Control of biofilm formation by poly-ethylene-co-vinyl acetate films incorporating nisin

The aim of this study was to evaluate the effect of poly-ethylene-co-vinyl acetate (EVA) films incorporating different concentrations (0.1%, 0.5% and 1%) of nisin on the biofilm-forming ability of Listeria monocytogenes ATCC 7644, Staphylococcus aureus 815 and Staphylococcus epidermidis ATCC 35984. Nisin was incorporated into two grades of EVA (EVA14 and EVA28) in the melt during a common film-blowing operation. The efficacy of EVA/nisin films was evaluated by biofilm biomass measurements and Live/Dead staining in combination with fluorescence microscopy. In order to evaluate whether the nisin incorporation could modify the film surface properties, contact angle measurements and scanning electron microscopy were performed. The results revealed the efficacy of EVA14/nisin films in reducing biofilm formation on their surfaces with more evident effect for S. epidermidis than L. monocytogenes and S. aureus strains. In contrast, EVA28/nisin films showed unsatisfactory activity. Fluorescence microscopy confirmed poor biofilm formation on EVA14/nisin films, also characterised by the presence of dead cells. The data presented in this study offer new potential applications for developing strategies aimed to improve the effect of antimicrobial agents.     

Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1�CE2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single‐ and double‐knockout mutations and RNA‐interference (RNAi)‐silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non‐viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma‐related 1 (RBR1)–E2FB complex and this is partly mediated by its N‐terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F‐dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient‐limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.     

Promotion of human adipose‐derived stem cell proliferation mediated by exogenous nucleosides

Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose‐derived stem cells), with or without co‐treatment with 5‐aza (5‐azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5‐aza induces a dose‐dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 μM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5‐Aza‐induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.     

Molecular and phylogenetic characterization of the sieve element occlusion gene family in <Emphasis Type="Italic">Fabaceae</Emphasis> and non-<Emphasis Type="Italic">Fabaceae</Emphasis>plants

Background  

The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the Fabaceae. Recently, the molecular characterization of three genes encoding forisome components in the model legume Medicago truncatula (MtSEO1, MtSEO2 and MtSEO3; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-Fabaceae.     

First report of Lutzomyia araracuarensis (Morales & Minter) (Diptera: Psychodidae) in Brazil

A male of Lutzomyia araracuarensis (Morales & Minter) and possibly six females of this same species were found in the northeastern area of Manacapuru county, Amazonas State. Samples were collected using light traps CDC, from April 2003 to June 2004.     

Density and structure of Saissetia oleae (Hemiptera: Coccidae) populations on citrus and olives: relative importance of the two annual generations

Saissetia oleae (Olivier) (Hemiptera: Coccidae) populations were studied and compared in citrus (Citrus spp.) and olive (Olea europaea L.) groves to determine the number of generations, crawler emergence periods and changes in population density during the year. Ten citrus and four olive groves were sampled regularly between March 2003 and December 2005 in eastern Spain, covering an area of 10,000 km2. Each sample consisted of 16 branches and 64 leaves. Saissetia oleae populations presented a similar trend in both crops during the three years of study. Populations peaked in July, when crawlers emerged after the egg-laying period, and decreased during several months due to mortality of first instars in summer. A second crawler emergence period, with lower numbers and more variability from year to year, occurred between October and March. Populations did not increase during this period, probably because most eggs and crawlers perished during the winter and also because females that gave rise to this fall-winter generation were half as big and fecund as spring females. No differences were found between the size of mature females that had developed on citrus and on olives during the spring. Considering this population pattern, the best seasonal period to apply pesticides to control S. oleae would be at the end of July, when populations are synchronous, all crawlers have already emerged, and first instars predominate.     

A large conformational change couples the ATP binding site of SecA to the SecY protein channel

In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magnesium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this conformationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel.     

Borna disease virus matrix protein is an integral component of the viral ribonucleoprotein complex that does not interfere with polymerase activity

We have recently shown that the matrix protein M of Borna disease virus (BDV) copurifies with the affinity-purified nucleoprotein (N) from BDV-infected cells, suggesting that M is an integral component of the viral ribonucleoprotein complex (RNP). However, further studies were hampered by the lack of appropriate tools. Here we generated an M-specific rabbit polyclonal antiserum to investigate the intracellular distribution of M as well as its colocalization with other viral proteins in BDV-infected cells. Immunofluorescence analysis revealed that M is located both in the cytoplasm and in nuclear punctate structures typical for BDV infection. Colocalization studies indicated an association of M with nucleocapsid proteins in these nuclear punctate structures. In situ hybridization analysis revealed that M also colocalizes with the viral genome, implying that M associates directly with viral RNPs. Biochemical studies demonstrated that M binds specifically to the phosphoprotein P but not to N. Binding of M to P involves the N terminus of P and is independent of the ability of P to oligomerize. Surprisingly, despite P-M complex formation, BDV polymerase activity was not inhibited but rather slightly elevated by M, as revealed in a minireplicon assay. Thus, unlike M proteins of other negative-strand RNA viruses, BDV-M seems to be an integral component of the RNPs without interfering with the viral polymerase activity. We propose that this unique feature of BDV-M is a prerequisite for the establishment of BDV persistence.     

Neutrophil effector functions triggered by Fc-gamma and/or complement receptors are dependent on B-ring hydroxylation pattern and physicochemical properties of flavonols

Tissue damage in autoimmune diseases involves excessive production of reactive oxygen species (ROS) triggered by immune complexes (IC) and neutrophil (PMN) interactions via receptors for the Fc portion of IgG (FcgammaR) and complement receptors (CR). Modulation of both the effector potential of these receptors and ROS generation may be relevant to the maintenance of body homeostasis. In the present study, the modulatory effect of four flavonols (myricetin, quercetin, kaempferol, galangin) on rabbit PMN oxidative metabolism, specifically stimulated via FcgammaR, CR or both classes of receptors, was evaluated by luminol- and lucigenin-dependent chemiluminescence assays. Results showed that flavonol inhibitory effect was not dependent on the cell membrane receptor class stimulated but related to the lipophilicity of the compounds (their apparent partition coefficient values were obtained by high-performance liquid chromatography), and was also inversely related to the number of hydroxyl groups in the flavonol B ring and the ROS-scavenger activity (assessed by the luminol--H2O2--horseradish peroxidase reaction). Under the experimental conditions the flavonols tested were not toxic to PMNs (evaluated by lactate dehydrogenase release and trypan blue exclusion) and did not interfere with IC-induced phagocytosis (evaluated by transmission electron microscopy). Our results suggested that inhibition of IC-stimulated PMNs effector functions by the flavonols tested herein was the result of cooperation of different cellular mechanisms.