Human papillomavirus-16 E7 interacts with Siva-1 and modulates apoptosis in HaCaT human immortalized keratinocytes.

The viral factor E7 plays a key role in the well-established association between "high-risk" Human Papillomavirus (HPV) infection and the development of epithelial malignant tumors, as uterine cervix and ano-genital cancer. To delve into the molecular mechanisms of HPV-mediated cell transformation, we searched for novel potential cellular targets of the HPV-16 E7 oncoprotein, by means of the yeast two-hybrid technique, identifying a protein-protein interaction between HPV-16 E7 and the pro-apoptotic cellular factor Siva-1. Using co-precipitation assays and the "PepSets" technique, we confirmed this physical interaction and mapped accurately, for both proteins, the amino acid residues involved.

Che-1 phosphorylation by ATM/ATR and Chk2 kinases activates p53 transcription and the G2/M checkpoint.

Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage.

Oligopeptides impairing the Myc-Max heterodimerization inhibit lung cancer cell proliferation by reducing Myc transcriptional activity.

Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression and a worse clinical outcome of cancer patients. The transcription factor Myc functions by heterodimerizing with its partner, Max. As a strategy to inhibit Myc activity, we have synthesized three small peptides corresponding to segments of the leucine zipper (LZ) region of Max.

Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates.

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines.

A biochemical approach for detecting interactions between peptides from the HIV gp120 glycoprotein and a CD4 sequence.

Peptides selected from the HIV viral protein gp120 bind to a synthetic peptide mimicking sequence 78-89 of the human lymphocyte CD4 molecule, linked to activated Sepharose. The binding of viral fragments to the CD4 peptide-Sepharose beads was ascertained either by aid of a ninhydrin reagent or by fluorescence microscopy. A suitable alignment of these HIV peptides with the CD4 fragment showed that multiple interactions might occur between hydrophobic or charged groups of the two molecules.

Responses of peptide-specific T cells to stimulation with polystyrene beads carrying HLA class I molecules loaded with single peptides.

Cell-sized microbeads carrying single peptide-loaded HLA class I molecules were prepared for HLA-A2 and HLA-B7 by a simple procedure which transfers single peptide-loaded HLA class I molecules from cultured cells to polystyrene beads using anti-peptide antibodies directed to an intracellular segment of HLA-A alpha chains. The surface density of peptide-loaded HLA class I molecules on beads was comparable to that on the peptide-loaded cells. HLA-A2 beads loaded with an HCV peptide HCV1073 were tested for stimulation activity on an HCV1073-specific CD8+ T cell clone NS3-1.

Increased resistance of peptides to serum proteases by modification of their amino groups.

The ability of synthetic protein fragments to survive the degradative action of aminopeptidases and serum proteolytic enzymes can be remarkably enhanced by slight modifications at their N-terminal alpha-amino group. This can be achieved by addition of beta-alanine or amino acids of the D-configuration, amino acids which are seldom found in a living organism. These modifications do scarcely modify the chemical and physical properties of the peptides, and should be preferred, especially for in vivo tests, to drastic alterations of peptides as produced by dinitrophenylation or dansylation of the amino groups.

Cross-reactivity between the major Parietaria allergen and rotavirus VP4 protein.

Background: The present study investigates immunological cross-reactivity between Par o 1, the major pollen allergen of Parietaria, and the VP4 protein of rotavirus, a microorganism that is world-wide the main etiological agent of gastroenteritis in children.

Methods: IgG and IgE cross-reactivity was assessed by direct binding and competitive inhibition assays (ELISA and DARIA), using recombinant VP4 from rhesus infectious rotavirus (RR), synthetic peptides and Par o 1-specific antibodies affinity purified from pooled and individual human sera.

Results: Antibodies specifically binding Par o 1, affinity purified from the sera of 35 individuals with skin test positivity to Parietaria and from 14 pools, were extensively cross-reactive with RRVP4.

Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb.

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo.

Anti-peptide antibodies that recognize conformational differences of HLA class I intracytoplasmic domains.

