Biomedical subjects

R Petracca

Publications and source records attributed to R Petracca.

At least 19 recordsLinked to original sources

PubMed2001

CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs.

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

Amino Acid Sequence↗

PubMed2001

Crystallization and preliminary crystallographic studies on the large extracellular domain of human CD81, a tetraspanin receptor for hepatitis C virus.

The large extracellular domain of CD81, a member of the tetraspanin family and a receptor protein for hepatitis C virus envelope E2 glycoprotein, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.6 A resolution were obtained at the ID14 beamline of the European Synchrotron Radiation Facility from a flash-frozen crystal at 100 K. The crystals belong to space group P2(1), with unit-cell parameters a = 31.5, b = 77.2, c = 38.5 A, beta = 107.4 degrees, and are likely to contain two extracellular domains (2 x 99 residues) per asymmetric unit.

Antigens, CD↗

PubMed2000

The comparison of the effect of LTR72 and MF59 adjuvants on mouse humoral response to intranasal immunisation with human papillomavirus type 6b (HPV-6b) virus-like particles.

Infections with genital human papillomaviruses (HPV) are likely to be neutralised more efficiently if a mucosal immune response can be elicited at the viral entry site. Local IgA antibodies are highly induced when antigens are co-administered with mucosal adjuvants, such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) which, however, are not expected to have wide application because of their pronounced toxicity. We have immunised mice intranasally with HPV-6b virus-like particles (VLPs) and a genetically modified LT-derived molecule with only residual toxicity, LTR72, and compared the humoral responses with those obtained following systemic immunisation with VLPs and the MF59 adjuvant. Titration of anti-HPV antibodies in sera and vaginal secretions established that LTR72 was able to elicit higher serum and mucosal IgA titers, in addition to IgG serum levels, comparable to those obtained by parenteral immunisation. These results confirm the potential of toxin-derived adjuvants and extend their use in combination with HPV antigens.

Adjuvants, Immunologic↗

PubMed2000

Influence of single base change in Shine-Dalgarno sequence on the stability of B. subtilis plasmid PSM604.

B. Subtilis expression plasmids generally require a stringent Shine-Dalgarno Sequence (SDS). Site-directed-mutagenesis was explored to change the Shine-Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.

Bacillus subtilis↗

PubMed2000

Structure-function analysis of hepatitis C virus envelope-CD81 binding.

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K(d) of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10(-9) M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.

Amino Acid Sequence↗

PubMed1998

Binding of hepatitis C virus to CD81.

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Amino Acid Sequence↗

PubMed1994

[Use of alpha interferon in microcondylomatosis of the female genitalia].

The Authors report the results of their research into the use of the alpha-interferon in the microcondylomatosis of the female genital apparatus. The therapy was successful in 18% of cases. Considering oncological risk connected with the permanency of the HPV in the uterine portio, the Authors consider they must continue the study on new diagnostic and therapeutic protocols.

Condylomata Acuminata↗

PubMed1993

Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer.

Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma. We report the nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease. The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates. The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene. Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin. An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseases and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease.

Amino Acid Sequence↗

PubMed1993

Detection in an enzyme immunoassay of an immune response to a recombinant fragment of the 128 kilodalton protein (CagA) of Helicobacter pylori.

The possibility of using a recombinant fragment of the CagA (128 kDa protein) for the diagnosis of Helicobacter pylori infection was evaluated. Following cloning of the gene coding for the CagA, a recombinant fragment of it was expressed in Escherichia coli, purified and used in Western blot and an EIA to screen sera from 82 patients with gastroduodenal disease who underwent endoscopic examination. In Western blot, good correlation was found between the serological data obtained with the recombinant antigen and those obtained using non-purified extracts of Helicobacter pylori. The EIA using the antigen showed a sensitivity of 96.2% and a specificity of 96.6% compared with Western blot. These data indicate that the recombinant protein is a reliable antigen for detection of infections with Helicobacter pylori strains that are associated with disease. The EIA assay described may be used in follow-up of the progression of the illness and the results of therapy.

