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Biomedical subjects

F Bonelli

Publications and source records attributed to F Bonelli.

At least 19 recordsLinked to original sources
PubMed2001

New coupled-particle light-scattering assay for detection of Ro/SSA (52 and 60 kilodaltons) and La/SSB autoantibodies in connective tissue diseases.

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sjögren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.

Antibodies, Antinuclear↗
PubMed2001

Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein.

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Amino Acid Sequence↗
PubMed2000

Reactivity to p52 and CM2 recombinant proteins in primary human cytomegalovirus infection with a microparticle agglutination assay.

We evaluated the reactivities of sera against p52 and CM2 recombinant antigens of human cytomegalovirus (HCMV), coated on microparticles, for the differentiation of primary HCMV infection from an established infection. Two different test formats of the CMV Multiplex Copalis assay were evaluated. The 214 serum samples tested were immunoglobulin M (IgM) positive or equivocal by our reference assay. Reactivities against p52 and CM2 antigens were tested for sera from 37 patients with a well-documented seroconversion within the preceding 3 months (119 serum specimens), 31 patients known to have had a seroconversion at least 8 months earlier (31 serum specimens), and 57 patients without a documented seroconversion (64 serum specimens). The assay had a sensitivity for the detection of a primary infection of 70 or 86% by the first test format and a sensitivity of 88 or 94% by the second test format, according to the criteria used to indicate a primary infection by this test. A good correlation of the results of the assay with our in-house avidity index was found. The specificity of the assay warrants further evaluation. With IgM-positive sera, the assay was not sufficiently specific to make a distinction between a primary infection and an established infection.

Agglutination Tests↗
PubMed1998

Large-scale production of peptides using the solid phase continuous flow method. Part 2: preparative synthesis of a 26-Mer peptide thrombin inhibitor.

A preparative method for the preparation of large peptides is described. An advantageous theoretical weight of peptide/weight of starting resin ratio (tPw/Rw) of about 0.3 was successfully experimented. The esterification of the first amino acid was realized with a racemization of less than 1%. The study of the coupling conditions led to the use of a diluted acylating mixture that allowed a 56% consumption of the amino acid derivatives (percentage use of amino acids) introduced in the synthesis. The cost analysis of the synthesis showed that the recovery of the amino acid derivatives was not worthwhile.

Amino Acid Sequence↗
PubMed1995

Synthesis and biological evaluation of novel NK-1 tachykinin receptor antagonists: the use of cycloalkyl amino acids as a template.

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the superimposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 A distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the pi stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed.

Amino Acids↗
PubMed1995

Structural characterization of a new class of penem beta-lactam antibiotics by triple quadrupole tandem mass spectrometry.

A series of new penem beta-lactam antibiotics, having at position 2 of the thiazoline ring an amido-substituted amino acid moiety spaced by a methylene group, was analyzed by collisionally activated dissociation (CAD) tandem mass spectrometry on a triple-quadrupole mass spectrometer, after ionization by cesium ion bombardment. This approach allowed complete structural characterization without any matrix interferences. All the compounds investigated underwent beta-lactam ring cleavage through a typical retro-Diels-Alder reaction, as well as cleavage of the 2-side chain at two different points, with charge retention on both sides of the molecule. Subsequent fragmentation reactions essentially involved combinations of the above processes.

Anti-Bacterial Agents↗
PubMed1995

Epitope mapping of the NS4 and NS5 gene products of hepatitis C virus and the use of a chimeric NS4-NS5 synthetic peptide for serodiagnosis.

Specific domains of the NS4 and NS5 gene products of hepatitis C virus have been identified using hydrophilicity profiles for the prediction of potential immunogenic regions, and epitope scanning techniques. Peptides synthesised on the basis of such data show excellent reactivity in the ELISA format. Introduction of a glycine-glycine spacer between two peptides (NS4-12 and NS5-44) to give a single chimeric peptides does not appear to impair immunoreactivity. An ELISA based on the chimeric peptide and a Core-NS3 recombinant protein correctly diagnoses a cohort of haemodialysed patients, three commercial HCV panels and the sera of a negative control population.

