Monosialoganglioside-GM1 triggers binding of the amyloid-protein salmon calcitonin to a Langmuir membrane model mimicking the occurrence of lipid-rafts

GM1 ganglioside is known to be involved in the amyloid-associated diseases and it is a crucial factor for the assembly of amyloid proteins on lipid-rafts, which are lipid structures located on the synaptic plasma membranes. Due to its slow aggregation rate, we employed salmon calcitonin (sCT) as a suitable probe representative of amyloid proteins, to study the interaction between this class of proteins and a membrane model. Here, we prepared a neuronal membrane model by depositing onto mica two Langmuir-Blodgett films in liquid-condensed phase: the outer monolayer was characterized by high content of GM1 (50%) and minority parts of cholesterol and POPC (25–25%), while the inner one by plain POPC. To deeply investigate the interaction of sCT with this model and the role-played by GM1, we prepared the outer leaflet adding sCT at a concentration such that the number of proteins equals that of GM1. Atomic Force Microscopy revealed the occurrence of two distinct kinds of flat surfaces, with globular aggregates localized exclusively on top of the highest one. To unravel the nature of the interaction, we studied by ζ-potential technique liposomes composed as the outer leaflet of the model. Results demonstrated that an electrostatic interaction sCT-GM1 occurred. Finally, to investigate the interaction thermodynamics between sCT and the outer leaflet, Langmuir films as the outer monolayer and containing increasing content of sCT were studied by compression isotherms and Brewster Angle Microscopy experiments. Based on the all body of results we propose an interaction model where GM1 plays a pivotal role.     

Functional Characterization of drim2, the Drosophila melanogaster Homolog of the Yeast Mitochondrial Deoxynucleotide Transporter

The CG18317 gene (drim2) is the Drosophila melanogaster homolog of the Saccharomyces cerevisiae Rim2 gene, which encodes a pyrimidine (deoxy)nucleotide carrier. Here, we tested if the drim2 gene also encodes for a deoxynucleotide transporter in the fruit fly. The protein was localized to mitochondria. Drosophila S2R⁺ cells, silenced for drim2 expression, contained markedly reduced pools of both purine and pyrimidine dNTPs in mitochondria, whereas cytosolic pools were unaffected. In vivo drim2 homozygous knock-out was lethal at the larval stage, preceded by the following: (i) impaired locomotor behavior; (ii) decreased rates of oxygen consumption, and (iii) depletion of mtDNA. We conclude that the Drosophila mitochondrial carrier dRIM2 transports all DNA precursors and is essential to maintain mitochondrial function.     

Molecular phylogenetics and taxonomic revision of Habenaria section Pentadactylae (Orchidaceae,Orchidinae)

As a step towards a revision of the sectional classification of Neotropical species of Habenaria, we focus here on section Pentadactylae. In its current delimitation, this is the largest of the 14 New World sections and embraces a group of 34 morphologically heterogeneous species. We expanded the sampling of Neotropical species currently placed in this section and performed Bayesian, maximum likelihood and parsimony analyses using nucleotide sequences from one nuclear (internal transcribed spacer, ITS) and three plastid (matK, trnK intron, rps16–trnK) DNA regions. In addition, morphological features of these species were reassessed. Based on our analyses, we propose that Habenaria section Pentadactylae should be recircumscribed to include only seven species: H. pentadactyla (the type species of the section), H. dutrae, H. ekmaniana, H. exaltata, H. henscheniana, H. megapotamensis and H. montevidensis. Thirty‐two species previously assigned to the section grouped within unrelated clades and are therefore excluded from the section. There are no unambiguous morphological synapomorphies for the section, but the group can be confidently recircumscribed and identified on the basis of a combination of diagnostic morphological vegetative and floral characters. Morphological floral features in Habenaria montevidensis are distinct from those of other species in the section, probably as a result of a shift to diurnal pollinators. Following a taxonomic revision of the group, H. crassipes is placed under the synonymy of H. exaltata and neotypes are designated for H. crassipes, H. montevidensis and H. recta (= H. ekmaniana). All species in the section live in marshes or wet grasslands from northern Argentina to central Brazil; most species are concentrated in southern Brazil. Most species are probably rare, and five may be threatened according to the World Conservation Union (IUCN) criteria. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 47–73.     