Adult↗

PubMed1993

Use of antibodies against a synthetic peptide of the E6 protein of human papillomavirus (HPV) type 16 for the diagnosis of genital HPV lesions.

The E6 open reading frame of the human papillomavirus (HPV) 16 codes for a protein of 158 amino acids with a theoretical molecular weight of 19.1 kD. A peptide corresponding to 147-158 amino acids (NH2-RSSRTRRETQLC-COOH) was synthesized, coupled to haemocyanin and used for immunization of rabbits. The antibodies obtained were used for the immunohistochemical analysis of a series of 41 paraffin-embedded biopsies including 29 cases of HPV 16-associated cervical lesions, five HPV 6, four HPV 11, and three HPV 18-associated genital warts. Expression of a reacting molecule (both intranuclear and cytoplasmic) could be demonstrated in 28/29 (96.6%) of the HPV 16 lesions, in 2/5 (40%) of the HPV 6, in 2/4 (50%) of the HPV 11, and in 3/3 of HPV 18 lesions. The intensity of the staining was weak in the HPV 6 and HPV 11 lesions, whereas it was classified as intense in 21/29 (72%) of the E6-positive HPV 16 lesions. Negative staining was almost invariably (5/6, 83%) confined to HPV-non-cervical intraepithelial neoplasia (HPV-NCIN) lesions, only 5/18 (27%) of which showed an intense staining. In contrast, intense staining was associated with CIN; 11/12 (92%) in HPV-CIN I and 8/11 (73%) in HPV-CIN II lesions. In the HPV 16 group, intense reactivity was more frequent in lesions shown to make clinical progression (8/9, 89%) as compared with those which underwent spontaneous regression (10/15, 66.7%). Topography of the immunostaining paralleled the localization of HPV DNA hybridization signals in 20/21 (95%) evaluable HPV 16 cases, in contrast to only 1/3 in HPV 18 cases and in none of the HPV 6 and HPV 11 lesions. These differences between HPV 16 and HPV 6/11 lesions in the staining with the HPV 16 E6 protein antibody might help explain at least some of the differences in their known biological behaviour.

Amino Acid Sequence↗

PubMed1992

Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products.

The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.

Amino Acid Sequence↗

PubMed1992

Adenosine receptors on human leukocytes. IV. Characterization of an A1/Ri receptor.

Adenosine (10(-9)-10(-6) mol/l) and R-phenylisopropyladenosine (10(-9)-10(-7) mol/l) partially inhibited the intracellular accumulation of cyclic AMP induced by isoproterenol, prostaglandin E1, histamine and 5'-N-ethylcarboxamidoadenosine in lymphocytes. In contrast, S-phenylisopropyladenosine, which is a poor agonist of the adenosine A1/Ri receptor, had essentially no inhibitory effect. 8-Phenyltheophylline, in low concentrations that do not inhibit cyclic AMP phosphodiesterase, completely blocked the inhibitory effect of R-phenylisopropyladenosine on the increase in cyclic AMP induced by prostaglandin E1. R-Phenylisopropyladenosine (10(-8)-10(-6) mol/l) also inhibited the cyclic AMP accumulation in lymphocytes induced by forskolin (10(-5) mol/l), which activates adenylate cyclase through direct interaction with the enzyme. We also investigated the presence of the adenosine A1/Ri receptor on human polymorphonuclear leukocytes. R-Phenylisopropyladenosine (3 x 10(-9)-10(-7) mol/l) abolished the stimulating effects of prostaglandin and forskolin on cyclic AMP accumulation in polymorphonuclear leukocytes. This effect was blocked by 8-phenyltheophylline and was not observed with the stereoisomer S-phenylisopropyladenosine. The results support the existence of an A1/Ri receptor that regulates cyclic AMP metabolism of human lymphocytes and polymorphonuclear leukocytes.

Adenylyl Cyclases↗

PubMed1990

Adenosine receptors of human leukocytes--II. Characterization of an inhibitory P-site.

