Healthcare professionals and patient information: a fresh look from the new italian law on consent.

ABSTRACT: This article discusses the importance of providing patients with adequate information and creating the care relationship, particularly focusing on the role of healthcare professionals in this activity. It examines the main legislative references on this topic at a European level and the new law, No. 219/2017, in Italy on informed consent that serves as a starting point for recalling important legal and ethical principles regarding the information to be provided to patients in the therapeutic relationship.

HCV genotype 3 and squamous cell carcinoma antigen (SCCA)-IgM are independently associated with histological features of NASH in HCV-infected patients.

Nonalcoholic steatohepatitis (NASH) enhances the risk of progressive liver disease. In chronic hepatitis C (CHC), liver steatosis is frequent, especially in genotype 3, but its clinical significance is debated. As squamous cell carcinoma antigen (SCCA)-IgM has been associated with advanced liver disease and risk of tumour development, we evaluated its occurrence in CHC and the possible relation with NASH at liver biopsy. Using a validated ELISA, serum SCCA-IgM was measured in 91 patients with CHC at the time of liver biopsy performed before antiviral treatment, at the end of treatment and 6 months thereafter, and in 93 HCV-negative patients with histological diagnosis of nonalcoholic fatty liver disease, as controls. SCCA-IgM was detected in 33% of CHC patients and in 4% of controls. This biomarker was found more elevated in CHC patients with histological NASH, and at multivariate analysis, SCCA-IgM and HCV genotype 3 were independently associated with NASH [OR (95% CI): 6.94 (1.21-40) and 27.02 (4.44-166.6)]. As predictors of NASH, HCV genotype 3 and SCCA-IgM had a specificity and a sensitivity of 97% and 44%, and of 95% and 27%, respectively. PPV and NPV were 80% and 86% for HCV genotype 3 vs 73% and 72% for SCCA-IgM. In patients with sustained virologic response to therapy, SCCA-IgM levels decreased significantly, while these remained unchanged in nonresponders. In conclusion, SCCA-IgM is detectable in one-third of patients with CHC and significantly correlates with histological NASH.

[Immunity and cancer: the role of PSA IgM Immune complexes for prostate cancer]. / Immunità e cancro: ruolo degli immunocomplessi PSA-IGM nel cancro della prostata.

Immunoediting is a new concept in cancer surveillance. Immunity is involved in detecting cellular waste, and taking off transformed cells. In particular, natural IgM antibodies play an important role in immunosurveillance mechanisms against transformed cells in humans.? Scientific evidence indicates that biomarkers for different types of cancer, such as liver and colorectal cancer, circulate in blood associated with immunoglobulin M (IgM) to form complexes that improve diagnosis in comparison to circulating free biomarkers. In prostate cancer it has been demonstrated that testing for serum levels of the PSA-IgM immune complex improves the diagnostic performance of total PSA. Preliminary reports indicate that the combination of PSA-IgM with total PSA is the best approach to reduce the number of negative prostatic mapping thus improving the diagnosis of prostate cancer.

Structural characterization of a Protein A mimetic peptide dendrimer bound to human IgG.

Understanding the chemical physical properties of protein binding sites is at the basis of the rational design of protein ligands. The hinge region of the Fc fragment of immunoglobulin G is an important and well characterized protein binding site, known to interact with several natural proteins and synthetic ligands. Here, we report structural evidence that a Staphylococcus aureus Protein A mimetic peptide dendrimer, deduced by a combinatorial approach, binds close to the Cgamma2/Cgamma3 interface of the constant fragment of a human IgG1 molecule, partially hindering the Protein A binding site. The X-ray analysis evidenced a primary binding site located between a terminal Arg residue of the ligand peptidic arm and a hydrophobic protein site consisting of Val308, Leu309, and His310. A molecular dynamic analysis of the model derived from the X-ray structure showed that in water at room temperature the complex is further stabilized by the formation of at least one more contact between a terminal Arg residue of the second arm of the peptide and the carboxylic group of a protein amino acid, such as Glu318, Asp312, or Asp280. It appears thus that stability of the Fc-dendrimer complex is determined by the synergetic formation of multiple bonds of different nature between the dendrimer arms and the protein accessible sites. The electrostatic and van der Waals energies of the complex were monitored during the MD simulations and confirmed the energetic stability of the two interactions.