Role of the feline immunodeficiency virus L-domain in the presence or absence of Gag processing: involvement of ubiquitin and Nedd4-2s ligase in viral egress

RNA-enveloped viruses bud from infected cells by exploiting the multivesicular body (MVB) pathway. In this context, ubiquitination of structural viral proteins and their direct interaction with cellular factors involved in the MVB biogenesis through short proline rich regions, named late domains (L-domains), are crucial mechanisms. Here we report that, in contrast with the human immunodeficiency virus (HIV), the feline immunodeficiency virus (FIV), a non-primate lentivirus, is strictly dependent for its budding on a "PSAP"-type L-domain, mapping in the carboxy-terminal region of Gag, irrespective of a functional viral protease. Moreover, we provide evidence that FIV egress is related to Gag ubiquitination, that is, linked to the presence of an active L-domain. Finally, although FIV Gag does not contain a PPxY motif, we show that the Nedd4-2s ubiquitin ligase enhances FIV Gag ubiquitination and it is capable to rescue viral mutants lacking a functional L-domain. In conclusion, our data bring to light peculiar aspects of FIV egress, but we also demonstrate that a non-primate lentivirus shares with HIV-1 a novel mechanism of connection to the cellular budding machinery.     

News and views from the 8th annual meeting of the Italian Society of Virology

The 8th annual meeting of the Italian Society of Virology (SIV) took place in Orvieto, Italy from the 21st to the 23rd of September 2008. The meeting covered different areas of Virology and the scientific sessions focused on: general virology and viral genetics; viral oncology, virus–host interaction and pathogenesis; emerging viruses and zoonotic, foodborne and environmental pathways of transmission; viral immunology and vaccines; viral biotechnologies and gene therapy; medical virology and antiviral therapy. The meeting had an attendance of about 160 virologists from all Italy. In this edition, a satellite workshop on “Viral biotechnologies” was organized in order to promote the role of virologists in the biotechnological research and teaching fields. A summary of the plenary lectures and oral selected presentations is reported. J. Cell. Physiol. 219: 797–799, 2009. © 2009 Wiley‐Liss, Inc.     

Antiphospholipid reactivity against cardiolipin metabolites occurring during endothelial cell apoptosis

We have recently shown that cardiolipin (CL) and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. In this study, we investigate the immunoreactivity to CL derivatives occurring during endothelial apoptosis in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). We compared the serum immunoreactivity to CL with that of its derivatives monolysocardiolipin (MCL), dilysocardiolipin (DCL), and hydrocardiolipin (HCL) by means of both enzyme-linked immunosorbent assay and thin-layer chromatography (TLC) immunostaining. In addition, we investigated the composition of phospholipid extracts from the plasma membrane of apoptotic endothelial cells and the binding of patients' sera to the surface of the same cells by using high-performance TLC and immunofluorescence analysis. The average reactivity to MCL was comparable with that of CL and significantly higher than that for DCL and HCL in patients studied, both in the presence or in the absence of beta2-glycoprotein I. Of relevance for the pathogenic role of these autoantibodies, immunoglobulin G from patients' sera showed an increased focal reactivity with the plasma membrane of endothelial cells undergoing apoptosis. Interestingly, the phospholipid analysis of these light membrane fractions showed an accumulation of both CL and MCL. Our results demonstrated that a critical number of acyl chains in CL derivatives is important for the binding of antiphospholipid antibodies and that MCL is an antigenic target with immunoreactivity comparable with CL in APS and SLE. Our finding also suggests a link between apoptotic perturbation of CL metabolism and the production of these antibodies.     

Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all enzymes retained their initial activities.     

Semi-synthetic and synthetic 1,2,4-trioxaquines and 1,2,4-trioxolaquines: synthesis,preliminary SAR and comparison with acridine endoperoxide conjugates

A novel series of semi-synthetic trioxaquines and synthetic trioxolaquines were prepared, in moderate to good yields. Antimalarial activity was evaluated against both the chloroquine-sensitive 3D7 and resistant K1 strain of Plasmodium falciparum and both series of compounds were shown to be active in the low nanomolar range. For comparison the corresponding 9-amino acridine analogues were also prepared and shown to have low nanomolar activity like their quinoline counterparts.