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Biomedical subjects

R Bazzo

Publications and source records attributed to R Bazzo.

At least 19 recordsLinked to original sources
PubMed2005

Cytological diagnosis of mandibular salivary gland adenocarcinoma in a dog.

A case of mandibular salivary gland adenocarcinoma in a 9-year-old female dog is described. Material collected by fine needle aspiration underwent cytological examination and after the diagnosis of salivary carcinoma the mass was surgically excised and then was processed for histological examination. The aim of this work is to describe the cytopathological features of this carcinoma and to emphasize the usefulness of fine needle aspiration technique, which is an effective, inexpensive and minimally invasive method of diagnosis that can be performed before incisional biopsy or even before surgical excision.

Adenocarcinoma↗
PubMed2003

Transitional cell carcinoma of the renal pelvis in two dogs.

Transitional cell carcinoma (TCC) of the renal pelvis was found in two dogs, a 7-year-old male English Setter and a 11-year-old female Shetland shepherd. Affected dogs were presented for clinical examination without any specific symptoms but haematuria in case 1 and occurrence of whitish material in the urine of case 2; neoplastic disorders were discovered with ultrasonographic investigation and fine needle aspiration biopsy. Histopathological examination was carried out after nephrectomy and ureterectomy of the affected kidney of both dogs, and confirmed the diagnosis of non-invasive and low grade TCC in case 1 and of infiltrating TCC in case 2. The clinical, gross, cytological and histopathological features of these rare tumours originating from transitional epithelium of the renal pelvis are reported.

Animals↗
PubMed2002

Sertoli cell tumour with Call-Exner-like bodies in a dog.

An histological diagnosis of Sertoli cell tumour and concurrent seminoma was formulated after complete resection of the right testicle in a 8-year-old Doberman. The unusual histological feature of abundant eosinophilic material with a extracellular location forming the Call-Exner-like bodies is reported.

Animals↗
PubMed2001

Sensitivity enhancement of a two-dimensional experiment for the measurement of heteronuclear long-range coupling constants, by a new scheme of coherence selection by gradients.

In this work we present a new pulse sequence for the measurement of long-range heteronuclear coupling constants in which the optimization of coherence selection by pulsed field gradients offers a net increase in sensitivity. This type of experiments is extremely valuable for conformational studies of molecules in natural abundance and in this context the use of gradients is essential for an efficient suppression of (12)C bound proton signals. A comparative analysis of the different gradient schemes available is presented with a conclusive elucidation of the relative sensitivities. Our gradient scheme could be advantageous as a building block for other related experiments.

Journal Article↗
PubMed2001

Improved sensitivity in indirect monitoring of chemical shifts of proton-heteronuclear spin pairs (1H-13C and 1H-15N) in 3D and 4D NMR spectroscopy.

In three-dimensional and four-dimensional experiments on doubly labelled proteins not only heteronuclear (13C or 15N) but also proton (1H) frequencies are often indirectly monitored, rather than being directly observed. In this communication we show how in these experiments by overlaying 1H and heteronuclear evolutions one can obtain decreased apparent relaxation rates of 1H signals, yielding improved sensitivity. The new method applies to spin pairs like 1H-15N, as in amide groups, or 1H-13C, as in methine groups of alpha or aromatic systems.

Carbon Isotopes↗
PubMed2001

Measurement of homonuclear three-bond J(H(N)Halpha) coupling constants in unlabeled peptides complexed with labeled proteins: application to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of hepatitis C virus (HCV).

A new isotope-filtered experiment has been designed to measure homonuclear three-bond J(H(N)Halpha) coupling constants of unlabeled peptides complexed with labeled proteins. The new experiment is based on the 3D HNHA pulse scheme, and belongs to the 'quantitative J-correlation' type. It has been applied to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of human hepatitis C virus (HCV).

Algorithms↗
PubMed2000

Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain.

Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N-terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His-Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His-Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes.

Aminobutyrates↗
PubMed1999

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism.

The solution structure of the hepatitis C virus (BK strain) NS3 protein N-terminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zinc-binding site exposed on the surface, subject to a slow conformational exchange process.

Amino Acid Sequence↗
PubMed1999

Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies.

The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.

