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P Neri

Publications and source records attributed to P Neri.

At least 37 records · Page 2Linked to original sources
PubMed1999

A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage.

We have aimed at developing a general methodology for the isolation of enzymatic activities from large repertoires of protein displayed on the surface of a filamentous phage. When selecting for protein binders by phage display, phage particles with suitable specificities are physically isolated by affinity capture and amplified by bacterial infection. Selection for catalysis mediated by enzymes displayed on filamentous phage is more difficult, as reaction products (which represent the biochemical memory of the reaction catalysed by the phage particle) diffuse away after the reaction is complete. We reasoned that if we were able to anchor the reaction products on the phage surface, the catalytically active phages could then be physically isolated using specific anti-product affinity reagents. We achieve the conditional anchoring of reaction substrates and products on phage by displaying enzyme-calmodulin chimeric proteins on filamentous phage as gene III fusions. Such phage particles can be targeted in a stable fashion (koff<10(-4) s(-1)) by chemical derivatives of a calmodulin-binding peptide. The peptide-phage complexes are stable in purification procedures such as capture with magnetic beads and polyethylene glycol precipitation, and can be conditionally dissociated by addition of calcium chelators. Glutathione-S-transferase and an endopeptidase were used in model selection experiments to demonstrate that it is possible to isolate catalytic activities from calmodulin-tagged enzymes displayed on filamentous phage, with enrichment factors >50 per round of selection.

Amino Acid Sequence↗
PubMed1999

Cerebellar contribution to spatial event processing: characterization of procedural learning.

Recently, we demonstrated the prevalent role of cerebellar networks in the acquisition of the procedural components of spatial information by testing hemicerebellectomized (HCbed) rats in a classical spatial task, the Morris water maze (MWM). As procedures used in the water maze are a mixture of different components (that is, general procedures, exploration procedures, direct reaching procedures), for optimally solving a spatial task all procedural components must be opportunely managed. Thus, severely impaired procedural learning of cerebellar origin can be better comprehended by fractionating the procedural facets. To this aim, a two-step water-maze paradigm was employed. Normal rats were first trained to search for a hidden platform moved to a different position in each trial, utilizing a water maze setting in which visual cues were abolished by heavy black curtains surrounding the tank. In this paradigm, normal animals solved the task by using general and exploration procedures, but they could not use direct reaching skills. A subgroup of these pretrained animals was then HCbed and, after recovery from cerebellar lesion, was tested in a water maze with normal environmental cues available, a paradigm in which normal animals develop abilities for reaching the target with very direct trajectories. Pretrained HCbed animals, however, did not display the typical spatial deficits of naive HCbed rats, persisted in exhibiting the scanning strategy learned during pretraining, and never displayed direct reaching skills. In conclusion, cerebellar networks appear to be involved in the acquisition of all procedural facets necessary for shifting behavior within the maze until direct reaching of the platform. The lack of flexibility in changing exploration strategies displayed by pretrained HCbed rats is interpreted by taking into account the well-known cerebellar frontal interplay sculpting a specific cerebellar role in the acquisition of spatial procedural steps.

Animals↗
PubMed1999

On the structural stability of a small bioactive peptide of potential use in biotechnology.

A tridecapeptide with the sequence CCEICCNPACFGC has been synthesized to reproduce the active moiety of a heat stable enterotoxin from Vibrio cholerae. The proton NMR analysis indicates, for the active synthetic fragment, a rigid secondary structure stabilised by three disulfide bridges. Such a rigid peptide, suitably detoxified and activated, could be a good candidate to be used as a carrier for linear bioactive peptides or other functional groups.

Biotechnology↗
PubMed1998

Seeing biological motion.

One of the more stunning examples of the resourcefulness of human vision is the ability to see 'biological motion', which was first shown with an adaptation of earlier cinematic work: illumination of only the joints of a walking person is enough to convey a vivid, compelling impression of human animation, although the percept collapses to a jumble of meaningless lights when the walker stands still. The information is sufficient to discriminate the sex and other details of the walker, and can be interpreted by young infants. Here we measure the ability of the visual system to integrate this type of motion information over space and time, and compare this capacity with that for viewing simple translational motion. Sensitivity to biological motion increases rapidly with the number of illuminated joints, far more rapidly than for simple motion. Furthermore, this information is summed over extended temporal intervals of up to 3 seconds (eight times longer than for simple motion). The steepness of the summation curves indicates that the mechanisms that analyse biological motion do not integrate linearly over space and time with constant efficiency, as may occur for other forms of complex motion, but instead adapt to the nature of the stimulus.

Humans↗
PubMed1998

Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel.

