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G Piccialli

Publications and source records attributed to G Piccialli.

16 recordsLinked to original sources
PubMed2004

Targeting duplex DNA with DNA-PNA chimeras? Physico-chemical characterization of a triplex DNA-PNA/DNA/DNA.

Targeting double-stranded DNA with homopyrimidine PNAs results in strand displacement complexes PNA/DNA/PNA rather than PNA/DNA/DNA triplex structures. Not much is known about the binding properties of DNA-PNA chimeras. A 16-mer 5'-DNA-3'-p-(N)PNA(C) has been investigated for its ability to hybridize a complementary duplex DNA by DSC, CD, and molecular modeling studies. The obtained results showed the formation of a triplex structure having similar, if not slightly higher, stability compared to the same all-DNA complex.

Chemical Phenomena↗
PubMed2003

Physico-chemical studies of a DNA triplex containing a new ferrocenemethyl-thymidine residue in the third strand.

The stability of a 16-mer DNA triple helix containing a 3-N(ferrocenemethyl)-thymidine residue in the third strand has been investigated in comparison with the unmodified triplex of the same sequence. A complete physico-chemical characterization of the two triple helices on changing the pH by means of calorimetry, circular dichroism and molecular modeling is therefore reported. The thermodynamic parameters were obtained in the pH range 5.5-7.2 by differential scanning calorimetry (DSC). For both triplexes the T(m) and Delta H degrees (T(m)) values increase on decreasing the pH. In the pH range 7.2-6.0 the triplex containing the ferrocenemethyl nucleoside is less stable than the unmodified one, whereas the modified triplex becomes more stable at pH 5.5. Such difference in stability at each pH value is overwhelmingly enthalpic in origin. CD spectra show conformational changes on decreasing the pH for both the triplexes. By spectroscopic pH titration the apparent pK(a) values of the cytosines in the two triplexes could be estimated, with the cytosines in the TFO containing the ferrocenemethyl residue having lower apparent pK(a) values. These results are consistent with the calorimetric data, showing a decrease of the thermodynamic parameters in the pH range 7.2-6.0 and an increase at pH 5.5 for the ferrocenylated triplex with respect to the unmodified one. The thermodynamic and spectroscopic data are also discussed in relation to molecular models.

Calorimetry, Differential Scanning↗
PubMed2001

Physico-chemical studies on DNA triplexes containing an alternate third strand with a non-nucleotide linker.

Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)m(pyrimidine)m-3'. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine-pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson-Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3'-3' phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P = phosphate and X = 1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.

Base Composition↗
PubMed2000

Studies toward the synthesis of pinolidoxin, a phytotoxic nonenolide from the fungus Ascochyta pinodes. Determination of the configuration at the C-7, C-8, and C-9 chiral centers and stereoselective synthesis of the C(6)-C(18) fragment.

The absolute stereochemistry at the C-7, C-8, and C-9 chiral centers of pinolidoxin (1) has been determined by chemical and spectral methods. First, the synthesis of four stereoisomeric fully benzoylated 2,3-erythro-1,2,3,4-heptanetetrols, corresponding to the C(6)-C(18) portion of the natural substance, has been accomplished starting from meso-tartaric acid. As next step, the selection of the synthetic tetrabenzoate possessing "natural" stereochemistry (10a'), suitable for absolute configuration determination, has been carried out by correlation with its "natural" homologue derived from degradation of pinolidoxin. Determination of the stereochemistry at the title chiral centers has been carried out by application of the Mosher's method both to 7a', a compound stereochemically related to 10a', and to pinolidoxin itself. The stereoselective synthesis of a protected form of the C(6)-C(18) portion of pinolidoxin, to be used in its total synthesis, has also been accomplished starting from commercially available D-erythronolactone.

Alkenes↗
PubMed2000

Hydrolysis of oleuropein by recombinant beta-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.

Bioreactors↗
PubMed2000

Effect of lengthening of peptide backbone by insertion of chiral beta-homo amino acid residues: conformational behavior of linear peptides containing alternating L-leucine and beta-homo L-leucine residues.

The synthesis and the solution behavior of the linear peptides containing a beta-homo (beta-H) leucine residue-Boc-Leu-beta-HLeu-Leu-OMe, Boc-beta-HLeu-Leu-beta-HLeu-Leu-OMe, and Boc-Leu-beta-HLeu-Leu-beta-HLeu-Leu-OMe-as well as the solid structure of the tripeptide, are reported. The conformational behavior of the peptides was investigated in solution by two-dimensional nmr. Our data support the existence in solution with different families of conformers in rapid interchange. The crystals of the tripeptide are orthorhombic, space group P2(1)2(1)2, with a = 15.829(1) A, b = 29.659(1) A, c = 6.563(1) A, and Z = 4. The structure has been solved by direct methods and refined to final R1 and wR2 indexes of 0.0530 and 0.1436 for 2420 reflections with I > 2sigma(I). In the solid state, the tripeptide does not present intramolecular H bonds, and the peptide backbone of the two leucine residues adopts a quasi-extended conformation. For the beta-HLeu residue, the backbone conformation is specified by the torsion angles straight phi(2) = -120.9(4) degrees, mu(2) = 56.7(4) degrees, psi(3) = -133.2(4) degrees. The side chains of the three residues assume the same conformation (g(-), g(-), trans), and all peptide bonds, except the urethane group at the N-terminus, are in the trans conformation. Preliminary conformational energy calculations carried out on the Ac-NH-beta-HAla-NHMe underline that the conformations with mu angle equal to 180 degrees and 60 degrees assume lower energy with respect to the others. In addition, we found a larger conformational freedom for the psi angle with respect to the straight phi angle.