Amino Acid Sequence↗
PubMed1999

Selective correlation of amide groups to glycine alpha protons and of arginine guanidine groups to delta protons in proteins by multiple quantum spectroscopy.

A new 3D pulse sequence correlates backbone amide proton and nitrogen with alpha proton resonances selectively for glycine residues in a fully doubly labeled (15N, 13C) protein. The excitation of multiple quantum coherences provides optimized resolution and sensitivity. Degenerate alpha proton groups can be promptly recognized. Correlation of guanidine NH groups to delta protons of arginine side chains is also obtained.

Amides↗
PubMed1998

The metal binding site of the hepatitis C virus NS3 protease. A spectroscopic investigation.

The NS3 region of the hepatitis C virus encodes for a serine protease activity, which is necessary for the processing of the nonstructural region of the viral polyprotein. The minimal domain with proteolytic activity resides in the N terminus, where a structural tetradentate zinc binding site is located. The ligands being been identified by x-ray crystallography as being three cysteines (Cys97, Cys99, and Cys145) and one histidine residue (His149), which is postulated to coordinate the metal through a water molecule. In this article, we present an analysis of the role of metal coordination with respect to enzyme activity and folding. Using NMR spectroscopy, the resonances of His149 were assigned based on their isotropic shift in a Co(II)-substituted protein. Data obtained with 15N-labeled NS3 protease were compatible with the involvement of the delta-N of His149 in metal coordination. pH titration experiments showed that the cooperative association of at least two protons is required in the protonation process of His149. Changes in the NMR signals of this residue between pH 7 and 5 are interpreted as evidence for a structural change at the metal binding site, which switches from a "closed" to an "open" conformation. Site-directed mutagenesis of His149 has shown the importance of this residue in the metal incorporation pathway and for achieving an active fold. The metal coordination of the protease was also investigated by circular dichroism and electronic absorption spectroscopies using a Co(II)-substituted enzyme. We show evidence for rearrangements of the metal coordination geometry induced by complex formation with an NS4A peptide cofactor. No such changes were observed upon binding to a substrate peptide. Also, CN- and N3- induced Co(II) ligand field perturbations, which went along with an 1.5-fold enhancement of protease activity.

Amino Acid Substitution↗
PubMed1997

Resonance assignment and secondary structure determination and stability of the recombinant human uteroglobin with heteronuclear multidimensional NMR.

Human uteroglobin (h-UG) or Clara cell 10kDa (cc10kDa) is a steroid-dependent, 17 kDa homodimeric, secretory protein with potent anti-inflammatory/immunomodulatory properties. However, the exact physiological role still remains to be determined. It has been hypothesised that its activity is exerted through the binding of a specific target represented by a small molecule (still unknown), and that the binding is regulated by the formation/disruption of two cysteine bonds. The binding properties of the reduced UG have been proved in vitro for several different molecules, but no in vivo data are available to date. However, binding has been observed between reduced rabbit UG and a protein of an apparent molecular mass of 90 kDa and, more recently, we found an h-UG-binding protein

Animals↗
PubMed1996

High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif.

We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the alpha-helical portion of a 26-residue 'consensus' peptide (CP1) belonging to the Cys(2)-Hys2 zinc-finger family. The result was shown to be a highly homogeneous alpha-helical library (Bianchi et al., 1995). The structures of the parent compound (CPI) and of a representative member (CPlm) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.

Magnetic Resonance Spectroscopy↗
PubMed1995

A conformationally homogeneous combinatorial peptide library.