We have investigated the effects of 2',5'-dideoxyadenosine (DDA), 9'-beta-D-arabinofuranosyladenine (ARA), and 9-beta-D-xylofuranosyladenine (XFA), which have been classified as P-site adenosine agonists, on the cyclic adenosine 3',5'-monophosphate (cAMP) metabolism of human lymphocytes and polymorphonuclear leukocytes (PMNs). DDA (10(-5)-2 x 10(-4) M), ARA and XFA caused a dose-dependent decrease in cAMP content of human lymphocytes. In addition to decreasing lymphocyte cAMP levels, DDA, ARA, and XFA markedly inhibited the effects of many adenylate cyclase-stimulating agents including beta-adrenergic stimuli, prostaglandin E1 (PGE1), histamine, adenosine, forskolin and cholera toxin. Theophylline and 3-isobutyl-1-methylxanthine, which are known antagonists of adenosine A1/Ri and A2/Ra receptors, did not modify the inhibiting effects of DDA. Mn2+ (1 mM) increased the sensitivity to inhibition of adenylate cyclase agonists by DDA. We also search for the presence of adenosine P-sites in human PMNs. DDA caused a significant decrease of PMN cAMP levels only at the highest concentrations used (2 x 10(-4) M). In contrast, even low concentrations of DDA (10(-6)-10(-4) M) concentration-dependently blocked the stimulatory effect of PGE1 and forskolin on PMN cAMP accumulation. The results support the existence of a purine P-site that regulates cAMP metabolism of human lymphocytes and PMNs.

Adenosine↗

PubMed1990

Topography-related expression of individual cytokeratins in normal and pathological (non-neoplastic and neoplastic) human oral mucosa.

Recently, regional changes of cytokeratin patterns in human normal non-keratinized or keratinized oral mucosa have been demonstrated and the expression of individual cytokeratin polypeptides in lesions of oral mucosa has been compared with that of normal tissues. In particular, the presence of cytokeratin 19 in the suprabasal cell layers of oral epithelia has been shown to be strongly correlated with premalignancy. In the present study, we describe the results of an immunohistochemical investigation performed using a monoclonal antibody specific for cytokeratin 1 on normal oral mucosa and benign or malignant oral lesions. We show the different distribution of this polypeptide in non-neoplastic lesions from different sites of oral mucosa and describe the presence of cytokeratin 19. Our results are in agreement with the data obtained previously. In the malignant cases we demonstrate that the distribution of the two cytokeratins is characterized by complementary patterns.

Carcinoma, Squamous Cell↗

PubMed1990

Expression of cytokeratin No. 19 polypeptide in genital papillomavirus lesions.

A series of 23 punch biopsies proved to contain human papillomavirus (HPV) type 16 and with established clinical course (including HPV-NCIN, HPV-CIN I, and HPV-CIN II lesion), and 18 additional biopsies of HPV 6-, 11-, 16- or 18-induced genital lesions were analyzed immunohistochemically for expression of cytokeratin No. 19 polypeptide. An immunoperoxidase-ABC technique was used with a polyclonal antibody raised against a synthetic nonapeptide corresponding to the residues 2-10 of the NH2-end, non-alpha-helical region. This polyclonal cytokeratin No. 19 antibody stained mainly (but not exclusively) the basal cells of the normal exocervical epithelium (heterogeneous pattern). Basal cell staining was intense slightly more frequently in HPV-CIN than HPV-NCIN lesions, i.e., ++ or more in 14/24 (58.3%) versus 8/17 (47.0%), respectively. The difference was more marked in the staining of the superficial cells, 70.8 and 58.8% showing intense expression of cytokeratin No. 19, respectively. In 6 (21.4%) of the 28 HPV 16 lesions, basal cell layer was intensely stained, as contrasted to none of the 13 HPV 6, 11 or 18 lesions. The most distinct feature was the well-defined granular staining pattern of the superficial layer in 8 out of 10 HPV 6/11 lesions, as contrasted to the homogeneous pattern in 24 out of 28 HPV-16-infected lesions. In superficial cells, regressed lesions exhibited intense staining in 9/13 (69.2%), as compared with only 4/10 (40%) of the progressed lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Cervix Uteri↗

PubMed1990

Immunolocalization of intermediate filament proteins using antibodies to synthetic peptides.

