Search PubMed⌕ Search

Biomedical subjects

A Tramontano

Publications and source records attributed to A Tramontano.

At least 55 records · Page 3Linked to original sources
PubMed1997

Temporal study of immunoglobin M seroreactivity to Borrelia burgdorferi in patients treated for Lyme borreliosis.

Forty-six patients with late Lyme disease who were considered improved or cured following treatment were monitored by immunoglobin M (IgM) immunoblotting (mean monitoring period, 27.6 months). There was a persistent IgM response in 32 (97%) of 33 initially positive patients. All but three showed a consistent number, type, and intensity of IgM bands over the entire follow-up period. IgM immunoblotting may not be useful for monitoring the response to treatment of Lyme borreliosis.

Adult↗
PubMed1997

The nonstructural proteins of the hepatitis C virus: structure and functions.

The hepatitis C virus is the major causative agent of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in cell culture, several of its features have been elucidated in the past few years. The viral genome is a single-stranded, 9.5kb long RNA molecule of positive polarity. The viral genome is translated into a single polyprotein of about 3000 amino acids. The virally encoded polyprotein undergoes proteolytic processing by a combination of cellular and viral proteolytic enzymes in order to yield all the mature viral gene products. The gene order of HCV has been determined to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The mature structural proteins, C, E1 and E2 have been shown to arise from the viral polyprotein via proteolytic processing by host signal peptidases. Conversely, generation of the mature nonstructural proteins relies on the activity of viral proteases. Thus, cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autoprotease encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine protease contained within the N-terminal region of NS3. Besides the protease domain, NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the protease. Whereas the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase, no function has yet been attributed to NS4B and NS5A. The latter is a cytoplasmic phosphoprotein and appears to be involved in mediating the resistance of the hepatitis C virus to the action of interferon.

Hepacivirus↗
PubMed1997

[Treatment of Hirschsprung's disease with the Soave-Coran technique: experience with newborns and children].

Authors report their experience with Soave endorectal pull-through (ERPT) procedure modified by Coran for Hirschsprung's disease. At the Santobono Children's Hospital of Naples 30 patients have been operated for Hirschsprung's disease; 21 of them (age 10 days-3 years and 7 months) have undergone a Coran's procedure: a single stage procedure was performed in 16, the other 5 underwent ERPT following colostomy. Four complications occurred: 2 intestinal obstruction and 2 anastomotic leaks. Excellent long-term results reported from literature and proved by Author's experience suggest that one stage Coran's procedure can be safely performed even in neonates avoiding colostomy complications and reducing total duration and costs of hospitalization.

Child, Preschool↗
PubMed1996

A zinc binding site in viral serine proteinases.

The NS3 protein of hepatitis C virus contains a chymotrypsin-like serine proteinase domain. We built a homology model of this domain which predicts the presence of a tetradentate metal binding site formed by three cysteines and one histidine. These residues are strictly conserved in all known hepatitis C viral genotypes as well as in other recently discovered related hepatitis viruses. We show that the hepatitis C virus enzyme does indeed contain a Zn2+ ion with S3N ligation and that the metal is required for structural integrity and activity of the enzyme. Strikingly, the residues forming the metal binding site are also conserved in the chymotrypsin-like 2A cysteine proteinases of picornaviruses. Remarkably, in these highly variable viral genomes the metal binding site is more conserved than the catalytic residues and thus allows us to define a novel class of zinc binding chymotrypsin-like proteinases and to identify a new attractive target for antiviral therapy.

Amino Acid Sequence↗
PubMed1996

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.

Amino Acid Sequence↗
PubMed1996

Update on protein structure prediction: results of the 1995 IRBM workshop.

Computational tools for protein structure prediction are of great interest to molecular, structural and theoretical biologists due to a rapidly increasing number of protein sequences with no known structure. In October 1995, a workshop was held at IRBM to predict as much as possible about a number of proteins of biological interest using ab initio prediction of fold recognition methods. 112 protein sequences were collected via an open invitation for target submissions. 17 were selected for prediction during the workshop and for 11 of these a prediction of some reliability could be made. We believe that this was a worthwhile experiment showing that the use of a range of independent prediction methods and thorough use of existing databases can lead to credible and useful ab initio structure predictions.

Animals↗
PubMed1996

Redesigning the substrate specificity of the hepatitis C virus NS3 protease.