[Psychoactive drugs and fitness to drive in the occupational field]. / Sostanze voluttuarie e idoneità alla guida in ambito lavorativo.

According to/After the conferences taken place on 30th October and on 18th December 2008, the company doctor functions have been enlarged: at present they include the duty of effectuating toxicological tests in order to find psychotropic and drug substances in some specific working classes, mainly in workers employed in public transports. Company doctors have the duty to report any possible positive matching in these workers'tests. From there, regulations in force are analyzed focusing to critical situations described in the complex and well articulated frame of reference.

Squamous cell carcinoma antigen-1 (SERPINB3) polymorphism in chronic liver disease.

BACKGROUND: The serpin squamous cell carcinoma antigen (SCCA, SERPINB3) has been found over-expressed in primary liver cancer and at lower extent in cirrhosis and chronic hepatitis. A novel SCCA-1 variant (SCCA-PD), presenting a single mutation in the reactive centre (Gly351Ala), has been recently identified (rs3180227). AIM: To explore SCCA-1 polymorphism in patients with HCV infection as single etiologic factor and different extent of liver disease. METHODS: One hundred and fourty-eight patients with chronic HCV infection (45 chronic hepatitis, 53 cirrhosis, 50 HCC) and 50 controls were evaluated. SCCA-1 polymorphism was studied by restriction fragment length polymorphism and confirmed randomly by direct sequencing. Circulating SCCA-IgM complex was determined by ELISA. RESULTS: SCCA-PD was detected with higher frequency in cirrhotic patients (45.3%, odds ratio=2.62; 95%CI 1.13-6.10, p=0.038) than in patients with chronic hepatitis or in controls (24.4% and 24%, respectively). Intermediate figures were found in hepatocarcinoma (36.0%). SCCA-IgM in serum was lower in patients carrying SCCA-PD than in wild type patients and the difference was statistically significant in cirrhotic patients (mean+/-S.D.=117.45+/-54.45 U/ml vs. 268.52+/-341.27 U/ml, p=0.026). CONCLUSIONS: The newly identified SCCA-PD variant was more frequently found in liver cirrhosis, suggesting that patients carrying this polymorphism are more prone to develop progressive liver fibrosis.

Tumour-specific induction of immune complexes: DCP-IgM in hepatocellular carcinoma.

BACKGROUND: In the sera of liver, colorectal and prostate cancer patients, several biomarkers may be detected as IgM immune complexes. To determine whether the presence of immune complexes was correlated to an increase of IgMs, we measured the IgM content in the sera of patients with hepatocellular carcinoma (HCC) and cirrhosis, and evaluated the occurrence of des-gamma-carboxy prothrombin (DCP) as immune complexes (DCP-IgM) compared to the levels of DCP and alpha-fetoprotein (AFP). PATIENTS AND METHODS: Serum samples from 31 patients with cirrhosis, 33 untreated HCC patients diagnosed by ultrasound, computed tomography and/or magnetic resonance and confirmed by histopathology, when indicated, and 30 healthy controls were analysed. Concentrations of IgM and DCP-IgM were determined by ELISAs. RESULTS: Circulating IgM in patients with HCC (median level = 1.79 mg mL(-1)) and cirrhosis (1.09 mg mL(-1)) were not significantly different (P = 0.1376) while DCP-IgM were significantly higher in HCC patients (median level = 2171.2 AU mL(-1)) than in those with cirrhosis (1152 AU mL(-1), P = 0.0047). No correlation was found between DCP-IgM and IgM in HCC (r = 0.227) and cirrhosis patients (r = 0.475). DPC-IgM was positive in 55% (18/33) of HCC patients and in 26% (8/31) of cirrhosis patients compared to 39% and 26% for DCP and 48% and 13% for AFP. DCP-IgM, DCP and AFP tests had 100% specificity in healthy controls. CONCLUSIONS: DCP-IgM in HCC patients was not associated with an increase in IgM concentration. DCP-IgM was more frequently detected in HCC patients than DCP and AFP, strengthening the diagnostic role of IgM immune complexes for liver cancer.

Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis.

About 30% of the patients with chronic hepatitis develop a progressive liver disease and one of the most intriguing issues is the detection of noninvasive markers for fibrosis stage and disease progression. High levels of squamous cell carcinoma antigen (SCCA)-immunoglobulin M (IgM) are detectable in hepatocellular carcinoma and their increase in cirrhotic patients can predict tumour development. As SCCA-IgM can also be detectable at low percentages in patients with chronic hepatitis, the aim of this study was to assess SCCA-IgM complexes in relation to disease outcome in this group of patients. An ELISA assay was used to determine the presence of SCCA-IgM in 188 patients with chronic hepatitis and in 100 controls. An additional serum sample was available after a median period of 6 years in 57 untreated patients: these patients were subdivided in group A, including eight patients with a fibrosis score increase > or =2 in a second liver biopsy and group B, including 49 patients without fibrosis progression during a similar follow up. SCCA-IgM complexes were detectable in 63 of 188 (33%) patients but in none of the controls. A significant increase of SCCA-IgM levels over time was observed in patients with fibrosis progression (mean +/- SD: 117 +/- 200 U/mL/year), but not in those without histologic deterioration (mean +/- SD: -8.8 +/- 31 U/mL/year, P < 0.0001). In conclusion, monitoring SCCA-IgM levels over time appears a useful approach to identify patients with chronic hepatitis at higher risk for cirrhosis development.

Expression pattern of squamous cell carcinoma antigen in oesophageal dysplasia and squamous cell carcinoma.

The aim of the present study was to evaluate the tissue expression of squamous cell carcinoma antigen (SCCA) in oesophageal dysplasia and squamous cell carcinoma (SCC) with reference to its clinico-pathologic and prognostic significance. Immunohistochemistry using SCCA polyclonal antibody was performed on SCCs from 61 surgical oesophagectomies. Fifteen cases of low-grade dysplasia (LGD) and 37 non-coexistent high-grade dysplasia (HGD) were also sampled from these materials, together with sixteen chronic cases of oesophagitis. SCCA immunoreactivity was present in the maturative compartments of all normal epithelia and oesophagitis. LGDs showed no SCCA immunoreactivity in the dysplastic proliferative component but only in the superficial normal layers. In 94.6% of HGDs, no SCCA immunoreactivity was detected throughout the thickness of the epithelium. In SCCs, SCCA expression higher than 25% was found in 54% of cases. SCCA positivity showed an inverse correlation with histological grade, whereas no statistically significant correlation was found with TNM classifications, stage, or survival. SCCA is not expressed in early oesophageal carcinogenesis but, in SCC, it represents an indicator of histologic differentiation. In differentiated SCC, SCCA may represent a negative factor for cancer invasiveness, through inhibition of proteases.

Radiographic evaluation of teeth subjected to high temperatures: experimental study to aid identification processes.

