Biomedical subjects

P Monaci

Publications and source records attributed to P Monaci.

At least 37 records · Page 2Linked to original sources

PubMed1995

Derivation of vaccines from mimotopes. Immunologic properties of human hepatitis B virus surface antigen mimotopes displayed on filamentous phage.

We have previously reported the identification, using human immune sera, of mimotopes of human hepatitis B virus surface Ag (HBsAg) displayed on filamentous phage. To test if these mimotopes could be useful in developing a vaccine against the human hepatitis B virus (HBV), we have compared the humoral immune response of animals immunized either with a recombinant HBsAg vaccine, or with mimotopes. Immunogens were prepared by fusing the mimotopes on different carrier molecules (phage coat protein pIII and pVIII, recombinant human H ferritin, HBV core peptide) and by synthesizing multiple antigenic peptides carrying the mimotopes' amino acid sequences. These immunogens were injected into mice and rabbits and sera were collected and tested for the presence of HBsAg-specific Abs. Our data confirm that mimotopes can induce a humoral immune response resembling that induced by the original Ag, and HBsAg mimotopes displayed on phage prove to be the best immunogens, inducing the most reproducible and potent immunization. Mimotopes that react as HBV subtype-specific Ags do not show this specificity as immunogen and induce a nonsubtype-restricted response. Furthermore, mimotopes displayed on phage elicit a strong response to HBsAg in a strain of mouse reported to show a low response to it. These results indicate that mimotopes identified from random peptide libraries through utilizing human immune sera could be important leads for the derivation of new vaccines.

Amino Acid Sequence↗

PubMed1995

A complex interplay of positive and negative elements is responsible for the different transcriptional activity of liver NF1 variants.

A full-length cDNA of the rat liver Nuclear Factor 1 (NF1L21) has been cloned and expressed in S. cerevisiae to analyse the architecture of its activation domain. NF1L21 displays a specific DNA-binding activity, as well as the ability to activate transcription from an artificial NF 1-responsive promoter in yeast. Interaction of two or more NF1L21 molecules with multiple sites on the same promoter activated transcription in a synergistic fashion. Functional analysis of the activation domain of NF1L21 reveals a tripartite structure. Two distinct positive elements are required for NF1L21 -mediated transcription activation. A proline-rich element sandwiched between these two positive domains attenuates their transactivation potential. A shorter NF1L variant (NFlL4) in which the distal positive element is replaced by a different sequence was also isolated. NF1L4 displays the same DNA-binding activity and dimerisation properties as NF1L21, but is unable to activate transcription in yeast.

Amino Acid Sequence↗

PubMed1995

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Amino Acid Sequence↗

PubMed1995

Peptide and protein display on the surface of filamentous bacteriophage.

The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics. Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences. Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques. The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines.

Amino Acid Sequence↗

PubMed1994

Monoclonal antibodies that recognise filamentous phage: tools for phage display technology.

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools to study phage assembly and structure. Furthermore, we describe two examples of how these mAb can be exploited in the construction and screening of peptide libraries displayed by the filamentous phase major coat protein. We have used one of these mAb to develop a sensitive ELISA with crude phage supernatants. This assay allows rapid screening of large numbers of clones from random peptide phage libraries. Some of the anti-phage mAb described here can interfere with wild-type phage propagation, while phage carrying modifications in their coat proteins are insensitive to growth inhibition. We have exploited this observation as a tool to favour the growth of phage displaying peptides fused to pVIII, with respect to vector phage.

Amino Acid Sequence↗

PubMed1994

Recognition by human sera and immunogenicity of HBsAg mimotopes selected from an M13 phage display library.

We used two mouse monoclonal antibodies (mAb) specific for the human hepatitis B virus surface antigen (HBsAg) to screen a random peptide library of 15 amino-acid residues displayed as a fusion to protein III of filamentous phage M13. By a combination of affinity selection, immuno-screening and ELISA techniques, we selected peptides that are recognized by the anti-HBsAg mAb and show aa similarity with the natural antigen. The selected phage-displayed epitopes (phagotopes) behave as antigenic mimics of HBsAg. One phagotope is specifically recognized by human sera from HBsAg-immunized individuals, pointing to the possible use of phagotopes as markers to detect the presence of specific Ab in the serum. The same phagotope also elicits Ab directed against HBsAg in mice, indicating that mAb-selected phagotopes can also be immunogenic mimics of the natural antigen. These findings demonstrate that it is possible to identify disease-specific epitopes that can be used as diagnostic reagents and as leads for the development of acellular vaccines.