Rabbit antibodies were raised against both long and short peptides derived from exon 7 sequences of human leukocyte antigen (HLA) class I alpha chains; anti-A/B against a 13-mer shared by most HLA-A alpha and HLA-B alpha chains, anti-C against a 15-mer characteristic of HLA-C alpha chains, anti-ACT against a 6-mer specific to HLA-A alpha chains, and anti-CCT against a 5-mer specific to HLA-C alpha chains. Binding activity of the antibodies was determined with peptides by enzyme-linked immunoabsorbent assay (ELISA) and with HLA class I transfectants and the parental cells by FACS analysis. Anti-A/B and anti-C were to a greater or lesser extent crossreactive with the long and short peptides, whereas anti-ACT and anti-CCT were specific to the corresponding short peptides.

T cell response to N-formylated peptides in humans.

We present the first evidence of a T lymphocyte response to N-formylated peptides in humans. N-formylated peptide sequences from self (mitochondrial) and foreign (microbial) antigens were used to isolate antigen-specific T cell clones from healthy individuals, including a set of monozygotic twins. The observed response differed from that previously described in mouse (CD4(+) phenotype and MHC class II restriction in humans vs.

Allele- and temperature-dependency of in vitro HLA class I assembly.

Allelic variations of in vitro HLA class I assembly have been investigated in both the absence and the presence of binding peptides by flow cytometry using human leukocyte antigen (HLA) class I alpha chains isolated by alkali treatment from cultured HLA homozygous B cells and polystyrene beads coated with anti-HLA class I alpha chain antibodies specific to the C-terminal segment (anti-HLA class I beads). The specificity of assembly was temperature dependent, while the stability of the assembled complex depended on the bound peptide. The efficiency of assembly was allele dependent and primarily ruled by the binding affinity of alpha chains with beta(2)m.

Elongation factor 1 beta/delta of Echinococcus granulosus and allergic manifestations in human cystic echinococcosis.

Allergic reactions, such as urticaria, itching and anaphylactic shock, often complicate the course of cystic echinococcosis (CE). To investigate the role of the IgE-immunoreactive recombinant Echinococcus granulosus elongation factor-1 beta/delta (EgEF-1 beta/delta) in the allergic disorders during CE we determined humoral and cell-mediated responses to this antigen in patients with CE grouped according to the clinical presence or absence of allergic reactions. Immunoblotting analysis showed that serum IgE-binding reactivity to EgEF-1 beta/delta differed significantly in patients with and without allergic reactions (38 of 42, 90% vs.

Polyclonal Antibodies Against gp185HER2 Peptides: Their Putative Role in the Identification of a Particular HER2 Status in Patients With Breast Cancer.

SUMMARY: The HER2 oncogene and its relative oncoprotein, gp185HER2, a transmembrane glycoprotein belonging to the epidermal growth factor receptor family, are overexpressed in a wide range of solid tumors including breast and ovarian cancer. In patients with breast cancer, both humoral and cell-mediated HER2 immune responses have been found as well as in some patients with gp185HER2 nonoverexpressing tumors. To establish whether peptide sequences identified as HLA-A2-restricted T-cell epitopes are expressed in breast tumor cell lines and tissues, we produced and characterized by different methodologic approaches polyclonal antibodies raised against four gp185HER2 peptides.

Polyclonal antibodies against gp185HER2 peptides: their putative role in the identification of a particular HER2 status in patients with breast cancer.

The HER2 oncogene and its relative oncoprotein, gp185HER2, a transmembrane glycoprotein belonging to the epidermal growth factor receptor family, are overexpressed in a wide range of solid tumors including breast and ovarian cancer. In patients with breast cancer, both humoral and cell-mediated HER2 immune responses have been found as well as in some patients with gp185HER2 nonoverexpressing tumors. To establish whether peptide sequences identified as HLA-A2-restricted T-cell epitopes are expressed in breast tumor cell lines and tissues, we produced and characterized by different methodologic approaches polyclonal antibodies raised against four gp185HER2 peptides.

Short synthetic peptides derived from viral proteins compete with HIV gp120 for the binding to CD4 receptors.

In the complex mechanism of adhesion, internalization, and infection of cells by human immunodeficiency virus (HIV) viral particles, a determinant role is played by the viral envelope glycoprotein gp120, which binds to CD4 receptors of T cells and monocytes. We tested the ability of a panel of 7- to 12-residue synthetic peptides, selected from the region 414-434 of the HIV-1 gp120, to inhibit the binding of the viral protein to CD4 receptors of cultured human lymphoid cells. The assay was based on the observation that the binding of gp120 to the receptors interferes with the binding of a specific anti-CD4 monoclonal antibody, as a result of the masking of the antibody epitope; thus, we tested whether preincubation of cells with the peptides before gp120 addition might restore the recognition of the CD4 molecule by the antibody.