The radiographic evaluation of dental remains represents a significant aspect in the forensic identification process, particularly after an exposure to fire. The aim of this "in vitro" study was to evaluate the radiographic features of unrestored, endodontically treated and restored teeth after exposure to an experimental range of high temperatures. Ninety human teeth were divided into two groups: (1) unrestored teeth, as a control group and (2) teeth endodontically treated (condensation technique) and restored with amalgam or composite fillings. Before testing the high temperatures, periapical radiographs of all teeth were performed. The tests of exposure to heat were carried out in an oven for six different temperatures (200, 400, 600, 800, 1000 and 1100 degrees C (392, 752, 1112, 1472, 1832, 2012 degrees F)). After each exposure, periapical radiographs of all the teeth were taken. The radiographic appearance of all the teeth before and after the thermal stresses were evaluated and the differences were recorded. The results of the radiographic examination showed that a number of significant radiographic details were conserved: the composite fillings were in place maintaining the shape till 600 degrees C (1112 degrees F), the amalgam fillings were in place maintaining the shape till 1000 degrees C (1832 degrees F) and the endodontic treatments were recognisable till 1100 degrees C (2012 degrees F).

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

Detection of circulating CEA-IgM complexes in early stage colorectal cancer.

We have recently shown that alpha fetoprotein (AFP) and squamous cell carcinoma antigen (SCCA), biomarkers associated with hepatocellular carcinoma, may be detected in patient sera as circulating immune complexes with IgM, and that assessment of serum levels of AFP-IgM and SCCA-IgM may be used for the detection of liver cancer. In this study we measured the levels of carcinoembryonic antigen (CEA) as free form (FCEA) and complexed to IgMs (CEA-IgM) in sera of patients affected by colorectal carcinoma (CRC) at different stages as well as in healthy subjects. FCEA levels were above the 5 ng/mL cutoff in 43% of CRC patients (31/72) and CEA-IgM levels were above the 200 AU/mL cutoff in 38% of CRC patients (27/72). Serum levels of CEA-IgM immune complexes (IC) and FCEA did not overlap and 64% of patients (46/72) were positive for at least one marker without compromising the detection specificity (94%). Early detection of CRC was significantly improved by CEA-IgM IC assay. CRC patients at an early stage (stage 1) had elevated CEA-IgM levels in 29% of cases (7/24), while FCEA levels were elevated in only 8% of cases (2/24). These results indicate that CEA-IgM is a complementary serological marker to FCEA which is much more sensitive for early stage CRC, and that the combination of these biomarkers may be useful in the early detection of colorectal cancer.

Improvement of liver cancer detection with simultaneous assessment of circulating levels of free alpha-fetoprotein (AFP) and AFP-IgM complexes.

We assessed the presence of alpha-fetoprotein (AFP) complexed with IgM (AFP-IgM IC) in serum of patients affected by hepatocellular carcinoma (HCC), cirrhosis and chronic hepatitis as well as in healthy subjects by means of a dedicated ELISA assay. The amount of AFP-IgM IC was expressed in arbitrary units (AU) on a reference standard curve. Free AFP (FAFP) levels were determined in parallel in each sample by means of an automated immunoassay system. The mean serum concentration of AFP-IgM IC was significantly higher in HCC patients (mean +/- SD: 1378.3 +/- 2935.7 AU/mL) than in cirrhotic patients (129.8 +/- 261.4 AU/mL) and in patients with chronic hepatitis (80.9 +/- 168.9 AU/mL) (p < 0.01). HCC patients had FAFP values above the 20 ng/mL cutoff in 44% of cases (22/50) and AFP-IgM IC values above the 120 AU/mL cutoff in 60% of cases (30/50). The occurrence of the free and IgM-complexed form of circulating AFP did not overlap, and 82% of patients (41/50) were positive for at least one marker. The results indicate that AFP-IgM IC is a complementary serological marker to FAFP and that the combination of these biomarkers may be useful in the diagnosis of liver cancer.

Surface expression of squamous cell carcinoma antigen (SCCA) can be increased by the preS1(21-47) sequence of hepatitis B virus.

