Selection from a peptide library of the antigenic determinants of a protein.
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Amino Acid Sequence↗
Biomedical subjects
L Castagnoli
Publications and source records attributed to L Castagnoli.
Hybrid Rop-pIII proteins for the display of constrained peptides on filamentous phage capsids.
In order to increase the versatility of phage display technology, it is desirable to be able to impose some structural constraints on the peptides that are presented by the phage particles. This is currently not feasible since the conformation of the capsid proteins, used to link the foreign peptide to the phage, are either unknown (pIII) or too simple (pVIII) to permit the engineering of peptide inserts into a constrained context. To reach this scope we have modified the amino-terminus of gene III by appending a well-characterized protein motif, the four-helix bundle of the bacterial protein Rop. Phage particles displaying Rop can be separated from wild-type (wt) particles by affinity purification with an antibody. Rop can be extensively modified by substituting its solvent-exposed residues and/or by inserting peptides either into the carboxy-terminal tail or into the bend region that connects the two alpha-helices of the monomer. These results open the possibility to construct peptide libraries where the peptides are constrained either into an omega-loop type conformation or an alpha-helix. Libraries formed by peptides inserted into the carboxy-terminus can also be constructed. Furthermore, the system that we have developed permits to produce large quantities of the elements of the libraries in the cytoplasm or to display them on the capsid of filamentous phages.
Bacterial Proteins↗
Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector.
Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.
Amino Acid Sequence↗
Control of ColE1 plasmid replication by antisense RNA.
One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.
Bacterial Proteins↗
Genetic and structural analysis of the ColE1 Rop (Rom) protein.
Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that controls the copy number of plasmid of the ColE1 family by increasing the affinity between two complementary RNAs. The Rop dimer is a bundle of four tightly packed alpha-helices that are held together by hydrophobic interactions. We have systematically altered, by site directed mutagenesis, most of the solvent exposed amino acids of the Rop bundle and we have identified the alterations that cause a decrease of the activity of the regulatory molecule. We conclude that Rop folding is rather insensitive to amino acid substitutions and to other mutations as drastic as deletions and insertions. Looking along the 2-fold symmetry axis the amino acid side chains whose alterations affect the function of Rop are all located on one side of the molecule. Furthermore they are clustered at the extremities of the alpha-helix bundle, the only exception being the aromatic ring of Phe-14.
Amino Acid Sequence↗
A strategy to optimize translation initiation in recombinant mRNA: application to the Rop gene.
Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E. coli. This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase. By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame. A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence. In this step, however, we use an oligonucleotide that has a degenerate sequence. That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence. The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest. We discuss the results obtained by applying the method to the Rop gene.
Bacterial Proteins↗
[Primary malignant lymphomas of the gastrointestinal system].
The Authors describe their experience regarding eight cases of primary malignant gastrointestinal lymphomas. After having briefly reexamined the epidemiology and the problems coming from the discordances that still exist on various systems of classification and of staging, they put in evidence that it is not always possible to reach a sure preoperative diagnosis on such pathology, and they explain some considerations about the diagnosis, the therapy and the prognosis.
Aged↗
[Synchronous and metachronous carcinoma of the large intestine].
The Authors report their experience about 20 cases of multiple large bowel adenocarcinomas, 9 of them synchronous and 11 metachronous. After developing a brief reexamination of epidemiology and etiopathogenesis, they explain their diagnostic and therapeutic considerations. The necessity of a real complete and exact radiological and endoscopical preoperative study, being also early, is at last emphasized, as well as an accurate postoperative follow-up.
Adult↗
Characterization of a promoter up mutation in the -35 region of the promoter of the primer for ColE1 replication.
A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized. This base change causes a promoter up phenotype. The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region. The promoter up phenotype is concluded to be due to a change in the primary structure of the -35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase.
Base Sequence↗
[Lymphoma of the thyroid (description of 2 cases)].
Primary lymphoma of the thyroid is a rare disease and it occurs in a low percentage of thyroid tumours. Nevertheless its frequency is increasing. We present two observed cases in this study. One of these is an exceptional extra-nodal localization of Burkitt's lymphoma. The role of surgery in these particular neoplasms and the importance of intraoperative histological examination for the rational therapeutic behaviour are stressed.
Burkitt Lymphoma↗
[Rupture of the pancreas after closed injury of the abdomen (presentation of a case, clinical and therapeutic notes)].
The authors observed a case of a complete breaking of pancreas between head and body associated to a serious lesion of the liver, favourably treated in emergency by means of a subtotal splenopancreatectomy and a suture of the hepatic lacerations, and that gave them the occasion to think over the serious risks linked with an insufficient evaluation of a pancreatic lesion and its most appropriate surgical treatment. In details, personal experience and the examination of Literature showed that in the laparotomy it is absolutely necessary to examine all the gland carefully and methodically in order to recognize and evaluate accurately the pancreatic lesion, which is often concealed by the massive manifestations caused by the concomitant lesions of other intra-abdominal viscera. Besides, among the suggested surgical techniques, the subtotal splenopancreatectomy proved to be the safest, bringing no consequences from the functional point of view, even when extended to more than 80% of the glandular tissue.
Accidents, Traffic↗
[Torsion of the greater omentum (considerations apropos of 2 clinical cases)].
AA. refer 2 cases of great omentum torsion, of the secondary unipolar type, and of the primary type, chronic course and pelvic palpable mass appearance. After a short revision of pathogenesis and clinical features, AA. emphasize the difficulty almost impossibility of a proper preoperative diagnosis, in consideration too of the relative rarity of this pathology.
Adult↗
[Submucosal lipomas of the colon (considerations apropos of 3 clinical cases)].
Submucous colonic lipoma is relatively rare and can be the cause of important diagnostic and consequently therapeutic problems. The AA. reexamine the anatomo pathological and clinical features and the diagnostic and therapeutic problems, and refer 3 cases observed one of which was localized in the ileo-cecal valve. The AA. remark that radiological and endoscopic investigations and laparotomy features also can simulate a malignant tumor and address to the surgical technics of the oncologic surgery.
Aged↗
Analysis of dominant copy number mutants of the plasmid pMB1.
We characterize two dominant copy number mutants of a derivative of plasmid pMB1. One of the two mutations maps in the -35 region of the primer promoter and results in increased promoter activity. The analysis of the secondary structure in the proximity of the mutant sequence suggests a possible mechanism which could be the basis of the promoter-up phenotype. By comparing the properties of the mutant and the wild type plasmid in an in vitro system, we confirm that the primer and not its coding sequence is the target of RNA I inhibition. The second mutation affects the sequence of the primer so that it is less sensitive to inhibition by RNA I. We propose that this mutation stabilizes a secondary structure necessary for primer formation.
Base Sequence↗
[Surgical treatment of gastro-esophageal reflux in hiatal hernia. I. Proposal for a technical modification of the Lortat-Jacob intervention].
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Diaphragm↗
[Surgical treatment of gastro-esophageal reflux caused by hiatal hernia. II: Results and discussion].
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Evaluation Studies as Topic↗
Control of pMB1 replication: inhibition of primer formation by Rop requires RNA1.
The rop gene participates in the control of plasmid copy number by interfering with transcripts originating from the primer promoter. Here we show that this inhibition mechanism requires RNA1 in trans. Mutations in the RNA1 coding sequence that result in plasmids with altered incompatibility properties do not affect the ability of the molecule to participate in the Rop inhibition mechanism. Furthermore we show that the target of the Rop-RNA1 inhibitory mechanism is located, at least in part, after the 52nd nucleotide of the sequence encoding the primer transcript.
Base Sequence↗