Proceedings of the fourth National Congress of the Italian Society of Virology

The aim of the yearly National Congress of the Italian Society of Virology (SIV) is to promote the discussion between senior and younger researchers to improve the knowledge and scientific collaboration among the various areas of Virology. The invited and selected lecturers of the fourth National Congress of SIV covered the following topics: general Virology and viral Genetics; virus host interactions and pathogenesis; viral immunology and vaccines; emerging and re-emerging viral diseases; antiviral therapy; innovative diagnostics; viral biotechnologies and gene therapy. As in the previous edition (Salata and Palù, 2004 J Cell Physiol 199:171-173), a specific topic was thoroughly covered in a roundtable. In this edition the overviewed topic was HCV, from epidemiology and genetic variability to immunology and antiviral therapy. The final program can be found at the web site http://www.siv-virologia.it. A summary of the oral presentations of the 2004 meeting is reported.     

Mitochondrial diversity in linguistic isolates of the Alps: a reappraisal

In Stenico et al. (1996) we reported unusually high levels of mitochondrial diversity in the Alps. In particular, two communities of Ladin speakers appeared the most extreme European mitochondrial outliers at that time. Recently, it has been observed that some rare nucleotide substitutions occur repeatedly among those sequences, raising the possibility of systematic sequencing errors. No biological material was left from the previous study, and hence we had to sample new individuals from the same communities. Here, we present the HVSI sequence variation, along with haplogroup assignment based on restriction fragment length polymorphism (RFLP), in 20 Ladin speakers of Colle Santa Lucia. None of the new sequences displays substitutions at the sites viewed as problematic. However, Ladins still show high levels of mtDNA diversity, both within their community and with respect to other Europeans, and they can still be considered one of the main European mitochondrial outliers.     

PKA independent and cell type specific activation of the expression of caudal homeobox gene Cdx-2 by cyclic AMP

Cdx-2 is a transactivator for the proglucagon gene in pancreatic and intestinal endocrine cells. Cdx-2 is also expressed in differentiated intestinal epithelia of nonendocrine origin. Cdx-2-/- mice are embryonic lethal, while Cdx-2+/- mutants show multiple malfunctions including the formation of intestinal polyps. Within the polyps, the remaining wild type Cdx-2 allele ceases its expression, while the expression of both Cdx-2 and proglucagon in the endocrine cells remains unaltered, indicating that Cdx-2 could be haplo-insufficient for nonendocrine cells, but not for proglucagon producing endocrine cells. We propose that mechanisms underlying Cdx-2 expression and auto-regulation [Xu F, Li H & Jin T (1999), J Biol Chem274, 34310-34316] differ in these two types of cells. We show here that forskolin and cAMP upregulate Cdx-2 expression in proglucagon producing cells, but not in colon cancer cells and primary intestinal cell cultures. It is unlikely that the activation is mainly mediated by PKA, because the activation was observed in a PKA deficient cell line. Co-transfecting a dominant negative Ras expression plasmid substantially repressed the Cdx-2 promoter, in contrast to a previous finding that Ras is a negative factor for Cdx-2 expression in colon cancer cells. Furthermore, forskolin activated ERK1/2 phosphorylation in the endocrine cells, and attenuation of ERK1/2 phosphorylation by its inhibitor is associated with attenuated Cdx-2 expression. Finally, an Epac pathway specific cAMP analogue stimulated both ERK1/2 phosphorylation and Cdx-2 expression. Taken together, our observations suggest that Cdx-2 expression is regulated by the second messenger cAMP, cell-type specifically, via the Epac pathway.     

ELF magnetic field exposure in a neonatal intensive care unit

In this paper, the magnetic flux density (MFD) distribution in a neonatal intensive care unit is described and MFD values inside a few open infant warming systems and incubators are reported. Typical measured values of the magnetic flux density at power frequency (50 Hz) in the "general environment" (the rooms of the unit) were lower than 0.2 microT, while higher MFD values were detected close to medical equipment and inside the open infant warming systems. In both cases, the magnetic flux density quickly decreases with increasing distance, so that measured values are reduced to "background" (i.e., general environment) levels 20-30 cm away from the sources. The total harmonic content over the 100-800 Hz frequency range was also evaluated. In the general environment, measured values in this band were negligible, while this was not the case close to medical equipment. Field levels inside the open and closed incubators depend on the position of the electronic control system, of the heating power generator and its winding conductor, and of the 220 V main plug. The magnetic flux density was also monitored for a prolonged period of time in a few types of open infant warming systems and incubators under standard intensive care unit operation with premature newborn present.     

Kinetics of Histidine Dissociation From the Heme Fe(III) in N-fragment (residues 1–56) of Cytochrome <Emphasis Type="Italic">c</Emphasis>

We have here investigated the dissociation kinetics of the His side chains axially ligated to the heme-iron in the ferric (1–56 residues) N-fragment of horse cyt c. The ligand deligation induced by acidic pH-jump occurs as a biexponential process with different pre-exponential factors, consistent with a structural heterogeneity in solution and the presence of two differently coordinated species. In analogy with GuHCl-denatured cyt c, our data indicate the presence in solution of two ferric forms of the N-fragment characterized by bis-His coordination, as summarized in the following scheme: His18–Fe(III)–His26 \rightleftharpoons His18–Fe(III)–His33. We have found that the pre-exponential factors depend on the extent of the pH-jump. This may be correlated with the different pK_a values shown by His26 and His33; due to steric factors, His26 binds to the heme–Fe(III) less strongly than His33, as recently shown by studies on denatured cyt c. Interestingly, the two lifetimes are affected by temperature but not by the extent of the pH-jump. The lower pK_a for the deligation reaction required the use of an improved laser pH-jump setup, capable of inducing changes in H⁺ concentration as large as 1 mM after the end of the laser pulse. For the ferric N-fragment, close activation entropy values have been determined for the two histidines coordinated to the iron; this result significantly differs from that for GuHCl-denatured cyt c, where largely different values of activation entropy were calculated. This underlines the role played by the missing segment (residues 57–104) peptide chain in discriminating deligation of the nonnative His from the sixth coordination position of the metal.     