Animals↗

PubMed1994

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease-specific epitopes from phage-displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease-specific phagotopes can be identified out of this pool through an 'antigen independent' procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease-related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Amino Acid Sequence↗

PubMed1994

Epitope discovery using peptide libraries displayed on phage.

Peptides displayed on phage, which mimic continuous and discontinuous epitopes, can be selected using purified antibodies or preparations of polyclonal serum. This review describes recent advances in this field, discusses the application of phage-display technology to the diagnosis of human diseases, and presents new ideas for the preparation of vaccines directed against specific epitopes on a pathogen.

Amino Acid Sequence↗

PubMed1993

A bipartite activation domain is responsible for the activity of transcription factor HNF1/LFB1 in cells of hepatic and nonhepatic origin.

HNF1/LFB1 is a transcription factor that controls the expression of several liver-specific genes. Previous in vitro experiments allowed us to identify two different regions in the carboxy-terminal portion of the protein responsible for most of the transcription activation potential: the first, ADI, between amino acids 546 and 628 and the second, ADII, between amino acids 281 and 318. To characterize the molecular anatomy of HNF1/LFB1 better, we have analyzed its trans-activating properties in vivo. Several HNF1/LFB1 deletion mutants were tested for their ability to induce transcription from HNF1/LFB1-dependent synthetic promoters in cells of hepatic and nonhepatic origin. These last recipient cells provide an HNF1/LFB1-deficient environment that is useful for a precise quantification of the recombinant protein. Our results confirm the importance of ADI and indicate that no activating property can be assigned to ADII in vivo. Moreover, a novel glutamine/proline-rich activation domain (ADIII) has been identified between amino acids 440 and 506. These findings are confirmed by domain-swapping experiments, carried out with the heterologous GAL4 DNA-binding domain, which also show that the activity of each individual activation domain is influenced by combining adjacent HNF1/LFB1 sequences. The data presented indicate that HNF1/LFB1 transcription activating potential relies on a complex structure and also provide important clues to understanding the different functions exerted by transcription factors of this family.

Amino Acid Sequence↗

PubMed1993

The X-ray structure of an atypical homeodomain present in the rat liver transcription factor LFB1/HNF1 and implications for DNA binding.

The transcription factor LFB1/HNF1 from rat liver nuclei is a 628 amino acid protein that functions as a dimer binding to the inverted palindrome GTTAATN-ATTAAC consensus site. We have crystallized a 99 residue protein containing the homeodomain portion of LFB1, and solved its structure using X-ray diffraction data to 2.8 A resolution. The topology and orientation of the helices is essentially the same as that found in the engrailed, MAT alpha 2 and Antennapedia homeodomains, even though the LFB1 homeodomain contains 21 more residues. The 21 residue insertion is found in an extension of helix 2 and consequent lengthening of the connecting loop between helix 2 and helix 3. Comparison with the engrailed homeodomain-DNA complex indicates that the mode of interaction with DNA is similar in both proteins, with a number of conserved contacts in the major groove. The extra 21 residues of the LFB1 homeodomain are not involved in DNA binding. Binding of the LFB1 dimer to a B-DNA palindromic consensus sequence requires either a conformational change of the DNA (presumably bending), or a rearrangement of the subunits relative to the DNA.

Amino Acid Sequence↗

PubMed1992

Trans-dominant inhibition of transcription activator LFB1.

Liver-enriched factor LFB1 (also named HNF1) is a dimeric transcription activator which is essential for the expression of many hepatocyte-specific genes. Here we demonstrate that LFB1 mutants in the POU A-like or in the homeo domains inhibit wild-type DNA binding by forming inactive heterodimeric complexes. Co-transfection of one of these mutants with wild-type LFB1 in HeLa cells eliminated LFB1 DNA binding and transcriptional activities through a trans-dominant mechanism. Expression of the same dominant negative mutant in human hepatoma HepG2 cells only partially inhibited endogenous LFB1 activity, due to stabilization of LFB1 dimers in these cells. Dimer stabilization in hepatoma cells is mediated by a heat-labile association with an 11kD polypeptide, analogous to the DCoH cofactor identified in rat liver by Mendel et al. (1). The property of stabilizing LFB1 dimers is also shared by HeLa cells which produce a HeLa homolog of DCoH. These results demonstrate that LFB1 dimer stabilization as well as the synthesis of 'stabilizing factors' are not restricted to cells expressing LFB1 or other members of its family.

