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Biomedical subjects

P Neri

Publications and source records attributed to P Neri.

At least 73 records · Page 4Linked to original sources
PubMed1989

Inhibition of experimental metastasis of murine fibrosarcoma cells by oligopeptide analogues to the fibronectin cell-binding site.

We have analyzed the effect of the synthetic oligopeptides Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) and Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), analogues to the fibronectin cell-binding sequence, on the formation of experimental lung metastasis. SR-BALB were injected alone or in conjunction with GRGDSP or GRGESP in the tail vein of BALB/c mice. Co-injection with GRGESP reduced by approximately 70% the number of metastatic colonies per mouse. However, co-injection with the closely related peptide GRGDSP, containing the conservative substitution Glu/Asp, did not affect metastatic behavior. GRGESP peptide anti-metastatic activity was not due to a toxic effect on tumor cells or on mice. In vitro adhesion assays testing for a possible effect of the peptide on cell-matrix interactions indicated that the GRGESP peptide did not affect cell adhesion to the matrix proteins tested.

Animals↗
PubMed1989

A monoclonal antibody to a synthetic fragment of rabies virus glycoprotein binds ligands of the nicotinic cholinergic receptor.

Rabies virus glycoprotein and snake venom curaremimetic neurotoxins share a region of high homology (30-45 for neurotoxins and 190-203 for the glycoprotein) in the regions that are believed to be responsible for binding the nicotinic acetylcholine receptor. Monoclonal antibodies raised to the 190-203 synthetic fragment of rabies virus glycoprotein were immobilized on a high performance affinity chromatography column and were able to bind neurotoxins. Toxins were displaced from the affinity column by elution at acidic pH and by affinity competition with acetylcholine at neutral pH. Furthermore, the affinity column proved to be useful for the purification of cholinergic ligands. Overall, these results indicate that the paratope of our monoclonal antibodies could behave as an 'internal image' of the nicotinic cholinergic receptor acetylcholine binding site.

Amino Acid Sequence↗
PubMed1988

High performance liquid chromatography immunoaffinity purification of antibodies and antibody fragments.

A rapid antibody purification method is described which combines high performance liquid chromatography (HPLC) resolution with affinity chromatography specificity. The antigen used as immobilized ligand is bound to the HPLC column matrix by reacting the amino groups of the protein with the active epoxy groups of the latter. Once the reaction has finished, the unreacted groups of the column are saturated with an appropriate scavenger. Unrelated proteins are then washed out and the specific antibodies recovered by lowering the pH of the elution buffer.

Animals↗
PubMed1987

Resolution of conformation equilibria in linear peptides by circular dichroism in cryogenic solvents.

The circular dichroism spectra of the synthetic peptide antigen, 209-222 of the surface glycoprotein of the rabies virus were recorded as a function of solvent composition and over the temperature range of +60 degrees C to -135 degrees C; beta-III and beta-II reverse turn conformations were found to exist in TFE/H2O (3:1) at room temperature and in ethanediol/H2O (2:1) below -110 degrees C respectively. Evidence, from comparison of observed and calculated spectra, is given to support the existence of a conformational equilibrium between a beta-II and a beta-III reverse turn. These data can serve as a basis for synthetic vaccine development and understanding the nature of polypeptide chain folding.

Circular Dichroism↗
PubMed1987

Immunogenicity of a free synthetic peptide: carrier-conjugation enhances antibody affinity for the native protein.

The synthetic peptide 166-174 of hCS sequence, corresponding to an antigenic determinant of the protein, was used to elicit MAbs to the native hCS molecule. The synthetic peptide was administered according to different immunization protocols to BALB/c mice, both in the free form or conjugated to carrier proteins. Spleens and popliteal lymph nodes from primed mice were fused with a myeloma cell line to produce MAbs, and selected clones were characterized for isotype and affinity. Spleen fusions gave rise to IgM MAbs, whereas lymph node fusions gave preferentially IgG MAbs. No correlation was found between antibody class and affinity since affinity is highly increased by carrier-conjugation, while it did not enhance IgG production. The free synthetic peptide showed a low immunogenicity: affinities of MAbs produced ranged from 10(5) to 10(7) l/mole, an average 1000-fold lower than the values obtained with carrier-conjugated peptide. In one case, however, carrier conjugation did not give rise to anti-hCS MAbs. Overall, these studies provide a rational approach to the production of anti-protein MAbs by synthetic peptide immunization and offer the opportunity to obtain MAbs of the desired isotype and affinity.