A variant of the serpin squamous cell carcinoma antigen (SCCA) has been identified as a hepatitis B virus binding protein and high expression of SCCA has recently been found in hepatocarcinoma. Since HBV is involved in liver carcinogenesis, experiments were carried out to examine the effect of HBV preS1 envelope protein on SCCA expression. Surface and intracellular staining for SCCA was assessed by FACS analysis. Preincubation of HepG2 cells and primary human hepatocytes with preS1 protein or with preS1(21-47) tetrameric peptide significantly increased the surface expression of SCCA, without modification of its overall cellular burden, suggesting a surface redistribution of the serpin. An increase in HBV binding and internalization was observed after pre-incubation of the cells with preS1 preparations, compared to cells preincubated with medium alone. Pretreatment of cells with DMSO, while not influencing SCCA basal expression, was responsible for an increase in the efficiency of HBV internalization and this effect was additive to that obtained after incubation with preS1 preparations. In conclusion, the HBV preS1(21-47) sequence is able to induce overexpression of SCCA at the cell surface facilitating virus internalization, while the increased efficiency of HBV entry following DMSO addition is not mediated by SCCA.

Overexpression of squamous cell carcinoma antigen variants in hepatocellular carcinoma.

Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (scoreor=2 (mean+/-s.d.: 2%+/-2.4 vs 7.5%+/-10.3, P<0.05). Squamous cell carcinoma antigen mRNA could be directly sequenced in 14 out of 18 liver tumours but in none of the corresponding nontumour samples. From sequence alignment, a novel SCCA1 variant (G(351) to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours.

Further study of restored and un-restored teeth subjected to high temperatures.

Forensic dentistry has been shown to be of fundamental importance in medico-legal investigations aimed at identifying human remains involving high temperature incidents because dental remains and prosthetic devices are resistant to quite high thermal change. In this project we studied teeth containing class I and V amalgam and composite fillings and compared them to un-restored teeth when exposed to high temperatures. Twenty five un-restored teeth, 25 teeth with class I amalgam restorations, 25 teeth with class V amalgam restorations and 25 teeth with class I composite fillings were placed in a furnace and heated at a rate of 30 degrees C/min. The effects at the predetermined temperatures 200, 400, 600, 800, 1000 and 1100 degrees C were examined macroscopically and then observed microscopically by means of a stereomicroscope. Our observations showed that the class I amalgam restorations at the different temperature levels remained in place, maintaining their shape despite disintegration of the crowns, whilst the class I composite restorations remained in place but in an altered shape. Comparing restored with un-restored teeth we observed different responses in crown disintegration at the different temperature levels.

Observations on dental prostheses and restorations subjected to high temperatures: experimental studies to aid identification processes.

In large scale disasters associated with fire the damage caused by heat can make medico-legal identification of human remains difficult. Teeth, restorations and prostheses, all of which are resistant to even quite high temperatures can be used as aids in the identification process. In this project the behaviour and morphology of teeth and dental prostheses exposed to a range of high temperatures was studied. Healthy teeth, dental restorations and prostheses were placed in a furnace and heated at a rate of 30 degrees C/min and the effects of the predetermined temperatures 200, 400, 600, 800, 1000 and 1100 degrees C were examined by stereomicroscopy and scanning electron microscopy (SEM). Our observations show that some prostheses and restorative materials resist higher temperatures than theoretically predictable and that even when a restoration is lost because of detachment or change of state its ante-mortem presence can be confirmed and detected by both stereomicroscopic examination and SEM of the residual cavity. We further conclude that a reasonably reliable estimation of the temperature of exposure can be made from an analysis of the teeth and restorative materials.

Novel ligands for the affinity-chromatographic purification of antibodies.

