Gennaro Piccialli

Hydrolysis of oleuropein by recombinant β-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus immobilised on chitosan matrix

The recombinant beta-glycosidase (EcS beta gly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70 degrees C). Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available beta-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor. The results of the biotransformation at 70 degrees C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction. Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate. The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h-1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h-1). For the hydrolysis of the oleuropein by bioreactor at 60 degrees C we used lower molar ratio oleuropein/enzyme only by the single step approach. In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h). The stability of the bioreactor at the operative temperatures showed a t1/2 of 30 days at 70 degrees C and a t1/2 of 56 days at 60 degrees C.

Solid phase synthesis of oligosaccharides

Abstract An efficient and stereocontrolled solid-phase chemical synthesis of oligosaccarides using a new type of insoluble support (a copolymer of polyethylene glycol and polystyrene) is described. Several Lewis acids were tested as activators of the trichloroacetimidate 12 , used as glycosyl donor, in the dimer 13 formation.

Aminosilane functionalizations of mesoporous oxidized silicon for oligonucleotide synthesis and detection

Direct solid phase synthesis of peptides and oligonucleotides (ONs) requires high chemical stability of the support material. In this work, we have investigated the passivation ability of porous oxidized silicon multilayered structures by two aminosilane compounds, 3-aminopropyltriethoxysilane and 3-aminopropyldimethylethoxysilane (APDMES), for optical label-free ON biosensor fabrication. We have also studied by spectroscopic reflectometry the hybridization between a 13 bases ON, directly grown on the aminosilane modified porous oxidized silicon by in situ synthesis, and its complementary sequence. Even if the results show that both devices are stable to the chemicals (carbonate/methanol) used, the porous silica structure passivated by APDMES reveals higher functionalization degree due to less steric hindrance of pores.

Tetra-end-linked oligonucleotides forming DNA G-quadruplexes: a new class of aptamers showing anti-HIV activity.

The biophysical and biological properties of unprecedented anti-HIV aptamers are presented. The most active aptamer (1L) shows a significant affinity to the HIV protein gp120.

Investigating the role of T7 and T12 residues on the biological properties of thrombin-binding aptamer: enhancement of anticoagulant activity by a single nucleobase modification.

An acyclic pyrimidine analogue, containing a five-member cycle fused on the pyrimidine ring, was synthesized and introduced at position 7 or 12 of the 15-mer oligodeoxynucleotide GGTTGGTGTGGTTGG, known as thrombin-binding aptamer (TBA). Characterization by 1H NMR and CD spectroscopies of the resulting aptamers, TBA-T7b and TBA-T12b, showed their ability to fold into the typical antiparallel chairlike G-quadruplex structure formed by TBA. The apparent CD melting temperatures indicated that the introduction of the acyclic residue, mainly at position 7, improves the thermal stability of resulting G-quadruplexes with respect to TBA. The anticoagulant activity of the new molecules was then valued in PT assay, and it resulted that TBA-T7b is more potent than TBA in prolonging clotting time. On the other hand, in purified fibrinogen assay the thrombin inhibitory activity of both modified sequences was lower than that of TBA using human enzyme, whereas the potency trend was again reversed using bovine enzyme. Obtained structure-activity relationships were investigated by structural and computational studies. Taken together, these results reveal the active role of TBA residues T7 and T12 and the relevance of some amino acids located in the anion binding exosite I of the protein in aptamer-thrombin interaction.

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

Abstract 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.

The Anti-Proliferative Effect of L-Carnosine Correlates with a Decreased Expression of Hypoxia Inducible Factor 1 alpha in Human Colon Cancer Cells

In recent years considerable attention has been given to the use of natural substances as anticancer drugs. The natural antioxidant dipeptide L-carnosine belongs to this class of molecules because it has been proved to have a significant anticancer activity both in vitro and in vivo. Previous studies have shown that L-carnosine inhibits the proliferation of human colorectal carcinoma cells by affecting the ATP and Reactive Oxygen Species (ROS) production. In the present study we identified the Hypoxia-Inducible Factor 1α (HIF-1α) as a possible target of L-carnosine in HCT-116 cell line. HIF-1α protein is over-expressed in multiple types of human cancer and is the major cause of resistance to drugs and radiation in solid tumours. Of particular interest are experimental data supporting the concept that generation of ROS provides a redox signal for HIF-1α induction, and it is known that some antioxidants are able to suppress tumorigenesis by inhibiting HIF-1α. In the current study we found that L-carnosine reduces the HIF-1α protein level affecting its stability and decreases the HIF-1 transcriptional activity. In addition, we demonstrated that L-carnosine is involved in ubiquitin-proteasome system promoting HIF-1α degradation. Finally, we compared the antioxidant activity of L-carnosine with that of two synthetic anti-oxidant bis-diaminotriazoles (namely 1 and 2, respectively). Despite these three compounds have the same ability in reducing intracellular ROS, 1 and 2 are more potent scavengers and have no effect on HIF-1α expression and cancer cell proliferation. These findings suggest that an analysis of L-carnosine antioxidant pathway will clarify the mechanism underlying the anti-proliferative effects of this dipeptide on colon cancer cells. However, although the molecular mechanism by which L-carnosine down regulates or inhibits the HIF-1α activity has not been yet elucidated, this ability may be promising in treating hypoxia-related diseases.

d(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K+ or NH4+ ions, through the 5′-5′ stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads.

New anti-HIV aptamers based on tetra-end-linked DNA G-quadruplexes: effect of the base sequence on anti-HIV activity

This communication reports on the synthesis and biophysical, biological and SAR studies of a small library of new anti-HIV aptamers based on the tetra-end-linked G-quadruplex structure. The new aptamers showed EC(50) values against HIV-1 in the range of 0.04-0.15 μM as well as affinities for the HIV-1 gp120 envelope in the same order of magnitude.

Solid phase synthesis of oligonucleotides tethered to oligo-glucose phosphate tails

Abstract Oligonucleotides conjugated at both 3′ and 5′-ends with glucose residues, 4,6-linked through a phosphodiester bridge, have been synthesized by sequential addition of a 6- O -DMT-glucose-4-phosphoramidite building block following a standard automated ODN assembly procedure. Two 3′,5′-bis-glycoconjugated 18-mers, designed for antisense experiments, have been prepared and their hybridization properties with a complementary DNA fragment evaluated by UV thermal analysis.