gating defects can be targeted by small molecules called generically correctors and potentiators, respectively. Aminoarylthiazoles (AATs) represent an interesting class of compounds that includes molecules with dual activity, as correctors and potentiators. With the aim to improve the activity profile of AATs, we have now designed and synthesized a library of novel compounds in order to establish an initial SAR that may provide indications about the chemical groups that are beneficial or detrimental for rescue activity. The new compounds were tested as correctors and potentiators in CFBE41o-expres...
Clinical Cancer Research, 2015
The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown t... more The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have anti-leukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current anti-leukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here we evaluated the anti-leukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n=6; B-CLL, n=19), and healthy leukocytes. Intracellular NAD+ and ATP levels, mitochondrial transmembrane potential (ΔΨm), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34+ progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD+ and ATP shortage, and induced ΔΨm dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including AML and B-CLL samples. Further evaluations of the combination of these agents in clinical setting should be considered.
Environmental Microbiology, 2015
The interactions of Vibrio aestuarianus 01/032 with haemolymph of the bivalves Mytilus galloprovi... more The interactions of Vibrio aestuarianus 01/032 with haemolymph of the bivalves Mytilus galloprovincialis and Crassostrea gigas were investigated to understand if haemolymph components (haemocytes and soluble factors) could be involved in the higher resistance to microbial infection shown by mussels in comparison with oysters. Although 01/032 bacteria adhered to haemocytes of both bivalves, they were sensitive to the bactericidal activity of whole haemolymph from mussel, but not from oyster; in addition, adhesion to mussel (but not oyster) haemocytes was affected by D-mannose. Mussel serum opsonins directed towards D-mannose-binding bacterial ligands were purified by affinity chromatography and were shown to mediate 01/032 interactions with M. galloprovincialis haemocytes. Nano-High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI-MS/MS) analysis showed that the purified opsonin matched the protein precursor of Mytilus edulis extrapallial protein (EP). In the presence of M. galloprovincialis EP protein (MgEP), C. gigas haemocytes killed V. aestuarianus 01/032 almost as efficiently as mussel phagocytes. These findings suggest that the different sensitivity of 01/032 strain to the antibacterial activity of oyster and mussel haemolymph might partly depend on the fact that C. gigas serum lacks MgEP-like opsonins. These results represent the basis for understanding the different sensitivity to microbial infections shown by the two bivalve species.
BMC Biotechnology, 2006
BACKGROUND: Taxol is an effective antineoplastic agent, originally extracted from the bark of Tax... more BACKGROUND: Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce
N-terminally truncated amyloid-b (Ab) peptides are present in early and diffuse plaques of indivi... more N-terminally truncated amyloid-b (Ab) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbN3(pE))40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Ab accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Ab1-40, Ab1-42, AbN3(pE))40 and AbN3(pE))42. Our data show that fibre morphology of Ab peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbN3(pE))40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbN3(pE))40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while fulllength peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance b-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.
Thrombosis and Haemostasis, 2006
Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, an... more Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, and standard chromatographic or capillary electrophoretic procedures are readily available. However, in the analyses of fibrinopeptide mixtures derived from the action of thrombin on human fibrinogen, a few unidentified peaks are usually present. The composition of these peaks was studied by reverse-phase HPLC/MS, revealing a single major anomalous peptide having a molecular mass of 1384.4. A further MS/MS analysis allowed the identification of this form, as a Nterminally truncated fibrinopeptide B (fpB) lacking the first two residues (pyroglutamic acid and glycine). This previously unidentified, relatively low-abundance form ( approximately 7%) has been found consistently in our fibrinopeptides preparations, and analysis of the parent Bbeta-chain suggest that it is likely present in circulating fibrinogen. In addition, deamidated forms of all fpB species (including desArgB), resulting from the conversion of asparagine to aspartic acid, were also identified. Overall, these previously unreported forms constitute a substantial amount of fpB (up to approximately 17% of the total), and should be taken into account for a reliable quantitative analysis of fpB release.
Journal of Proteome Research, 2009
Biosilica is an amazing example of natural order and complexity. Siliceous sponge spicules, in pa... more Biosilica is an amazing example of natural order and complexity. Siliceous sponge spicules, in particular, are characterized by a large variety of dimensions and shapes, with an ultrastructure based on silica nanoparticles strictly packaged around an axial filament constituted by a family of proteins called silicateins. These peculiar proteins have a high sequence homology with cathepsins and they play a double role of enzyme and template in the control of biosilica precipitation. However, their natural structural organization inside the spicules is far from being understood in details. In this work, axial filaments extracted from spicules of Petrosia ficiformis have been extensively analyzed by mass spectrometry, exploiting MALDI and ESI analysis of both the intact protein and the peptides coming from digestion of the axial filament with different proteases. Results demonstrate that P. ficiformis spicules contain almost only silicatein beta. Several post-translational modifications, like methylations at the N-terminal region, three phosphorylation sites, and the oxidation of a histidine and of a cysteine to cysteic acid, are described.
Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed t... more Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA transla- tion. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a
Escherichia coli is a robust, economic and rapid expression system for the production of recombin... more Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284:26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.
Experimental Parasitology, 2005
The Sec61α protein is defined as a highly conserved essential integral component of the endoplasm... more The Sec61α protein is defined as a highly conserved essential integral component of the endoplasmic reticulum in eukaryotic cells. We report a detailed immunolocalization of the Entamoeba histolytica homologue of the Sec61α subunit (EhSec61α), which shows an irregular pattern throughout the cell and is also found on the cell surface, its effective down-regulation by means of antisense peptide nucleic acids
Proceedings of The National Academy of Sciences, 2005
ADP-ribosyl cyclases are ubiquitous enzymes responsible for synthesis from NAD+ of the intracellu... more ADP-ribosyl cyclases are ubiquitous enzymes responsible for synthesis from NAD+ of the intracellular calcium-releasing signal molecules cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP+). Here, we show that cyclases from lower and higher Metazoa also synthesize three adenylic dinucleotides from cADPR and adenine: diadenosine diphosphate and two isomers thereof. These dinucleotides are present and metabolized in mammalian cells
Bioorganic & Medicinal Chemistry, 2015
The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants... more The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds.
Journal of Biological Chemistry, 2014
Background: Much evidence indicates that giant viruses, including Megavirus chilensis, possibly e... more Background: Much evidence indicates that giant viruses, including Megavirus chilensis, possibly encode autonomous glycosylation systems. Results: The Megavirus genome encodes proteins involved in the synthesis of 2-acetamido-2,6-dideoxy hexoses. Conclusion: N-Acetyl-rhamnosamine that is found in the virion glycans is produced by a novel viral biosynthetic pathway. Significance: These results will help in understanding the origin and function of the virally encoded glycosylation machineries.
Tetrahedron Letters, 2003
A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) ... more A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.