Antioxidant activity of phenolic and related compounds: a density functional theory study on the O-H bond dissociation enthalpy

We report here on calculations at the hybrid DFT/HF (B3-LYP/6-31G(d, p)) level of the O-H bond dissociation enthalpy (O-H BDE) of phenylpropenoic acids (caffeic, ferulic, p-coumaric and cinnamic) and phenolic acids and related compounds (gallic, methylgallate, vanillic and gentisic) in order to gain insight into the understanding of structure-antioxidant activity relationships. The results were correlated and discussed mainly on the basis of experimental data in a companion work (Galato D, Giacomelli C, Ckless K, Susin MF, Vale RMR, Spinelli A. Antioxidant capacity of phenolic and related compounds: correlation among electrochemical, visible spectroscopy methods and structure-antioxidant activity. Redox Report 2001; 6: 243-250). The O-H BDE values showed remarkable dependence on the hydroxyl position in the benzene ring and the existence of additional interaction due to hydrogen bonding. For parent molecules, the experimental antioxidant activity (AA) order was properly obeyed only when intramolecular hydrogen bonding was present in the radicalized structures of o-dihydroxyl moieties. In structurally related compounds, excellent correlation with experimental data was in general observed (0.64 < rho < 0.99). However, it is shown that excellent correlation can also be obtained for this series of compounds considering p-radicalized structures which were not stabilized by intramolecular hydrogen bonding, but this had no physical meaning. These findings suggested that the antioxidant activity evaluation of phenolic and related compounds must take into consideration the characteristics of each particular compound.     

Testing the vibrational exciton and the local mode models on the instructive cases of dicarvone,dipinocarvone, and dimenthol vibrational circular dichroism spectra

The vibrational circular dichroism (VCD) spectra of dicarvone ( 1 ), dipinocarvone ( 2 ), and dimenthol ( 3 ) have been recorded in the range 900–3200 cm⁻¹, encompassing the mid-infrared (mid-IR), the CO stretching, and the CH-stretching regions. For compound 3 also, the fundamental and the first overtone OH stretching regions have been investigated by IR/NIR absorption and VCD. Density functional theory (DFT) calculations allow one to interpret the IR and VCD spectra and to confirm the configuration/conformational studies previously conducted by X-ray diffraction. The most intense VCD signals are associated with the vibrational normal modes involving symmetry-related groups close to the CC bond connecting covalently the two molecular units. The vibrational exciton (VCDEC) model is fruitfully tested on the VCD data of compounds 1 and 2 for the spectroscopic regions at ~1700 cm⁻¹, and the local mode model is tested on compound 3 at ~3500 and ~6500 cm⁻¹. For compounds 1 and 2 also, ECD spectra are reported, and the exciton mechanism is tested also there, and connections to the VCDEC model are examined.     

Living on the edge: The use of fruit-feeding butterflies to evaluate edge effect on subtropical assemblages

This study evaluated how the edge effect influences the structuration of fruit-feeding butterfly assemblages in swamp forest fragments of the subtropical Atlantic Forest, Southern Brazil. Sampling was carried out twice in 10 fragments using baited traps placed in sampling units both at the forest edge and 50 m within the forest interior, with the habitats being defined by a set of environmental variables. Richness and abundance were higher for edge habitats with an effect of temperature depending on humidity and luminosity. The subfamily/tribe composition of fruit-feeding butterflies was segregated between edge and interior and was predicted by wind speed and the interaction between humidity and luminosity. Fifty meters within the forest interior is not sufficient to cause homogenization of butterfly composition between the edge and interior of swamp forest fragments, indicating distinct assemblages in each habitat. The interior harboured forest-loving butterfly groups while the edge harboured generalist sun-loving and common butterflies associated with disturbed areas, suggesting resistance to the effects of habitat fragmentation. We highlight the importance of using fruit-feeding butterfly groups, instead of species, to evaluate edge effects. We also suggest that a heterogeneous matrix with native habitats and distinct semi-natural land-use systems be maintained to manage subtropical areas by increasing connectivity within the landscape. Considering the impacts that the Atlantic Forest suffers, increased knowledge of modifications caused at small and regional scales is crucial for the maintenance of ecological processes and represents a tool for conservation planning and environmental agendas.     

Differing modes of interaction between monomeric Aβ(1-40) peptides and model lipid membranes: an AFM study

Membrane interactions with β-amyloid peptides are implicated in the pathology of Alzheimer's disease and cholesterol has been shown to be key modulator of this interaction, yet little is known about the mechanism of this interaction. Using atomic force microscopy, we investigated the interaction of monomeric Aβ(1-40) peptides with planar mica-supported bilayers composed of DOPC and DPPC containing varying concentrations of cholesterol. We show that below the bilayer melting temperature, Aβ monomers adsorb to, and assemble on, the surface of DPPC bilayers to form layers that grow laterally and normal to the bilayer plane. Above the bilayer melting temperature, we observe protofibril formation. In contrast, in DOPC bilayers, Aβ monomers exhibit a detergent-like action, forming defects in the bilayer structure. The kinetics of both modes of interaction significantly increases with increasing membrane cholesterol content. We conclude that the mode and rate of the interaction of Aβ monomers with lipid bilayers are strongly dependent on lipid composition, phase state and cholesterol content.