Binding Sites↗

PubMed1991

Alteration in L-type pyruvate kinase gene expression is not associated with the LF-B1 mRNA level.

The relation of expression of the LF-B1 gene with the L-type pyruvate kinase (L-PK) mRNA level in rat liver and hepatoma cells was investigated. The L-PK mRNA level in rat liver changed after partial hepatectomy, during development and on intake of a high carbohydrate diet, while the level of LF-B1 mRNA remained unchanged or altered reciprocally. Dedifferentiated AH-130 cells, which did not express L-PK mRNA, expressed LF-B1 mRNA. These results suggest that transcription of the pyruvate kinase L gene is not simply regulated by the level of LF-B1 mRNA.

Animals↗

PubMed1991

Promoter elements and factors involved in hepatic transcription of the human ApoA-I gene positive and negative regulators bind to overlapping sites.

DNase I footprinting analysis of the proximal apoA-I promoter sequences with rat liver nuclear extracts identified four protected regions: A, -22 to +17; B, -128 to -77; C, -175 to -148; and D, -220 to -190. Region D (-220 to -190) binds at least two distinct activities, designated AID1 and AID2, respectively, which can be separated by ion exchange chromatography. Region C (-175 to -148) forms five DNA protein complexes. Three of the complexes (2, 4, and 5) originate from the binding of more than one heat-stable nuclear factor, and two (1 and 3), from the binding of two heat-labile factors. The heat-stable factors bind in the -175 to -148 region and can be distinguished from C/EBP, which recognizes the same region, with DNA binding gel electrophoretic assays. Both factors 1 and 3 bind in the -168 to -148 apoA-I region. Despite the lack of a CCAAT motif in this region, the binding of factor 1 is competed out by oligonucleotides containing the binding sites of NFY and NFY*. Mutagenesis of the promoter region showed that mutations in the -171 to -166 and -158 to -153 regions diminished the binding of the heat-stable factors and reduced hepatic transcription to 14 and 8% of control, respectively. In contrast, a mutation in the -164 to -159 region abolished the binding of factor 1 and was associated with a 4.6-fold increase in hepatic transcription. These findings suggest that the heat-stable factors act as positive regulators, whereas factor 1 acts as a negative regulator in apoA-I gene transcription.

Animals↗

PubMed1991

LFB3, a heterodimer-forming homeoprotein of the LFB1 family, is expressed in specialized epithelia.

We have cloned and characterized a mouse cDNA coding for LFB3, a DNA binding protein containing an extra-large homeodomain. The first 315 amino acids of LFB3 are highly homologous to the DNA binding domain of LFB1, a regulatory protein involved in the expression of several liver-specific genes. LFB3 is a transcriptional activator which binds to DNA as a dimer and forms heterodimers with LFB1 both in vitro and in vivo. However, LFB3 expression seems not to be directly correlated with the liver-specific phenotype, since it is detected in dedifferentiated hepatoma cell lines which express neither LFB1 nor several liver-specific genes. LFB3 expression starts before that of LFB1 during mouse and rat development, and is strongly increased upon retinoic acid induced differentiation of F9 embryonic carcinoma cells. LFB3 and LFB1 are expressed in the epithelial component of many organs of endodermal and mesodermal origin, suggesting that they may play a more general role associated with the differentiation of specialized epithelia.

Amino Acid Sequence↗

PubMed1990

Identification and characterization of hepatocyte-specific regulatory regions of the rat pyruvate kinase L gene. The synergistic effect of multiple elements.