Animals↗
PubMed1986

Determination of antigen-specific immunoglobulin content in ascitic fluids and antisera.

A rapid and sensitive method is described for the determination of the antigen-specific immunoglobulin contents of ascitic fluids and/or antisera. Immunoglobulins are quantitatively separated from the biological fluid by immunoaffinity chromatography on the insolubilized antigen using the Millipore Millititer filtration system to perform the simultaneous assay of multiple samples. The specific immunoglobulins are then eluted at acidic pH and their concentration is estimated by solid-phase competitive radioimmunoassay. Radioimmunoassay values, corrected for dilutions, give the antigen-specific immunoglobulin concentration of ascitic fluids and/or antisera.

Ascitic Fluid↗
PubMed1986

Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication.

Pertussis toxin, a protein composed of five different subunits (S1, S2, S3, S4, and S5), is the major virulence factor of Bordetella pertussis. We have cloned and sequenced a DNA fragment of 4.7 kilobases that contains the genes coding for the five subunits. The genes are clustered within 3.2 kilobases in the following order: S1, S2, S4, S5, and S3. A sequence closely resembling Escherichia coli promoters is found only before the S1 gene, and a possible termination signal is present at the end of the S3 gene, which suggests that the pertussis toxin genes are organized in a single operon. A possible Shine-Dalgarno sequence is present before the S1 gene but not before the other four genes that 8-12 nucleotides upstream from the ATG codon show a new consensus sequence, 5'TCC(T)GG3', possibly involved in the regulation of translation. We have also found sequence homology between the S2 and S3 genes and their protein products indicating that gene duplication played a major role in the evolution of pertussis toxin.

Amino Acid Sequence↗
PubMed1985

Identification of a mixture of two different monoclonal antibodies by nonlinear regression analysis after solid phase radioimmunoassay with labeled antigen.

The results of competitive solid phase radioimmunoassay with labeled antigen, when analyzed by nonlinear regression, may reveal the presence of two different monoclonal antibodies, occasionally produced in a hybridoma culture. In fact, a hybridoma-derived antihuman chorionic somatomammotropin antibody prepared in our laboratories showed an unsatisfactory curve fitting when the experimental data were elaborated on the basis of a monoclonal model. The antibody was subjected to recloning, and gave two separate homogeneous monoclonal antibodies with different affinity constants.

Antibodies, Monoclonal↗
PubMed1985

Production and characterization of monoclonal antibodies to anti-human chorionic somatomammotropin by immunization with two free synthetic peptides.

The peptides corresponding to the fragments 135-140 and 166-174 of human chorionic somatomammotropin (hCS) were synthesized, and used to raise monoclonal antibodies to the native hCS molecule. The synthetic peptides were injected into BALB/c mice in the free form, i.e. not conjugated to a carrier, and the spleens were fused with Sp2/01Ag8 myeloma line to produce monoclonal antibodies. The antibodies produced belonged to the IgM and IgG classes and, once purified by affinity chromatography on hCS-Sepharose, they were covalently coupled to macroporous polystyrene beads and characterized by competitive radioimmunoassay. Their affinity constants were determined by elaborating the radioimmunoassay data by nonlinear regression analysis and they were found to range from 10(5) to 10(6) M-1. The evaluation of the affinity constant of the antibodies produced is always important as a measure of the immunogenicity of an antigen, particularly when synthetic peptides are used as immunogens.

Animals↗
PubMed1984

Antigenic structure of human chorionic somatomammotropin: four epitopes revealed by monoclonal antibodies.

