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M Zanetti

Publications and source records attributed to M Zanetti.

At least 19 recordsLinked to original sources
PubMed1992

cDNA sequence analysis of an antibiotic dodecapeptide from neutrophils.

The full-length cDNA of a neutrophil antibiotic dodecapeptide has been cloned by reverse transcription/PCR from bovine bone marrow RNA. This peptide was originally isolated from bovine neutrophils, and shown to exert a potent antimicrobial activity in vitro on both Escherichia coli and Staphylococcus aureus. The cDNA codes for a polypeptide of 155 amino acid residues with a predicted mass of 17,629 Da and a pI of 8.03. The deduced sequence comprises a putative signal peptide of 29 amino acids, a 114 residue pro-region, and a carboxy-terminal dodecapeptide corresponding to the mature antibiotic. The pro-sequence displays extensive identity to corresponding regions of other structurally unrelated antibiotic peptides of bovine neutrophils recently cloned.

Amino Acid Sequence↗
PubMed1992

cDNA cloning of the neutrophil bactericidal peptide indolicidin.

A structurally novel, tryptophan-rich antimicrobial tridecapeptide amide, named indolicidin, has recently been purified from bovine neutrophils (Selsted et al. (1992) J. Biol. Chem. 267, 4292-4295). Here we describe the molecular cloning of this endoantibiotic, which is synthesised in bone marrow cells as a 144 amino acid residue precursor. The encoded protein has a predicted mass of 16479 Da and a pI of 6.51. A putative signal peptide of 29 amino acids precedes a 101 residue pro-region. The mature peptide is at the 3' end of the open reading frame. A glycine, not found in purified indolicidin, is present at the carboxyl terminus of the deduced sequence and is very likely involved in post-translational peptide amidation.

Amino Acid Sequence↗
PubMed1992

Synthetic peptides of human CD4 enhance binding of Ig to monocyte/macrophage cells. I. Characterization and mapping studies.

Human T cell glycoprotein CD4 binds to class II MHC molecules and to HIV envelope protein gp120. We have shown that CD4 and synthetic peptides corresponding to amino acid residues 21-49 of the first extracellular domain of CD4, also bind Ig and, with greater avidity, antibody:Ag complex. We investigated the effect of CD4 synthetic peptides on the binding and uptake of human Ig by monocyte/macrophage U937 cells. We found that a synthetic peptide corresponding to amino acid residues 21-49 enhanced binding to U937 cells of both aggregated and nonaggregated Ig. The enhancement was concentration dependent, occurred both in normal and low ionic strength conditions, and varied with the time and the temperature of the preincubation step. The enhancement was maximal after preincubation for 3 h at 37 degrees C. A peptide concentration of 20 micrograms/ml was sufficient for optimal binding of both nonaggregated and aggregated Ig. CD4 peptide 21-49 also enhanced binding of Ig to Staphylococcus aureus protein A. These studies open a new perspective in the way monocyte/macrophage cells handle Ig, antibody:Ag or Id:anti-Id complex, in particular when present at threshold amounts in a nonprecipitating form.

Amino Acid Sequence↗
PubMed1992

Antigenized antibodies.

A new process, antigenization of antibodies, consisting of the expression of oligopeptides in the hypervariable loops of an antibody molecule is described. The potential applications of antigenized antibodies are discussed.

Animals↗
PubMed1992

A method to analyze the interaction between gp120 of human immunodeficiency virus and CD4.

The study presents a new in vitro method to investigate the interaction between the glycoprotein (gp)120 of human immunodeficiency virus (HIV) and its receptor, CD4. The method is based on the binding of soluble recombinant CD4 to a human T cell line, 8E5, which constitutively expresses gp120 at its surface as a result of infection with HIV (LAV) and lacks reverse transcriptase activity. The binding of CD4 to gp120 on the cell surface is revealed by immunofluorescence using a murine monoclonal antibody to CD4. Binding can be inhibited by different substances like dextran sulfate, heparin, pentosan polysulfate, but not Leu3a. The reasons for this discrepancy are discussed. We propose this assay as a simple, reproducible, and rapid new method to screen new, pharmacological inhibitors of the gp120/CD4 interaction.