BACKGROUND: Hepatitis C Virus (HCV) non-structural protein 3 (NS3) encodes a trypsin-like serine protease that catalyzes the cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions in the viral polyprotein and that shows a preference for a cysteine as the P1 residue. RESULTS: We describe here a partial model of the HCV NS3 protease which allowed us to predict the position of the secondary structure elements of the enzyme and of the residues involved in its specificity. By replacing these with the corresponding residues of Streptomyces griseus protease B1 we obtained a protease that, similar to the bacterial protein and unlike the wild-type enzyme, is able to cleave a substrate containing a phenylalanine in the P1 position. CONCLUSIONS: These results confirm the reliability of our model and represent one of the few examples of redesign of a serine protease substrate specificity directed by molecular modelling.

Amino Acid Sequence↗
PubMed1995

Modelling antibody-antigen interactions: ferritin as a case study.

In this work, we propose a model for the structure of the antigen-antibody complex formed by human H-ferritin and an antibody that specifically recognizes it. We cloned and sequenced the antibody gene, predicted the antibody three-dimensional structure, and reconstructed the H-ferritin-antibody complex using an automated docking procedure previously validated on known complexes. This procedure allowed us to identify one putative complex which we carefully analysed, in order both to evaluate its likelihood, in light of a set of experimental results described in the literature, and to predict precisely which are the sites of interaction between the two molecules. Our model is compatible with the experimentally determined characteristics of the complex. Some of the residues that form the predicted antigenic site of ferritin can be found in the amino acid sequence of peptides selected from a random peptide library because of their affinity for the ferritin monoclonal antibody. Furthermore, the structural difference between the antigenic site in human H-ferritin and the corresponding region in other species permits us to rationalize the inability of the antibody to recognize human L-ferritin and rat, chicken and mouse H-ferritin. Through the analysis of our model complex, we identify a number of other residues putatively involved in the interaction. This multidisciplinary approach shows that synergy between computational and experimental methods may bring further insight into the understanding of antibody-antigen recognition rules.

Amino Acid Sequence↗
PubMed1995

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Amino Acid Sequence↗
PubMed1995

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability.

The alcohol dehydrogenase gene from the thermophilic archaeum Sulfolobus solfataricus has been subcloned and expressed in Escherichia coli under the control of the T7 inducible promoter. The recombinant protein shows properties analogous to those of the native enzyme, including thermostability, despite the fact that E.coli does not post-translationally modify two lysine residues which are N-epsilon-methylated in the native enzyme. We constructed a 3-D model of the S.solfataricus alcohol dehydrogenase using the known structure of its isozyme from horse liver as a template. Our analysis of the structural zinc binding site suggested that this site is present and functional in the S.solfataricus enzyme and that a glutamate ligand can contribute to thermostability by influencing electrostatic interactions around the metal centre. To investigate this hypothesis, we constructed, expressed and characterized a mutant where the glutamate is replaced by a cysteine, thus restoring the zinc binding site of mesophilic alcohol dehydrogenases. The mutant shows the same activity but a reduced thermostability with respect to the wild-type recombinant protein, as suggested by our model.

Alcohol Dehydrogenase↗
PubMed1995

Identifying a putative common binding site shared by substance P receptor and an anti-substance P monoclonal antibody.

Substance P G-protein coupled receptor and the antigen recognition site of a monoclonal antibody raised against substance P share a stretch of five contiguous identical amino acids. This observation prompted us to build an atomic model of both the receptor and the antibody and to analyse their common features. In particular, we report here that a pocket of similar size and composition is present in both proteins, strongly suggesting a similarity in the mode of binding of both macromolecules to substance P. From the analysis of our models, the available data on the mode of binding of the antibody to substance P and recent data on substance P receptor mutants, we concluded that the pocket is very likely to be involved in binding of the C-terminal 'message sequence' of the tachykinin. This allowed us to suggest specific site-directed mutants of the receptor which should shed some light on the mechanism of peptide recognition by G-protein coupled receptors.

Amino Acid Sequence↗
PubMed1995

PLANET: a phage library analysis expert tool.

In recent years random peptide libraries displayed on filamentous phage have been widely used and new ideas and techniques are continuously developing in the field (1-5). Notwithstanding this growing interest in the technique and in its promising results, and the enormous increase in usage and scope, very little effort has been devoted to the implementation of software able to handle and analyze the growing number of phage library-derived sequences. In our laboratory, phage libraries are extensively used and peptide sequences are continuously produced, so that the need arose of creating a database (6) to collect all the experimental results in a format compatible with GCG sequence analysis packages (7). We present here the description of an XWindow-based software package named PLANET (Phage Library ANalysis Expert Tool) devoted to the maintenance and statistical analysis of the database.

Bacteriophages↗