Amino Acid Sequence↗
PubMed1994

Identification of human urinary metabolites of isbufylline by high-performance liquid chromatography/thermospray mass spectrometry.

Analysis of urinary metabolites of isbufylline (1,3-dimethyl-7-(2-methylpropyl)xanthine) in healthy male volunteers after oral administration of a single dose of 320 mg was undertaken by high-performance liquid chromatography/mass spectrometry (HPLC/MS with thermospray ionization. Filtered urines were directed injected into the HPLC/MS system, equipped with a Lichrospher 100 RP 18 analytical column. The mobile phase was 0.1 M, pH 3.7 ammonium acetate in water and acetonitrile; the composition was varied linearly from 5% to 40% of the organic modifier in 40 min with a flow rate of 1 ml min-1. Three more chromatographic peaks appeared in urines from treated subjects as compared to untreated ones; their probable quasi-molecular ions were at m/z 253, 239 and 267 respectively, while the original drug, of 236 Da, was not present in appreciable quantity. The collisionally activated daughter ion spectra of the above ions allowed identification of 1,3-dimethyl-7-(3-hydroxy-2-methylpropyl)xanthine (D3OHMPX), 1-methyl-7-(2-hydroxy-2-methylpropyl)xanthine (M2OHMPX), and 1,3-dimethyl-(2-carboxypropyl)xanthine (D2CMPX), the first one being present as glucuronic acid conjugate.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Expression in E. coli and purification of a chimeric p22-NS3 recombinant antigen of hepatitis C virus (HCV).

A recombinant antigen (p22-NS3), possessing putative HCV nucleocapsid protein (p22) and non-structural protein 3 (NS3) epitopes, was heavily expressed in E. coli and purified. The p22-NS3 purified recombinant antigen strongly reacts with sera containing human antibodies directed against p22 and NS3 providing a starting point for the design of an HCV single all-encompassing antigen for a blood screening assay.

Antigens, Viral↗
PubMed1993

Purification of recombinant hepatitis delta antigen expressed in E. coli cells.

Recombinant DNA technology enables the massive production of recombinant hepatitis delta antigen (recHDAg) retaining immunological properties and transport functions. However, purification procedures of the recombinant delta antigen have, to date, not been described in the literature. We present a purification procedure allowing one to obtain highly purified recHDAg from bacterial cells expressing the hepatitis delta antigen.

Antigens, Viral↗
PubMed1993

Properties of a human monoclonal antibody specific for the NS4 region of hepatitis C virus.

A human monoclonal IgG1(kappa) antibody (hmAb) specific for a sequential epitope comprised within the 5-1-1 fragment of the NS4 region of hepatitis C virus (HCV) has been recently generated (1). In this study, B-cell clone supernatant containing the hmAb was purified by passage over a protein A affinity column. Preincubation with synthetic oligopeptides containing the epitope recognized by the hmAb resulted in complete inhibition of binding to the whole recombinant protein, attesting to its specificity. Calculation of the dissociation constant (Kd) using a synthetic 20-mer as antigen gave a value of 3.3372 x 10(-8) M, consistent with that of a human IgG. We predict that this hmAb will be helpful in characterizing the as yet unidentified native NS4 protein of HCV.

Antibodies, Monoclonal↗
PubMed1993

Characterization of RNA-binding domains of hepatitis delta antigen.

Hepatitis delta antigen (HDAg), the only protein encoded by the hepatitis delta virus (HDV), binds specifically genomic and antigenomic strands of the HDV RNA. In a previous study, three recombinant HDAg subdomains were synthesized, covering residues 11 to 78, 79 to 163 and 164 to 212, and only the middle domain was shown to be responsible for the binding to HDV RNA. To investigate HDAg sequences involved in HDV RNA binding, we synthesized five peptides, 15 to 29 residues in length, and tested their ability to bind HDV RNA using a simple non-radioactive ELISA with digoxigenin-labelled HDV genomic or antigenomic RNA probes. The specificity of interactions was demonstrated by comparison with control peptides and non-HDV RNA probes, and with an inhibition assay using recombinant HDAg. The HDAg-binding domain found within the middle region (79 to 163) of HDAg was more finely mapped: it is located between residues 79 and 107. In addition, another domain (residues 2 to 27) of HDAg was also found to bind specifically to HDV RNA. These two peptides share sequence similarities at residues 2 to 10 and 97 to 107 with other RNA-binding domains.