We report the construction and the use of a phage display human antibody library (>3 x 10(8) clones) based on principles of protein design. A large repertoire of functional antibodies with similar properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto the two antibody germ line segments most frequently found in human antibodies. With this strategy we concentrated sequence diversity in regions of the antibody structure that are centrally located in the antigen binding site, while leaving residues in more peripheral positions available for further mutagenesis aimed at improving the affinity of the selected antibodies. In addition, the library was tested by selecting antibodies against six biologically relevant antigens. Using only 0.3 microg of antigen eluted from a two-dimensional gel spot, we isolated binders specific for the ED-B domain of fibronectin, a marker of angiogenesis. These antibodies recognize the native antigen with affinities in the 10(7)-10(8) M-1 range, and perform well in immunosorbent assays, in two-dimensional Western blotting and in immunohistochemistry. The affinity of one anti-ED-B antibody was improved by 27-fold by combinatorially mutating six strategically selected residues in the heavy chain variable domain. A further 28-fold affinity improvement could be achieved by mutating residues 32 and 50 of the light chain. The resulting antibody, L19, bound to the ED-B domain of fibronectin with very high affinity (Kd = 54 pM), as determined by real-time interaction analysis with surface plasmon resonance detection, band shift analysis, and by competition experiments with electrochemiluminescent detection.

Amino Acid Sequence↗
PubMed1998

Identification of critical residues of staphylococcal enterotoxin B for lymphomonocyte proliferation and cytokine production.

Superantigens bind to major histocompatibility complex class II molecules and stimulate large numbers of T cells expressing particular Vbeta elements of the T-cell receptor. Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that causes food poisoning and toxic-shock syndrome. The toxicity of SEB is thought to be mediated by T-cell stimulation and cytokine production. Different regions of the SEB molecule are important for mitogenic activity. To identify critical residues of SEB in the region 124-1 54, which competitively inhibits the mitogenic activity of the toxin, we used the synthetic peptide approach and alanine scanning mutagenesis as a probe. We synthesized eight peptides with alanine replacement of all residues in the SEB sequence 131-138 and tested them for the capacity to inhibit both SEB-induced proliferation of human lymphomonocytes and the production of tumor necrosis factor alpha and interferon gamma. Mutation to alanine of the residue Thr 133 improved the inhibition of SEB-induced proliferation and cytokine production, whereas the substitution of Ser 131 also increased the inhibition, albeit to a lesser degree. The peptide obtained by substitution of Val 136 with alanine was unable to inhibit SEB-induced proliferation and cytokine production, suggesting that Val 136 is essential for mitogenic activity. Thus hydrophobic interactions apparently are very important for mitogenic activity. The identification of critical residues in this active site in the SEB and the computer modeling based on crystal X-ray data contribute to a better understanding of the molecular mechanism of the superantigen and may be useful for therapeutical applications.

Alanine↗
PubMed1997

Molecular mimicry between the rabies virus glycoprotein and human immunodeficiency virus-1 GP120: cross-reacting antibodies induced by rabies vaccination.

The 160-170 sequence of human immunodeficiency virus (HIV)-1 gp120 mimics a nicotinic receptor-binding motif of rabies virus glycoprotein and snake neurotoxins. This sequence has been proposed to be involved in the binding of HIV-1 gp120 to the acetylcholine binding sites of nicotinic receptors. By using biomolecular interaction analysis (BIA) technology we have found that HIV-1 gp120 can bind to detergent-extracted nicotinic receptor from fetal calf muscle. The binding is inhibited by nicotine and by a synthetic peptide reproducing the gp120 160-170 sequence. The molecular mimicry between gp120 and rabies virus glycoprotein is confirmed by cross-reacting antibodies. We have found that vaccination against rabies can induce the production of anti-HIV-1 gp120 antibodies in humans. The cross-reacting antibodies are directed to the gp120 sequence involved in the mimicry with the rabies virus glycoprotein. The cross-reactivity between the rabies virus and HIV-1 has important implications in transfusion medicine. Moreover, the presence of cross-reacting antibodies between the nicotinic receptor binding site of rabies virus glycoprotein and a fragment of HIV-1 gp120 strengthens the hypothesis about the possible role of nicotinic receptors as potential receptors for HIV-1 in the central nervous system.

Antibodies, Viral↗
PubMed1997

Hierarchical affinity maturation of a phage library derived antibody for the selective removal of cytomegalovirus from plasma.