Crystallography, X-Ray↗
PubMed2000

Glycosylations of inosine and uridine nucleoside bases and synthesis of the new 1-(beta-D-glucopyranosyl)-inosine-5',6"-diphosphate.

Gluco- and ribosylation of the bases of sugar protected inosine and uridine were investigated, obtaining only adducts with beta-configuration at the new glycosidic carbon; stereospecific insertion of a sugar moiety at the 1-N of inosine was achieved either using a Mitsunobu approach (for ribosylation) or by direct coupling of 1-alpha-bromoglucose 13 with 2',3',5'-tri-O-acetylinosine for glucosylation. 1-(beta-D-glucosyl)-inosine, chosen as starting substrate for glucosylated analogs of cyclic IDP-ribose, was phosphorylated at the primary hydroxyls and tested in intramolecular pyrophosphate bond formation.

Carbohydrate Conformation↗
PubMed1999

A new solid-phase synthesis of oligonucleotides 3'-conjugated with peptides.

A convenient 'on line' solid-phase synthesis of oligonucleotides conjugated at the 3'-end with peptides by means of a polymeric support linking the first nucleoside via the base has been developed. A 17-mer designed for antisense experiments against HIV-1, linking at the 3'-terminus the tripeptide Gly-Gly-His, was prepared in good yields and characterized by MALDI-TOF mass spectrometry.

Chemistry↗
PubMed1997

Identification of the active site nucleophile in the thermostable beta-glycosidase from the archaeon Sulfolobus solfataricus expressed in Escherichia coli.

Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli was fully inactivated at 65 degrees C, according to pseudo-first-order kinetics, by [3H]conduritol B epoxide (DL-1,2 anhydro-myo-inositol) synthesized as the active site directed inhibitor by a slight modification of Legler's procedure [Legler, G. (1977) Methods Enzymol. 46, 368-381]. The determination of kinetic constants for the inactivation showed that the process took place through the formation of a stabilized inhibitor-enzyme intermediate. Inactivation and reactivation studies suggested that the inhibitor-enzyme intermediate complex was formed more rapidly and hydrolyzed at a lower rate than it was for other glycosidases. Moreover, the stoichiometry of the binding, determined by electrospray mass spectrometric analysis, revealed that one molecule of the inhibitor was covalently bound to each enzyme subunit. The binding site for [3H]conduritol B epoxide was identified by the isolation and partial sequence analysis of the radioactive peptide obtained by cyanogen bromide and pepsin digests. Electrospray tandem mass analysis of the labeled peptide showed that the inhibitor was covalently bound to E387. This result, in agreement with data obtained from sequence alignments of S. solfataricus beta-glycosidase with other gluco- and galactosidases of the glycosyl hydrolase family 1 [Henrissat, B. (1991) Biochem. J. 280, 309-316], indicates that the conserved E387 is the nucleophilic amino acid residue in the active site of the enzyme.

Amino Acid Sequence↗
PubMed1996

A new pattern for helix-turn-helix recognition revealed by the PU.1 ETS-domain-DNA complex.

The Ets family of transcription factors, of which there are now about 35 members regulate gene expression during growth and development. They share a conserved domain of around 85 amino acids which binds as a monomer to the DNA sequence 5'-C/AGGAA/T-3'. We have determined the crystal structure of an ETS domain complexed with DNA, at 2.3-A resolution. The domain is similar to alpha + beta (winged) 'helix-turn-helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8 degrees. The domain contacts the DNA by a novel loop-helix-loop architecture. Four of amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix-turn-helix' motif, which binds downstream and on the opposite strand.

Amino Acid Sequence↗
PubMed1994

Interaction of the high-affinity inhibitor tetrahydro-dUMP with the allosteric enzyme deoxycytidylate aminohydrolase.

Tetrahydro-dUMP, an analog of the putative transition state in aminohydrolysis of deoxycytidine monophosphate (dCMP) inhibits the allosteric enzyme deoxycytidylate aminohydrolase with high affinity. The inhibition is reversible, and its kinetics is consistent with the analog binding at the substrate site only to one and the same conformation that binds the substrate dCMP. Such kinetics is what would be expected for a transition state analog interacting in an allosteric "K system."

Allosteric Regulation↗
PubMed1992

The dual-mode quaternary structure of seminal RNase.

Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.

Animals↗
PubMedDate not supplied

Synthesis and DNA binding properties of DNA-PNA chimeras.

A systematic study to evaluate the ability of 5'-DNA-3'-p-(N)-PNA-(C) chimeras to form triple helix structures has been undertaken. Preliminary results carried out on a 16-mer chimera with three PNA monomers at the 3'-end showed the formation of a stable DNA-PNA/DNA/DNA triplex, having similar conformational behaviour to a canonical DNA/DNA/DNA triplex.

Binding Sites↗