Identification of a LFA-1 region involved in the HIV-1-induced syncytia formation through phage-display technology.

We have identified a peptide region on CD18 molecule (the beta subunit of the LFA-1 molecule) involved in syncytia formation of HIV-1-infected lymphocytes. Several phage clones mimicking an epitope of the CD18 cell-surface determinant were isolated from two 9-mer random peptide phage-displayed libraries via their binding to the CD18-specific monoclonal antibody (mAb) MHM23, which in in vitro assay inhibits syncytia formation in HIV-1-infected cells. The peptide sequences displayed on phages that blocked immunolabeling of this mAb on LFA-1-expressing cells were used to identify the epitope recognized by mAb MHM23 by sequence comparison.

Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry.

Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-microm polystyrene beads through the Fc portion by the use of protein A.

Global immune disregulation in multiple sclerosis: from the adaptive response to the innate immunity.

Increasing evidences show a global immune disregulation in multiple sclerosis (MS). The possible involvement of myelin and non-myelin (auto-)antigens in the autoaggressive process as well as the disregulation of both adaptive and innate immunity challenge the concept of specific immunotherapy. T cells at the boundary between innate and adaptive immunity, whose immunoregulatory role is becoming increasingly clear, have recently been shown to bear relevance for MS pathogenesis.

Selective 'in synthesis' labeling of peptides with biotin and rhodamine.

A new method is described for the selective 'in synthesis' labeling of peptides by rhodamine or biotin at a single, predetermined epsilon-amino group of a lysine residue. The alpha-amino group and other lysyl residues of the peptide remain unmodified. Peptides are assembled by the Fmoc approach, which requires mild operative conditions for the final deprotection and cleavage, and ensures little damage of the reporter group.

Flexibility of amino acid residues at position four of nonapeptides enhances their binding to human leucocyte antigen (HLA) molecules.

The binding affinity of synthetic nonapeptides to human leucocyte antigens (HLA) molecules of the A0201 allotype, the most common in Caucasian, is enhanced or reduced by suitable amino acid substitutions at position 4, as a result of increased or decreased chain flexibility. A higher flexibility of the bond at this position correlates with an easier accommodation of the fragment into the HLA groove, while rigidity of the peptide chain appears to interfere. These data are based on two lines of evidence: a) most natural high affinity ligands for HLA-A0201 possess, at position 4, flexible residues b) substitutions of such residues by rigid amino acids results in a decrease of binding affinity.

Possible role of the plasminogen receptor as a site of interaction of the human immunodeficiency virus p24 immunosuppressive heptapeptide Ch7 with the host immune system.

Previous work from our laboratory demonstrated that a synthetic heptapeptide (Ch7: RGSDIAG), corresponding to a conserved sequence of human immunodeficiency virus (HIV) core protein p24 (amino acids 232- 238), was able to specifically abrogate antigen-induced responses in cultures of normal human peripheral blood mononuclear cells (PBMC), probably acting at the level of monocytes. The Ch7 peptide displays sequence homology to human plasminogen. In the present report we show that a compound (6-aminoexanoic acid), known to prevent plasminogen binding to monocyte-like cells, greatly reduced the immunosuppressive capacity of Ch7.

Characterization of a dodecapeptide containing a dominant epitope of Par j 1 and Par o 1, the major allergens of P. judaica and P. officinalis pollen.

The pollen of Parietaria, a weed of the Urticaceae family, is a major cause of respiratory allergy in Europe, where the most common species are P. judaica and P. officinalis.

Epitope mapping of the monoclonal antibody MM12.10 to external MDR1 P-glycoprotein domain by synthetic peptide scanning and phage display technologies.

Epitope mapping of MDR1-P-glycoprotein using specific monoclonal antibodies (mAbs) may help in delineating P-glycoprotein topology and hence in elucidating the relationship between its structural organization and drug-efflux pump function. In this work, by using synthetic peptide scanning and phage display technologies, the binding sites of the mAb MM12.10, a novel antibody to intact human multidrug resistant (MDR) cells, were studied.

Isolation, characterization and comparison of antipeptide and antiprotein rabbit antibodies to the pi-isoform of glutathione S-transferase.

The main linear epitopes of pi-glutathione transferase (pi-GST, EC 2.5.1.