Recombinant antibody fragments can be produced in large quantities using bacterial expression systems and could potentially be useful for the generation of biofilters for the selective removal of viral particles from fluids. A human single chain-Fv antibody library, derived from synthetic repertoires of germ line VH-gene segments rearranged in vitro and paired to a single light chain (Nissim et al., 1994, EMBO J., 13, 692-698), has recently been used to isolate hundreds of different binding specificities by panning with antigen. Antibodies from this library typically have affinities in the 10(6)-10(7) M-1 range. Occasionally, better binders are isolated but at other times the affinities recovered are poor. In the latter situation binding cannot be detected with soluble antibodies, but only by high-avidity display of multiple copies of antibodies on phage. By panning with human cytomegalovirus (HCMV)-coated immunotubes, we have isolated a number of antibody clones from this library that bound to the antigen only if displayed on the filamentous phage, but not in soluble form. One of these clones was selected for an affinity maturation procedure, achieved by combinatorial mutagenesis of the complementarity determining region 3 (CDR3) of the antibody light chain, followed by selection of the resulting library for HCMV binding. By this means, we were able to isolate a number of binders, some of which exhibited specific HCMV binding in soluble form. The clone that gave the strongest ELISA signal was expressed in bacteria, purified in solution, characterised using a novel capture methodology with surface plasmon resonance detection on a BIAcore instrument and used for the production of an immunofilter for the removal of HCMV form human serum. The filter removed more than 99% of applied HCMV in 10 min circulation time, while the amount of HCMV retained non-specifically in a cartridge derivatised with a non-specific antibody was less than 10% under similar conditions.

Antibodies, Viral↗
PubMed1997

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

Amino Acid Sequence↗
PubMed1996

Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement.

Calmodulin is a calcium-binding protein that regulates a wide range of enzymes. It is also one of the few examples of a small protein capable of binding to peptides with very high affinity, and is therefore an interesting candidate for biotechnological applications and a good model system for studying how proteins associate. We have synthesized a complete series of peptides derived from the recognition sequence of skeletal muscle myosin light-chain kinase, corresponding to single-point amino acid mutations to alanine. These peptides bind to calmodulin with a biphasic kinetic: a fast association step followed by a slow intramolecular isomerisation. We have measured the isomerisation rate (k(isom)) of these peptides for calmodulin by stopped-flow analysis, and their association and dissociation kinetic constants (k(on) and k(off)) by real-time interaction analysis using surface plasmon resonance detection. In addition, k(off) constants were measured by competition experiments using a high-sensitivity luminescence analyser and native polyacrylamide gels. We have observed that all the alanine-scanning peptides bound to calmodulin with better affinity than the wild-type. In one case, a Asn-->Ala substitution resulted in a 1000-fold improvement in affinity, owing to a slower off-rate. Our results indicate that naturally occurring calmodulin binders may have evolved to have high affinities, but far from the maximum. Our affinity data are in contrast with recently published predictions of interactions responsible for high-affinity calmodulin binding based on modelling and energy calculations.

Alanine↗
PubMed1996

Recombinant anti-human melanoma antibodies are versatile molecules.

The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

Amino Acid Sequence↗
PubMed1995

Calmodulin as a versatile tag for antibody fragments.

Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner. We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments. Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose). The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis. Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.

Amino Acid Sequence↗
PubMed1994

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.

Amino Acid Sequence↗
PubMed1993

Characterization of immunoreactive octapeptides of human-cytomegalovirus gp58.

We have mapped continuous epitopes, for positions 591-673 of the human cytomegalovirus 58-kDa glycoprotein using overlapping synthetic peptides and human sera. This region contains a fragment previously described as including the dominant site for induction of human-cytomegalovirus antibodies. Since the selected sequence is highly conserved among herpes viruses, we have considered the possible presence of antigenic cross-reactivity, particularly with the Epstein-Barr virus. Several peptides in the studied region were antigenic and two main continuous epitopes have been identified. Serological cross-reactions observed with Epstein-Barr virus are discussed, focusing on the possible implications of structural features and sequence similarity between human-cytomegalovirus and Epstein-Barr-virus glycoproteins.

Amino Acid Sequence↗
PubMed1993

A model of the rabies virus glycoprotein active site.

The glycoprotein from the neurotropic rabies virus shows a significant homology with the alpha neurotoxin that binds to the nicotinic acetylcholine receptor. The crystal structure of the alpha neurotoxins suggests that the Arg 37 guanidinium group and the Asp 31 side-chain carboxylate of the erabutoxin have stereochemical features resembling those of acetylcholine. Conformational studies on the Asn194-Ser195-Arg196-Gly197 tetrapeptide, an essential part of the binding site of the rabies virus glycoprotein, indicate that the side chains of Asn and Arg could also mimic the acetylcholine structure. This observation is consistent with the recently proposed mechanism of the viral infection.

Amino Acid Sequence↗
PubMed1992

Binding of HIV-1 gp120 to the nicotinic receptor.

We previously described a significant sequence homology between HIV-1 gp120 and the functional sites responsible for the specific binding of snake curare-mimetic neurotoxins and rabies virus glycoprotein to the nicotinic acetylcholine receptor. Here we report findings about the existence of a mechanism of functional molecular mimicry which could enable the binding of HIV-1 gp120 to nicotinic acetylcholine receptors in muscle cells and neurons.

Acetylcholine↗