CD4 Antigens↗
PubMed1992

Ontogeny of the immune system and the invisible frontier to immune regulation.

The data presented focus on three topics: 1) Self-reactivity of early B cells as a constitutive feature of the immune system; 2) Self-reactive V regions and their possible involvement in immune regulation; and 3) Autoantibodies directed at T cell surface molecules as a new form of direct regulation of the B cell repertoire on the T cell compartment. Evidence is provided for lack of substantial difference in the reactivity of neonatal hybridomas from normal and autoimmune mice, and the proposal is made that the immune systems of normal and autoimmune neonatal mice start with similar characteristics implying that avoidance of autoimmune disease is matter of active regulation through a process learned in ontogeny. Two general possibilities for immune regulation are discussed. One is based on the V regions of self-reactive antibodies and their antigenic determinants. The other is through natural autoantibodies able to interfere with the state of activation of T cells. It is concluded that the role of highly conserved structures like self antigens is to maintain immunoglobulin genes and favor their expression in the incipient immune system so that simple patterns of regulation can be set in motion and made available.

Animals↗
PubMed1991

Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides.

The antimicrobial polypeptides Bac7 and Bac5 (bactenecins) are stored in the large granules of bovine neutrophils as precursor forms, or probactenecins. Maturation of probactenecins has been investigated by studying the effects of stimulus-induced degranulation on this process. Stimulation of neutrophils with PMA, which is a secretagogue for specific and large granules but not for azurophils, induces a substantial discharge of uncleaved probactenecins in the extracellular medium, as revealed by Western blot analysis. When neutrophils are exposed to opsonized bacteria in the presence of cytochalasin B, resulting in exocytosis of the content of azurophils in addition to that of specific and large granules, probactenecins are secreted and rapidly converted into the corresponding mature antimicrobial peptides. Such a conversion is prevented if serine proteases, stored in the azurophils, are inhibited by pretreatment of neutrophils with PMSF. Phagocytosis, while causing a rapid discharge of the contents of azurophil and of the large granules into phagocytic vacuoles, as indicated by immunogold electron microscopy, also induces cleavage of probactenecins into mature peptides, as revealed by Western blot analysis. We conclude that the final processing of the storage forms of bactenecins arises from their interaction with the serine protease(s) of azurophils during bacteria-induced degranulation of neutrophils.

Animals↗
PubMed1991

Immunogenicity of an engineered internal image antibody.

We engineered an antibody expressing in the third complementarity-determining region of its heavy chain variable region a "foreign" epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP) of the circumsporozoite protein of Plasmodium falciparum parasite, one of the etiologic agents of malaria in humans. A monoclonal antibody to P. falciparum specific for the (NANP)n amino acid sequence bound to the engineered antibody, and a synthetic (NANP)3 peptide blocked this interaction. Immunization of rabbits and mice with the engineered antibody resulted in the elicitation of a humoral response to (NANP)3 synthetic peptide and P. falciparum parasite. In mice, in which immunity to the (NANP)n epitope is highly restricted by immune response genes, antibodies were induced in responder and nonresponder haplotypes of the major histocompatibility complex. Rabbit antibodies efficiently inhibited the in vitro invasion of cultured liver cells by P. falciparum parasite. Collectively, this study indicates that immunity to malaria in the absence of the parasite can be induced using antibody variable regions engineered to mimic the parasite's molecular structure. In general terms, the results suggest that antibody (idiotype) mimicry of an exogenous antigen is possible and may only require a discrete stretch of identity between the two molecules. The implication for the preparation of antibody-based vaccines and idiotype regulation of immunity are discussed.

Amino Acid Sequence↗
PubMed1991

CD4/immunoglobulin interaction: implications for immune physiology and autoimmunity.