Synthetic peptides corresponding to amino acid sequences of amino terminal non-alpha helical domains of human cytokeratin 18 and to low molecular weight human neurofilament subunit were used to obtain monospecific antisera. The results of our immunohistochemical investigations confirmed in general the data previously published on the distribution of cytokeratin 18 in human, rat, and calf tissues. The reactivity of the antiserum was abolished after formalin fixation of specimens. Immunolocalization of the neurofilament subunit using our monospecific antiserum was quite variable from species to species in cells of the central and peripheral nervous systems, and also varied as the result of the tissue fixation procedures. In particular, formalin fixation destroyed the immunoreactivity of the recognized epitope. We discuss the advantages and limits of the use of synthetic peptides as immunogens to produce polyclonal antibodies against intermediate filament proteins, with particular attention to the epitope masking phenomena in cytokeratin polypeptides and the phosphorylation of epitopes in neurofilament subunits.

Amino Acid Sequence↗

PubMed1990

Immunohistochemical localization of filaggrin in benign and malignant lesions of the human oral mucosa.

Filaggrin is a protein normally present in the granular and horny layer of stratified squamous epithelia. We studied the presence of this protein in 83 benign lesions and in 73 cases of malignant epithelial tumours of the oral cavity and investigated its possible role as an immunohistochemical marker. The immunohistochemical technique was based on the P.A.P. method. The results in benign lesions show a distribution of filaggrin similar to that observed in the normal mucosa. By contrast, an irregular distribution of filaggrin is observed in areas of leukoplakia with parakeratosis and in papillomas. In malignant lesions the expression of this protein is closely related to the degree of differentiation of the cellular elements, being positive in more differentiated and negative in anaplastic areas. Therefore in some types of benign lesions filaggrin testifies an alteration of the normal process of keratinization. Filaggrin is more significant in malignant lesions in which its presence if any permits an evaluation of the degree of differentiation of the tumour.

Carcinoma, Squamous Cell↗

PubMed1988

Altered expression of filaggrin in human papillomavirus (HPV) lesions of the uterine cervix.

A series of 64 punch biopsies collected from women prospectively followed-up for cervical Human papillomavirus (HPV) infections (with and without CIN), and 38 control biopsies (normal epithelia, and classical CIN) were analysed for expression of filaggrin (a histidine-rich protein constituent of keratohyalin granules) using the ABC technique and polyclonal antibody. HPV typing was completed using the in situ hybridization technique with DNA probes for HPV 6, 11, 16, 18 and 31. Three patterns of filaggrin distribution were differentiated: pattern I, all layers above the basal cells stained positive regularly; pattern II, all layers above the basal cells stained irregularly, and pattern III, scattered superficial cells stained positive. There was a significant difference between HPV-noCIN and HPV-CIN lesions in their filaggrin patterns, pattern I being present in the majority (77.7%) of HPV-noCIN lesions, as contrasted to HPV-CIN lesions, where pattern III was the predominant one (43.5%), followed by pattern II (32.6%). In HPV-CIN as well as in CIN lesions, pattern I was inversely related to the grade of CIN, being entirely absent in HPV-CIN III and CIN III. A significant difference exists between CIN and HPV-CIN lesions, concerning the presence of pattern III (4.3% and 43.5%, respectively, P less than 0.001). The difference was less dramatic with regard to pattern I (30.4% and 21.7%, respectively, P less than 0.05). In the lesions containing HPV 6, 11 or 31 DNA, filaggrin distribution was shown to be more close to that of the normal epithelium (I 36.7%, and II 34.7%), while in the HPV 16 and 18-infected cases, pattern III was the predominant one (46.7%). The assessment of filaggrin pattern in HPV lesions might be of help in evaluating the severity of the disturbance of keratinocyte differentiation induced by the progression of HPV infections.

Cervix Uteri↗