The rat pyruvate kinase L (PKL) gene produces the L- and R-type isozymes by alternative transcription that is regulated in a tissue-specific manner. To investigate which DNA elements are involved in hepatocyte-specific expression of the L-type isozyme, we performed transient DNA transfer experiments with PKL/chloramphenicol acetyltransferase fusion genes. We found three positive regulatory regions required for expression of the L-type isozyme in adult rat hepatocytes by functional analyses of a series of 5' and internal deletion constructs of the fusion genes. These regions, designated as PKL-I, PKL-II, and PKL-III, were located between nucleotides -76 and -94, -126 and -149, and -150 and -170, respectively. PKL-I showed enhancer-like activity alone, whereas PKL-II and PKL-III did not have any independent effect. Combinations of L-I + L-II and L-II + L-III, but not of L-I + L-III, showed synergistic enhancer activities when oriented in the same direction. The inclusion of all three elements oriented in the same direction had the maximum synergistic effect, indicating that these elements function as a unit. This unit enhanced expression from heterologous as well as homologous promoters in a manner that was independent of its orientation and position relative to the cap site. The activity of the unit was not detected in HeLa cells or K562 erythroleukemia cells, suggesting that this unit possessed cell-type specificity. PKL-I consists of a palindrome sequence 5'-CTGGTTATACTTTAACCAG-3', which contain a sequence homologous to the LF-B1-binding site. PKL-II contains the sequence 5'-TTCCTGGACTCTGGCCCCCAGTGT-3', which is similar to that of the LF-A1-binding site. PKL-III contains a palindrome sequence 5'-CCACGGGGCACTCCCGTGG-3', which include a sequence homologous to the binding site of the adenovirus major late transcription factor. Gel retardation assay indicated that the different trans-acting factors interacted with three elements and that the transacting protein bound to PKL-I was in fact LF-B1. However, the trans-acting factors bound to PKL-II and PKL-III were different from LF-A1 and major late transcription factor, respectively. Thus, we conclude that three cis-acting elements are very important for specific expression of the PKL gene in hepatocytes and that LF-B1 and two unknown factors bound to these elements interact with each other to cause a synergistic effect.

Animals↗

PubMed1990

A myosin-like dimerization helix and an extra-large homeodomain are essential elements of the tripartite DNA binding structure of LFB1.

The transcription activator LFB1 is a major determinant of hepatocyte-specific expression of many genes. To study the mechanisms underlying LFB1 transcriptional selectivity, we have initiated its biochemical characterization. By in vitro complementation assays we have defined two distinct regions required for high levels of transcription, which resemble previously described activation domains. In contrast, the region of LFB1 necessary for DNA binding displays several novel features. The DNA binding domain is tripartite, including a homeodomain of unusual length (81 amino acids) and an N-terminal helix similar to part of myosin. This helical region mediates dimerization, which is shown to be essential for DNA binding.

Amino Acid Sequence↗

PubMed1990

Synergistic trans-activation of the human C-reactive protein promoter by transcription factor HNF-1 binding at two distinct sites.

The promoter region of the human C-reactive protein (CRP) gene comprises two distinct regions (APREs, for Acute Phase Responsive Elements) each one containing information necessary and sufficient for liver specific and IL-6 inducible expression in human hepatoma Hep3B cells. In this paper we show that both APREs contain a low affinity binding site for the liver specific transcription factor HNF-1/LF-B1. The two sites are separated by approximately 80 bp. Mutations in either of the two sites abolish inducible expression. The same effect is specifically obtained in cotransfection competition experiments when the human albumin HNF-1 site is used as competitor. However, HNF-1 is not the intranuclear mediator of IL-6 because synthetic promoters formed by multimerized copies of different HNF-1 binding sites are not transcriptionally activated by this cytokine. An expression vector encoding full length HNF-1 is capable of trans-activating transcription from the wild-type CRP promoter but not from mutants which have lost the ability to bind HNF-1. Moreover, the level of trans-activation observed with the natural promoter containing both HNF-1 binding sites is far greater than the level of mutated variants containing only one of the two sites. This result strongly suggests that two HNF-1 molecules bound simultaneously to sites distant from each other can act synergistically to activate gene expression.

Base Sequence↗

PubMed1989

The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain.

The nuclear protein LF-B1 (also referred to as HNF-1) is a transcription activator required for the expression of several liver-specific genes. LF-B1 has been purified to homogeneity from rat liver nuclear extracts. The sequence of the protein has been partially determined and, subsequently, overlapping cDNA clones containing the entire open reading frame of LF-B1 were isolated. The full-length cDNA encodes a 628 amino acid protein and directs the synthesis in vitro of a protein capable of binding DNA with the same specificity as LF-B1. The cDNA was recombined into a vaccinia virus vector and active LF-B1 was obtained from infected HeLa cells. Addition of the vaccinia recombinant protein to rat spleen extracts results in activation of transcription of an LF-B1-dependent promoter. The DNA binding domain of LF-B1 is located in the amino-terminal part of the protein and displays distant structural similarity to the homeobox domain. The distribution of LF-B1 mRNA is restricted to liver, which correlates with the tissue-specific expression of its target genes.

Amino Acid Sequence↗