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Biomedical subjects

A Tramontano

Publications and source records attributed to A Tramontano.

At least 19 recordsLinked to original sources
PubMed2002

A model for recognition of polychlorinated dibenzo-p-dioxins by the aryl hydrocarbon receptor.

Ligand binding by the aryl hydrocarbon receptor (AhR), a member of the bHLH-PAS family of transcriptional regulatory proteins, has been mapped to a region within the second 'PAS' domain, a conserved sequence motif first discovered in the Per-ARNT-Sim family of proteins. In addition to the bacterial photoactive yellow protein (PYP), which had been proposed as a structural prototype for the three dimensional fold of PAS domains, two crystal structures of the PAS domain have recently been determined: the human potassium channel HERG and the heme binding domain of the bacterial O(2) sensing FixL protein. The three structures reveal a highly conserved structural framework in evolutionary rather distant PAS domains, provide a more general view of how these domains can recognize their ligands and suggest a structure-function relationship that we exploited to build a three-dimensional model of the ligand binding domain (LBD) of the mouse aryl hydrocarbon receptor (mAhR). The model allowed us to putatively identify the residues responsible for the recognition of polychlorinated dibenzo-p-dioxins (PCDDs) by AhR receptors and to formulate an hypothesis on the signal transduction mechanism.

Amino Acid Sequence↗
PubMed2001

Phosphonate ester probes for proteolytic antibodies.

The reactivity of phosphonate ester probes with several available proteolytic antibody (Ab) fragments was characterized. Irreversible, active site-directed inhibition of the peptidase activity was evident. Stable phosphonate diester-Ab adducts were resolved by column chromatography and denaturing electrophoresis. Biotinylated phosphonate esters were applied for chemical capture of phage particles displaying Fv and light chain repertoires. Selected Ab fragments displayed enriched catalytic activity inhibitable by the selection reagent. Somewhat unexpectedly, a phosphonate monoester also formed stable adducts with the Abs. Improved catalytic activity of phage Abs selected by monoester binding was evident. Turnover values (kcat) for a selected Fv construct and a light chain against their preferred model peptide substrates were 0.5 and 0.2 min(-1), respectively, and the corresponding Michaelis-Menten constants (Km) were 10 and 8 microm. The covalent reactivity of Abs with phosphonate esters suggests their ability to recapitulate the catalytic mechanism utilized by classical serine proteases.

Animals↗
PubMed2001

Analysis and assessment of comparative modeling predictions in CASP4.

We describe here the results of our analysis of the comparative modeling predictions submitted to the fourth round of Critical Assessment of Structure Prediction (CASP4). On the basis of a numerical evaluation of the models, we assessed their ability to predict the overall fold correctly, the relative orientation of domains in multidomain proteins, the conformation of the side chains, the loop regions, and the biologically important residues of the targets. We also discuss the performance of automatic prediction servers and compare the results of CASP4 with those obtained in CASP3.

Automation↗
PubMed2001

Mimotopes of the hyper variable region 1 of the hepatitis C virus induce cross-reactive antibodies directed against discontinuous epitopes.

Hepatitis C virus (HCV) is a major cause worldwide of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and the development of an effective vaccine represents a high priority goal. The hyper variable region 1 (HVR1) of the second envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. To be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1, selected from a specialized phage library using HCV patients' sera. Some of the cross-reacting anti-mimotope antibodies elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

Amino Acid Sequence↗
PubMed2001

Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1.

A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building.

Amino Acid Sequence↗
PubMed2001

Induction of cross-reactive humoral immune response by immunization with mimotopes of the hypervariable region 1 of the hepatitis C virus.

Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, worldwide, and the development of an effective vaccine represents a high priority goal. The Hyper Variable Region 1 (HVR1) of the second Envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. Thus, to be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1. selected from a specialized phage library using HCV patients' sera. At least some of the cross-reacting anti-mimotope antibodies, elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

Amino Acid Sequence↗
PubMed2001

[Biliary atresia syndrome and splenic malformation. A case report].

Biliary atresia is a severe neonatal malformation in which the entire extrahepatic biliary tract or part of it is absent and replaced by fibrosclerotic tissue. Having been described for the first time by Thomson in 1891, biliary atresia has an incidence of 1:10,000-15,000 live neonates with a slight predominance in the female sex; it is associated with other malformations in 25% of cases. In 1993 Davenport et al. identified a subgroup of patients with biliary atresia who presented a splenic malformation. This condition was known as biliary atresia and splenic malformation syndrome (BASM). The cases of biliary atresia and BASM are still not clear and a number of etiopathogenetic hypotheses have been put forward. The treatment is surgical and includes a first stage of hepatic-porto-enteroanastomosis using a defunctionalised Roux loop (Kasal), followed by the definitive operation comprising liver transplant. We report the case of a boy who was brought to our attention with jaundice, polypnea and hepatosplenomegalia; instrumental tests allowed the diagnosis of biliary atresia, median liver, stomach and spleen on the right, polysplenia, atresia of the intrahepatic tract of the inferior vena cava, presence of a superior vena cava to the left that drained into the upper left portion of the common atrium. Moreover, the cardiac apex was present on the right, extensive DIA of the common atrium type, extensive DIV and right aortic arch.

Biliary Atresia↗
PubMed2000

Genome sequences and great expectations.