CD4 has an important role in T cell activation events that depend on its binding to non-polymorphic MHC class II determinants on antigen-presenting cells. Here, we provide evidence that CD4 also interacts with immunoglobulins (Ig). The Ig-binding region lies within residues 21-49 of V1 domain of CD4. Immunochemical studies suggest that this property of CD4 does not depend on the three-dimensional folding of the CD4 molecule. Synthetic peptides (p) encompassing amino acid residues 16-49 and 21-49 of CD4 bind immunoglobulins in comparable way to the intact molecule. In vitro p 16-49 enhances significantly idiotype/anti-idiotype and some weak antigen-antibody interactions. Antigen antibody complexes formed in antigen excess bind CD4 peptides with much higher avidity then non-complexed antibodies. The possible role of the CD4/Ig interaction in T-B cell cooperation is discussed.

Animals↗
PubMed1991

Idiotypic analysis of human anti-topoisomerase I autoantibodies.

Anti-topoisomerase I autoantibodies (anti-topo I) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's phenomenon. Polyclonal anti-idiotypic sera were raised against affinity-purified anti-topo I from 2 patients with scleroderma (EM, SG) and 1 healthy individual (NM). All 3 anti-topo I preparations expressed immunodominant private Ids in or near the antigen binding site of the autoantibody. Further analysis of Id-EM showed isotypic restriction to IgG and a stable Id-expression over the course of 9 years. Id-SG and Id-NM were expressed on IgG and on IgA. The idiotypic character of anti-topo I closely resembles that of anti-centromere autoantibodies which are associated with the CREST syndrome of scleroderma. The data suggest an antigen-driven process in the origin of autoantibodies in scleroderma.

Antigens↗
PubMed1991

Idiotypic analysis of human anticentromere autoantibodies.

The idiotypes (Ids) of anticentromere antibodies (ACA) have been studied using a fusion protein obtained from cloned cDNA of the major centromere antigen, CENP-B, for isolation of the autoantibodies. IgG-ACA were affinity purified from 4 patient sera and anti-Ids prepared in rabbits. Analysis revealed the existence of two distinct types of immunodominant Ids. One Id is near the antibody combining site and one is framework associated. A longterm longitudinal study of Id expression in a patient who seroconverted from ACA (-) to ACA (+) when she developed Raynaud's phenomenon showed a close correlation between Id expression and ACA titers (r = 0.94). These results may be interpreted as evidence for an autoantigen driven process in the anticentromere immune response.

Adult↗
PubMed1990

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography.

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.

Anisoles↗
PubMed1990

Human CD4 binds immunoglobulins.

T cell glycoprotein CD4 binds to class II major histocompatibility molecules and to the human immunodeficiency virus (HIV) envelope protein gp120. Recombinant CD4 (rCD4) bound to polyclonal immunoglobulin (Ig) and 39 of 50 (78%) human myeloma proteins. This binding depended on the Fab and not the Fc portion of Ig and was independent of the light chain. Soluble rCD4, HIV gp120, and sulfated dextrans inhibited the CD4-Ig interaction. With the use of a panel of synthetic peptides, the region critical for binding to Ig was localized to amino acids 21 to 38 of the first extracellular domain of CD4. CD4-bound antibody (Ab) complexed with antigen approximately 100 times better than Ab alone. This activity may contribute to the Ab-mediated enhancement of cellular HIV interaction that appears to depend on a trimolecular complex of HIV, antibodies to gp120, and CD4.

Amino Acid Sequence↗
PubMed1990

Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules.

