Organophosphate-resistant forms of acetylcholinesterases in two scallops--the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.     

Development of an autotrophic culture system for the in vitro mycorrhization of potato plantlets

An autotrophic culture system was developed for the in vitro mycorrhization of potato plantlets. Roots of micropropagated plantlets were associated to an arbuscular mycorrhizal fungus under in vitro conditions, while shoots developed under open air conditions. Several thousand spores, an extensive extraradical mycelium and an abundant root colonization were obtained. Spores were able to colonize new plantlets under the same conditions. These results support the capacity of the autotrophic culture system to continuously culture arbuscular mycorrhizal fungi and may serve as a powerful tool to investigate various aspects of the symbiosis for which a source-sink relationship and photosynthetic active tissues are necessary.     

The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

Reconciling deep calibration and demographic history: bayesian inference of post glacial colonization patterns in Carcinus aestuarii (Nardo, 1847) and C. maenas (Linnaeus, 1758)

A precise inference of past demographic histories including dating of demographic events using bayesian methods can only be achieved with the use of appropriate molecular rates and evolutionary models. Using a set of 596 mitochondrial cytochrome c oxidase I (COI) sequences of two sister species of European green crabs of the genus Carcinus (C. maenas and C. aestuarii), our study shows how chronologies of past evolutionary events change significantly with the application of revised molecular rates that incorporate biogeographic events for calibration and appropriate demographic priors. A clear signal of demographic expansion was found for both species, dated between 10,000 and 20,000 years ago, which places the expansions events in a time frame following the Last Glacial Maximum (LGM). In the case of C. aestuarii, a population expansion was only inferred for the Adriatic-Ionian, suggestive of a colonization event following the flooding of the Adriatic Sea (18,000 years ago). For C. maenas, the demographic expansion inferred for the continental populations of West and North Europe might result from a northward recolonization from a southern refugium when the ice sheet retreated after the LGM. Collectively, our results highlight the importance of using adequate calibrations and demographic priors in order to avoid considerable overestimates of evolutionary time scales.     

Pneumococcal pili are composed of protofilaments exposing adhesive clusters of Rrg A

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2-3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.     

Chemical–Physical Changes in Cell Membrane Microdomains of Breast Cancer Cells After Omega-3 PUFA Incorporation

Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and progression of breast cancer. However, no clear data are present in literature that could demonstrate how n?-?3 PUFA can interfere with breast cancer growth. It is suggested that these fatty acids might change the structure of cell membrane, especially of lipid rafts. During this study we treated MCF-7 and MDA-MB-231 cells with AA, EPA, and DHA to assess if they are incorporated in lipid raft phospholipids and are able to change chemical and physical properties of these structures. Our data demonstrate that PUFA and their metabolites are inserted with different yield in cell membrane microdomains and are able to alter fatty acid composition without decreasing the total percentage of saturated fatty acids that characterize these structures. In particular in MDA-MB-231 cells, that displays the highest content of Chol and saturated fatty acids, we observed the lowest incorporation of DHA, probably for sterical reasons; nevertheless DHA was able to decrease Chol and SM content. Moreover, PUFA are incorporated in breast cancer lipid rafts with different specificity for the phospholipid moiety, in particular PUFA are incorporated in PI, PS, and PC phospholipids that may be relevant to the formation of PUFA metabolites (prostaglandins, prostacyclins, leukotrienes, resolvines, and protectines) of phospholipids deriving second messengers and signal transduction activation. The bio-physical changes after n?-?3 PUFA incubation have also been highlighted by atomic force microscopy. In particular, for both cell lines the DHA treatment produced a decrease of the lipid rafts in the order of about 20-30?%. It is worth noticing that after DHA incorporation lipid rafts exhibit two different height ranges. In fact, some lipid rafts have a higher height of 6-6.5?nm. In conclusion n?-?3 PUFA are able to modify lipid raft biochemical and biophysical features leading to decrease of breast cancer cell proliferation probably through different mechanisms related to acyl chain length and unsaturation. While EPA may contribute to cell apoptosis mainly through decrease of AA concentration in lipid raft phospholipids, DHA may change the biophysical properties of lipid rafts decreasing the content of cholesterol and probably the distribution of key proteins.     

Ethylene dimerization catalyzed by mixed phosphine–iminophosphorane nickel(II) complexes: a DFT investigation

A computational study utilizing density functional theory (DFT) was performed to analyze the mechanism of ethylene dimerization catalyzed by (P,N) nickel(II) complexes, where (P,N) is a mixed phosphine–iminophosphorane ligand. Two plausible reaction pathways were considered, namely the Cossee and metallacycle pathways, for three model systems. The fundamental role of ligand assymetry and the importance of steric and trans effects were elucidated. In order to discriminate between both mechanisms, the activation of the precatalyst by trimethylaluminum was modeled. The results obtained allow the establishment of useful guidelines for creating new specifically tailored nickel-based catalysts for controlled dimerization.