In search for a rational way to convert the information encoded in peptide structures into peptidomimetics, major progress could be made by coupling the power of selection methods, now enormously increased in number as a result of the development of combinatorial peptide libraries, with the rational design of structure-inducing templates for the selectable sequences. The availability of libraries of peptides with predetermined structure would enable selection-driven peptidomimetic design, whereby a conformational model for the peptide pharmacophore would be directly derived from the screening, allowing the design of a suitable non-peptidic scaffold to replace the peptide backbone. We describe here the first example of a conformationally homogeneous combinatorial peptide library, which yields ligands with the expected structure upon selection. The library was built by randomising five positions in the alpha-helical portion of a 26 amino acid Cys2His2 consensus "zinc-finger" motif. Since in zinc-fingers metal coordination and folding are coupled, in our library metal-dependent binding represents a built-in control against the selection of structurally undefined sequences. The alpha-helical library was produced as both fusion with the pVIII protein of filamentous phage and soluble peptides by chemical synthesis, the latter enabling the expansion of the selectable repertoire by the inclusion of non-coded amino acids. The two libraries were independently screened with the same receptor (a monoclonal IgA reactive against the lipopolysaccharide of the human pathogen Shigella flexneri), yielding a very similar consensus. In particular, the peptides defined by both methods showed very strong, zinc-dependent binding to the IgA. The geometrical arrangement of the side-chains of the selected peptide pharmacophore was shown by circular dichroism, Co(II)-complex absorption and high-resolution NMR to be structurally invariant with respect to the parent zinc-finger.

Amino Acid Sequence↗
PubMed1991

The conformational effects of N-glycosylation on the tailpiece from serum IgM.

1H-NMR spectroscopy has been used to study the conformation and dynamics of the isolated tailpiece from human serum immunoglobulin M, a 22-residue peptide containing a single asparagine glycosylation site. The peptide is isolated as a set of glycoforms, varying only in the sequence of the oligosaccharide attached at the glycosylation site. The oligosaccharides present have the general formula (Man)n(GlcNAc)2, with 45% having n = 6, 45% having n = 8 and 10% having n = 7 and/or 9. They have been identified and their NMR parameters compared to those found for the isolated oligosaccharides in free solution. The conformation and dynamics of the peptide component have also been studied, using NOE data and hydrogen-exchange experiments, and the results compared to those obtained from the aglycosyl peptide of the same sequence. The presence of the peptide is found to have no measurable effect on the conformation of the oligosaccharides. However, the presence of oligosaccharide causes a decrease in the conformational mobility of the backbone and sidechains of the peptide in the region of the glycosylation site. This is proposed to result from interactions between the oligosaccharide core and the amino acid side chains. Further, the conformation of the N-glycosidic linkage has been shown to be both rigid and planar. Thus, the conformational space available to an N-linked oligosaccharide in a glycoprotein relative to the protein may depend to a large extent upon the flexibility of the asparagine side chain. Various roles for the different glycoforms of the tail peptide are discussed.

Amino Acid Sequence↗
PubMed1991

Contribution of proline-14 to the structure and actions of melittin.

The structure and dynamic properties of bee venom melittin and a synthetic analogue, [Ala14]-melittin (melittin P14A), are compared, using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and amide exchange measurements in methanol. P14A is shown to adopt a regular, stable alpha-helical conformation in solution without the flexibility around the Pro-14 residue found in melittin. P14A has twice the hemolytic activity of melittin but is less able to induce voltage-dependent ion conductance in planar bilayers. The results indicate that helix flexibility afforded by the Pro-14 residue promotes the ability of melittin to adopt the transbilayer associates thought to underlie ion translocation.

Alanine↗
PubMed1991

Solution structure of human calcitonin gene-related peptide by 1H NMR and distance geometry with restrained molecular dynamics.

The structure of human calcitonin gene-related peptide 1 (hCGRP-1) has been determined by 1H NMR in a mixed-solvent system of 50% trifluoroethanol/50% H2O at pH 3.7 and 27 degrees C. Complete resonance assignment was achieved by using two-dimensional methods. Distance restraints for structure calculations were obtained by semiquantitative analysis of intra- and interresidue nuclear Overhauser effects; in addition, stereospecific or X1 rotamer assignments were obtained for certain side chains. Structures were generated from the distance restraints by distance geometry, followed by refinement using molecular dynamics, and were compared with experimental NH-C alpha H coupling constants and amide hydrogen exchange data. The structure of hCGRP-1 in this solvent comprises an amino-terminal disulfide-bonded loop (residues 2-7) leading into a well-defined alpha-helix between residues 8 and 18; thereafter, the structure is predominantly disordered, although there are indications of a preference for a turn-type conformation between residues 19 and 21. Comparison of spectra for the homologous hCGRP-2 with those of hCGRP-1 indicates that the conformations of these two forms are essentially identical.

Amino Acid Sequence↗