Amino Acid Sequence↗
PubMed1991

Multiple antigen peptides (MAPs) as candidate vaccines against malaria.

Multiple Antigen Peptides (MAPs), branched molecules where multiple copies of a desired antigenic sequence are assembled on a small peptide core, have been recently described as an alternative approach to the synthesis of high molecular weight immunogens. In comparison with conventional peptide-carrier conjugates, the MAPs show several advantages, including chemical unambiguity and ease of synthesis. A MAP based on the sequence of the repetitive domain of P. malariae sporozoites was immunogenic in a large number of mouse strains. When covalently linked to the corresponding sequence of the P. falciparum circumsporozoite protein, [NANP]40, the resulting conjugate showed the properties of a multivalent vaccine, overcoming the severe genetic restriction of the [NANP] sequence. A second generation of MAPs including both sequences, with more desirable chemical properties, was equally effective. These compounds represent a promising step towards the development of synthetic, multivalent peptide vaccines against human malaria.

Amino Acid Sequence↗
PubMed1990

A multiple antigen peptide from the repetitive sequence of the Plasmodium malariae circumsporozoite protein induces a specific antibody response in mice of various H-2 haplotypes.

The major repetitive epitopes of the surface circumsporozoite (CS) protein of malaria sporozoites represent candidates for the development of subunit vaccines against malaria. However, previous experimental work has shown that repetitive peptides from the CS proteins of Plasmodium falciparum, P. vivax, P. yoelii and P. berghei are immunogenic only in mice with the H-2b or H-2k haplotype. This led to the conclusion that strong T helper epitopes from the non-repetitive CS sequences were required in the design of sporozoite vaccines. In the present study, we investigated the immunogenicity in mice of a octa-branched multiple antigen peptide (MAP) containing repeats of the CS protein of the human malaria parasite, P. malariae, [MAP8(NAAG)6], and found that mice with an H-2b, H-2d, H-2k, H-2f, H-2q, and H-2s haplotype produced anti-peptide antibodies after immunization and that only H-2r mice were nonresponsive. This antibody response, not induced in athymic H-2b nu/nu mice, was directed against the (NAAG) sequence, but not against the lysine core of the MAP construct. Finally, when covalently linked to a synthetic polymer of the repetitive (NANP) sequence of the P. falciparum CS protein, [MAP8(NAAG)6] behaved as a carrier molecule for the production of anti-(NANP)n antibodies in H-2d and H-2k mice, genetically nonresponder to the (NANP)n sequence. Should this wide immunogenicity of the P. malariae CS (NAAG) repetitive sequence also apply to humans, it might be considered for the design of multivalent subunit malaria vaccines.

Adjuvants, Immunologic↗
PubMed1990

Major antigenic domain recognized by monoclonal antibodies maps within the carboxy-terminal moiety of a recombinant human immunodeficiency virus-1 p24 protein.

Antigenicity in mice of a recombinant polypeptide including the complete amino acid sequence of mature human immunodeficiency virus type 1 p24 protein was studied by induction of monoclonal antibodies (MAbs). A panel of nine recloned hybridomas secreting MAbs with anti-p24 reactivity was isolated and further characterized. Competitive inhibition experiments suggested that the MAbs could be grouped into four epitopic classes corresponding to at least two distinct determinants. Analysis of reactivity to recombinant p24 deletion variants indicated that all the recognized epitopes are localized within a carboxy-terminal domain (amino acids 168-208) which should be largely exposed in recombinant as well as authentic antigen. Lack of response to N-terminal and central portions of p24 suggests that the antigenicity of those regions in the natural polypeptide is strongly conformation-dependent.

Antibodies, Monoclonal↗