To assess how automatic function assignment will contribute to genome annotation in the next five years, we have performed an analysis of 31 available genome sequences. An emerging pattern is that function can be predicted for almost two-thirds of the 73,500 genes that were analyzed. Despite progress in computational biology, there will always be a great need for large-scale experimental determination of protein function.

Animals↗
PubMed2000

Enzyme mimicry by the antiidiotypic antibody approach.

The concept of "internal image" of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches.

Amino Acid Sequence↗
PubMed2000

A model for the hepatitis C virus envelope glycoprotein E2.

Several experimental studies on hepatitis C virus (HCV) have suggested the envelope glycoprotein E2 as a key antigen for an effective vaccine against the virus. Knowledge of its structure, therefore, would present a significant step forward in the fight against this disease. This paper reports the application of fold recognition methods in order to produce a model of the HCV E2 protein. Such investigation highlighted the envelope protein E of Tick Borne Encephalitis virus as a possible template for building a model of HCV E2. Mapping of experimental data onto the model allowed the prediction of a composite interaction site between E2 and its proposed cellular receptor CD81, as well as a heparin binding domain. In addition, experimental evidence is provided to show that CD81 recognition by E2 is isolate or strain specific and possibly mediated by the second hypervariable region (HVR2) of E2. Finally, the studies have also allowed a rough model for the quaternary structure of the envelope glycoproteins E1 and E2 complex to be proposed. Proteins 2000;40:355-366.

Amino Acid Sequence↗
PubMed2000

Monoclonal antibody light chain with prothrombinase activity.

Prothrombin is the precursor of thrombin, a central enzyme in coagulation. Autoantibodies to prothrombin are associated with thromboembolism, but the mechanisms by which the antibodies modulate the coagulation processes are not understood. We screened a panel of 34 monoclonal antibody light chains isolated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identified, and one of the light chains was characterized further. The prothrombinase activity eluted from a gel-filtration column run in denaturing solvent (6 M guanidine hydrochloride) at the characteristic positions of the light chain dimer and monomer. A constant level of catalytic activity was observed across the width of the light chain monomer peak, assessed as the cleavage of IEGR-methylcoumarinamide, a peptide substrate corresponding to residues 268-271 of prothrombin. Hydrolysis of this peptide by the light chain was saturable and consistent with Michaelis-Menten-Henri kinetics (K(m) 103 microM; k(cat) of 2.62 x 10(-)(2)/min). Four cleavage sites in prothrombin were identified by N-terminal sequencing of the fragments: Arg(155)-Ser(156), Arg(271)-Thr(272), Arg(284)-Thr(285), and Arg(393)-Ser(394). The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under conditions that readily permitted detectable prothrombin cleavage. Two prothrombin fragments (M(r) 55 000 and 38 000), were isolated by anion-exchange chromatography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroanilide. Conversion of fibrinogen to fibrin was accelerated by the prothrombin fragments generated by the light chain. These finding suggest a novel mechanism whereby antibodies can induce a procoagulant state, i.e., prothrombin activation via cleavage of the molecule.

Antibodies, Monoclonal↗
PubMed2000

Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics.

We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.

Amino Acid Sequence↗
PubMed2000

Analysis of a cDNA sequence encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii.

A spleen cDNA library was constructed from the Antarctic teleost Trematomus bernacchii and immunoscreened with rabbit IgG specific for T. bernacchii Ig heavy chain. Eleven cDNA clones, varying in size and encoding the entire heavy chain or parts of it, were isolated. Here the complete nucleotide and deduced amino acid sequences of clone 2C2 encoding the secretory IgH chain form are reported. Comparison of the amino acid sequence of the entire constant region of the T. bernacchii Ig heavy chain with those from other teleosts and two holostean fish showed percent identity ranging 53.6-60.6%, with the highest values found for Salmoniformes. The multiple sequence alignment revealed the presence of two remarkable insertions: one at the VH-CH1 boundary and a second one, not found in any other IgM heavy chain, localised at the CH2-CH3 boundary. The latter occurred in the region proposed to act as a 'hinge', and resulted in a CH2-CH3 hinge peptide longer than any other IgM hinge. Differences were also found in the number and position of putative N-glycosylation sites of the compared sequences. It is suggested that the unusual features found in the T. bernacchii Ig heavy chain might contribute to the flexibility of the Ig molecule and help understand more about the adaptation of Ig molecules to the polar sea environment.

Amino Acid Sequence↗
PubMed2000

Antibody modeling: implications for engineering and design.

Our understanding of the rules relating sequence to structure in antibodies has led to the development of accurate knowledge-based procedures for antibody modeling. Information gained from the analysis of antibody structures has been successfully exploited to engineer antibody-like molecules endowed with prescribed properties, such as increased stability or different specificity, many of which have a broad spectrum of applications both in therapy and in research. Here we describe a knowledge-based procedure for the prediction of the antibody-variable domains, based on the canonical structures method for the antigen-binding site, and discuss its expected accuracy and limitations. The rational design of antibody-based molecules is illustrated using as an example one of the most widely employed modifications of antibody structures: the humanization of animal-derived antibodies to reduce their immunogenicity for serotherapy in humans.

Antibodies↗
PubMed2000

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries.

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

Antibodies↗
PubMed1999

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses.

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.

Animals↗