Bactenecins are highly cationic polypeptides of bovine neutrophil granules and exert in vitro a potent antimicrobial activity. We have previously purified two bactenecins, designated in an abbreviated form Bac7 and Bac5 from their approximate molecular masses of 7 and 5 kD (Gennaro, R., B. Skerlavaj, and D. Romeo. 1989. Infect. Immun. 57:3142-3146). Here we have studied the biosynthesis, processing, and localization of precursors of Bac7 and Bac5 in bovine bone marrow cells of the myeloid lineage. In vitro translation directed by mRNA isolated from these cells has shown that the primary translation products are preprobactenecins of 23.5 and 21 kD, and are processed to polypeptides of 20 and 15.8 kD, respectively. The 20-kD polypeptide is the granule storage form of Bac7, or proBac7, as also demonstrated by Western blot analysis of lysates of peripheral neutrophils. Between 15 and 50 min from the beginning of its biosynthesis the 15.8-kD polypeptide is converted into the 15-kD granule storage form of Bac5, or proBac5. As shown by immunogold EM, proBac7 and proBac5 are sorted and targeted to the matrix of the so called large granules, which are the predominant organelles in the cytoplasm of bovine neutrophils and are the exclusive store of the nonoxidative antimicrobial system of these cells. Solubilization of granules with Triton X-100 with concomitant unmasking of proteases leads to cleavage of the proforms to Bac7 and Bac5. Experiments performed with protease inhibitors suggest that the proteolytic cleavage is catalyzed in detergent-solubilized neutrophils by neutral serine protease(s), very likely derived from the azurophil granules.

Animals↗
PubMed1990

Structural definition by antibody engineering of an idiotypic determinant.

Using computer-aided techniques for predicting molecular structure, we constructed an atomic model of the variable domain of a murine anti-thyroglobulin antibody whose immunodominant idiotypic determinant (Id62) was mapped by site-directed mutagenesis and immunochemical analysis. We previously showed that under experimental conditions this idiotype activates anti-idiotypic B cells and T cells, and modulates the response to thyroglobulin in mice. Because idiotype interactions are considered of physiological importance for immune regulation, we studied this idiotype as a model to understand the relationship between function and structure. To determine the contribution of heavy- and light-chain variable domains to the idiotype structure, we constructed chimeric expression vectors and introduced them into the (non-secreting) P3X63Ag8.653 myeloma cell line. Mutants of the heavy-chain variable domain were obtained by site-directed mutagenesis and transfected into the murine (lambda 1) light-chain producer J558L cell line. The expressed proteins were purified from culture supernatants of transfected cells and characterized. We provide evidence that the third hypervariable loop (D region) of the heavy-chain variable domain is the structural correlate of the idiotypic determinant of this autoantibody and is independent from the nature of the associated light chain. Substitution of residues of the first and second complementarity-determining regions do not affect idiotype expression. The results described here are discussed in relation to our understanding, at a molecular level, of the interaction of idiotopes with B- and T-cell compartments.

Amino Acid Sequence↗
PubMed1990

Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding.

Immunoglobulins bind antigens and express individual antigenic specificities mainly through residues located in hypervariable loops of their N-terminal domains. Hypervariable loops are kept in place by a molecular scaffold organized in a sandwich-like structure with two beta-sheets stabilized by a disulfide bridge (the immunoglobulin fold). This structural feature, together with the possibility of obtaining high level expression, extracellular secretion, easy purification and stability of the protein product, render immunoglobulin an ideal 'molecular vehicle' for the expression of exogenous peptides. Here we report on the engineering of an immunoglobulin expressing an exogenous epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)3. By recombinant DNA techniques, we inserted three copies of the tetrapeptide (NANP)3 in the third hypervariable loop (D region) of an immunoglobulin heavy chain variable domain. We show that the engineered antibody was properly assembled and secreted. A panel of polyclonal and monoclonal antibodies, including anti-synthetic peptides and anti-(NANP)n antibodies, were used to study the molecular configuration of the engineered domain's surface. The results indicate that (i) the exogenous sequence did not appreciably alter the overall fold of the variable domain; and (ii) the inserted epitope folded with a configuration immunologically similar to the one assumed in the native protein, suggesting that short- and medium- rather than long-range interactions stabilized the structure of the (NANP)3 peptide in the folded protein. We propose this system for the expression of peptidic sequences, and their structural and functional analysis.

Amino Acid Sequence↗