Affinity chromatography represents one of the most powerful fractionation techniques for the large-scale purification of biotechnological products. Despite its potential, the use of this methodology is limited by the availability of specific ligands for each target. Combinatorial chemistry and molecular modeling, often combined, have become interesting and innovative methods for generating novel ligands, tailored to specific biotechnological needs. One of the greatest area of application has been the discovery of novel ligands for the purification of antibodies, which represent an emerging but very important class of innovative therapeutic agents for the treatment of a vast array of diseases. Naturally available affinity ligands, such as Protein A or G for IgG purification or lectins for IgA and IgM purification, which are obtained from microorganisms or genetically modified bacteria through complex and expensive procedures, are not well suited for large-scale purification and require moreover time-consuming analytical controls to check for the presence of contaminants which may affect the safety of the purified antibody for clinical purposes. Recent results suggest that the application of combinatorial technologies and molecular modeling for the discovery of synthetic ligands may open new avenues for the development of more efficient, less expensive and--more importantly--safer procedures for antibody purification at the industrial level.

Cloning and expression of a novel hepatitis B virus-binding protein from HepG2 cells.

A direct involvement of the hepatitis B virus (HBV) preS1-(21-47) sequence in virus attachment to cell membrane receptor(s) and the presence on the plasma membranes of HepG2 cells of protein(s) with receptor activity for HBV have been suggested by many previous experiments. In this study, by using a tetravalent derivative of the preS1-(21-47) sequence, we have isolated by affinity chromatography from detergent-solubilized HepG2 plasma membranes a 44-kDa protein (HBV-binding protein; HBV-BP), which was found to closely correspond to the human squamous cell carcinoma antigen 1 (SCCA1), a member of the ovalbumin family of serine protease inhibitors. Comparison of SCCA1 sequence with the sequence of the corresponding HBV-BP cDNA, cloned by polymerase chain reaction starting from RNA poly(A)(+) fractions extracted from HepG2 cells, indicated the presence of only four nucleotide substitutions in the coding region, leading to three amino acid changes. Intact recombinant HBV-BP lacked inhibitory activity for serine proteases such as alpha-chymotrypsin and trypsin but inhibited with high potency cysteine proteases such as papain and cathepsin L. Direct binding experiments confirmed the interaction of recombinant HBV-BP with the HBV preS1 domain. HepG2 cells overexpressing HBV-BP after transfection of corresponding cDNA showed a virus binding capacity increased by 2 orders of magnitude compared with untransfected cells, while Chinese hamster ovary cells, which normally do not bind to HBV, acquired susceptibility to HBV binding after transfection. Native HBV particle entry was enhanced in transfected cells. Both recombinant HBV-BP and antibodies to recombinant HBV-BP blocked virus binding and internalization in transfected cells as well as in primary human hepatocytes in a dose-dependent manner. Our findings suggest that this protein plays a major role in HBV infection.

N-terminal myristylation of HBV preS1 domain enhances receptor recognition.

The N-terminal portion of the large envelope protein of the human hepatitis B virus (HBV), the preS1 domain, plays a fundamental role in cell attachment and infectivity. Recent investigations have suggested that myristylation of preS1 Gly2 residue is essential for viral infectivity, but the importance of this post-translational modification on HBV-receptor interaction has not been elucidated completely. In this study we produced, using stepwise solid-phase chemical synthesis, the entire preS1[1-119] domain (adw2 subtype), and compared its receptor binding activity with the myristylated form, myristyl-preS1[2-119] in order to define the importance of fatty acid modification. Both synthetic proteins were fully characterized in terms of structural identity using TOF-MALDI mass spectrometry and analysis of tryptic fragments. Circular dichroism measurements indicated a low content of ordered structure in the preS1 protein, while the propensity of the myristylated derivative to assume a conformationally defined structure was more evident. HBV-receptor binding assays performed with plasma membranes preparations from the hepatocyte carcinoma cell line HepG2 clearly showed that the preS1[1-119] domain recognizes the HBV receptor, and confirmed that binding is occurring through the 21-47 region. The myristylated derivative recognized HBV receptor preparations with higher affinity than the preS1 domain, suggesting that the conformational transitions induced in the preS1 moiety by fatty acid post-translational modification are important for efficient attachment of viral particles to HBV receptors.