The characterization of epitope specificity of a panel of 15 monoclonal antibodies against human chorionic somatomammotropin (hCS), previously prepared in our laboratory, allowed us to distinguish 4 antigenic determinants on the hCS molecule. Experiments were carried out with competition and sandwich assays. The results allowed us to divide our MAbs into 4 groups, recognizing 4 different epitopes, and to select 4 MAbs each distinguishing a different epitope in order to pursue further studies on the antigenic structure of hCS.

Antibodies, Monoclonal↗
PubMed1984

1H-13C selective NOE studies of the decapeptide gramicidin S.

The cyclic decapeptide gramicidin S has been used as a model biopolymer to test the reliability of a structural method which is based on a relaxation analysis of heteronuclear selective NOEs. The observation of through-the-space dipolar couplings between intra- and inter residue amide protons and carbonyl carbons, perfectly consistent with the well established peptide solution conformation, confirms the effectiveness of this structural approach. As a corollary of the latter, carbonyl carbon resonances are unequivocally assigned. Moreover, a direct experimental proof of a Orn-NH2----Phe C = O hydrogen bonding is here given.

Biopolymers↗
PubMed1984

Characterization of monoclonal antibodies against human chorionic somatomammotropin: competitive screening and determination of the affinity constants.

A simple screening method has been developed for the characterization of monoclonal antibodies (MAbs) against human chorionic somatomammotropin (hCS) produced in our laboratory. The method is easily adaptable to MAbs against other soluble antigens. Our proposed method allows evaluation of the degree of binding of the antibodies to the antigen, and of cross-reaction with human growth hormone and ovine prolactin. The positive clones have been further characterized by solid-phase radioimmunoassay in order to determine their affinity constants, which ranged from 10(7) to 10(9)/M as determined by non-linear regression analysis.

Animals↗
PubMed1984

Identification and synthesis of an antigenic determinant common to human chorionic somatomammotropin and human growth hormone.

Human chorionic somatomammotropin (hCS) and human growth hormone (hGH), despite their different biological activities, show a remarkable degree of homology in their primary structure, which explains their immunological cross-reactivity. On the basis of the hydrophilicity profiles, we predicted that the sequence 165-174 would correspond to an antigenic determinant common to both hormones. The sequence 165-174 was synthesized in the solid phase and the ability to bind antibodies to hCS was tested by a radioimmunoassay at each step of the synthesis, without detaching the peptide from the resin. We found that the immunological sequence able to bind antibodies to hCS is that corresponding to 167-174. In similar experiments, we showed that the sequence 166-174 is able to bind antibodies to hGH. In the plaque-forming cell test using the synthetic fragment 166-174 bound to sheep red blood cells, we also observed that the ratio between the number of cells forming antibodies to hCS and to the 166-174 peptide was always between 0.45 and 0.73, thus suggesting that the 166-174 peptide represents a major determinant of hCS.

Amino Acid Sequence↗
PubMed1983

Purification of diphtheria toxin by chromatography on Cibacron Blue-Sepharose.

Diphtheria toxin binds to Cibacron Blue-Agarose and may be eluted by increasing the ionic strength of the elution buffer. Experiments using difference spectroscopy showed that the interaction between toxin and dye is ionic rather than hydrophobic, and therefore it is of a different nature with respect to that usually found in nucleotide-requiring enzymes.

Anthracenes↗
PubMed1983

[Behavior of serum immunoglobulins in boutonneuse fever].

The behaviour of serum immunoglobulins in 35 patients suffering from Mediterranean exanthematous has been studied. No particular quantitative or qualitative change lieve noted. Supported by the scanty and contrasting data in existing literature, it is maintained that aspecific immunological serum doses are of little use, but research is urged into specific antibodies by means of indirect immunofluorescence and the Elisa method.

Adult↗
PubMed1983

A simple method for the estimation of amino groups on insoluble matrix beads.

A method for the determination of free amino groups on solid supports is proposed. The method is based on the reaction of the matrix with excess 2,4,6-trinitrobenzenesulfonic acid (TNBS) and subsequent quantitative determination of unreacted TNBS by reaction with glycine. The method is simple and reproducible and does not require the solubilization of the matrix, therefore proving suitable even for crosslinked insoluble supports.

Amines↗