Identification and genetic diversity analysis of strain Pseudomonas syringae S5: A pathogen responsible for bacteriosis in Avena sativa plants

The genus Pseudomonas includes pathogenic species P. syringae, which can be found in various agricultural environments and which can affect a wide variety of plants, causing significant economic losses when the environmental conditions for its proliferation are optimal. Comprehensive characterizations of phytopathogenic bacteria belonging to the genus Pseudomonas are scarce in Argentina. In this work, the tabtoxin‐producing strain Pseudomonas S5, isolated from oat, was identi?ed as a P. syringae through biochemical tests such as the LOPAT test, and genetic tests such as the analysis of the small subunit ribosomal RNA gene (16S rRNA) sequence and repetitive elements, using BOX and ERIC primers. It was also determined that this phytopathogen is potentially capable of infecting other crops of agricultural importance for our region, such as soybean. This ability to infect different hosts gives it an adaptive advantage that allows it to endure seasonal changes in the environment where it lives. Our work contributes to the physiological classification of the phytopathogen P. syringae S5 isolated from our region, as well as to the knowledge about its range of potential hosts.     

Prevailingly cationic agmatine-based amphoteric polyamidoamine as a nontoxic, nonhemolytic, and "stealthlike" DNA complexing agent and transfection promoter

AGMA1, a prevailingly cationic amphoteric polyamidoamine obtained by polyaddition of (4-aminobutyl)guanidine (agmatine) to 2,2-bis(acrylamido)acetic acid, was studied as a potential DNA carrier and transfection promoter. Fluorescein-labeled AGMA1 was prepared by conjugation with fluorescein isothiocyanate and its cell uptake, blood permanence, and body distribution studied. In spite of its cationic character, AGMA1 is neither toxic nor hemolytic in the pH range 4.0-7.4, circulates for a long time in the blood without preferentially localizing in the liver, easily enters HT-29 cells, gives stable complexes with DNA, and is endowed with good transfection efficiency, suggesting the ability to transport in the cytoplasm a DNA payload without any measurable membranolytic activity. If compared with other transfection promoters, including polyamidoamines of different structures, AGMA1 is apparently endowed with a unique combination of desirable requirements for a nonviral DNA polymer carrier and warrants potential as a transfection agent in vivo.     

Bimodal loop-loop interactions increase the affinity of RNA aptamers for HIV-1 RNA structures

Multiple loop-loop interactions between adjacent RNA hairpins regulate gene expression in different organisms. To demonstrate that such natural interactions could be mimicked for generating RNA ligands that are able to recognize simultaneously at least two structured RNA targets, a double kissing complex model was designed. The target consisted of two HIV-1 transactivating responsive (TAR) RNA variants, BRU and MAL, connected by a non-nucleotidic linker. The double ligand was generated by combining the corresponding hairpin aptamers, R06BRU and R06MAL, identified previously by in vitro selection [Ducongé, F., and Toulmé, J. J (1999) RNA 5, 1605-1614]. The resulting interaction was analyzed by thermal denaturation monitored by UV spectroscopy, electrophoretic mobility shift assays (EMSAs), and surface plasmon resonance (SPR) experiments. The bimodal complex was characterized by a binding equilibrium constant increased by at least 1 order of magnitude compared to that of the complexes between the individual parent hairpins. This resulted from a slower dissociation rate. We then made use of such a strategy for targeting two structured functional motifs of the folded 5' untranslated region (5'UTR) of HIV-1. Two bivalent RNA ligands were designed that targeted simultaneously the TAR and dimerization initiation site (DIS) hairpins or the TAR and poly(A) ones. The results show that these ligands also displayed enhanced affinity for their target compared to the individual molecules. The work reported here suggests that bimodal structured RNA ligands might provide a way of increasing the affinity of aptamers for folded RNA targets.     

Growth and shape transformations of giant phospholipid vesicles upon interaction with an aqueous oleic acid suspension

The interaction of two types of vesicle systems was investigated: micrometer-sized, giant unilamellar vesicles (GUVs) formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and submicrometer-sized, large unilamellar vesicles (LUVs) formed from oleic acid and oleate, both in a buffered aqueous solution (pH 8.8). Individual POPC GUVs were transferred with a micropipette into a suspension of oleic acid/oleate LUVs, and the shape changes of the GUVs were monitored using optical microscopy. The behavior of POPC GUVs upon transfer into a 0.8 mM suspension of oleic acid, in which oleic acid/oleate forms vesicular bilayer structures, was qualitatively different from the behavior upon transfer into a 0.3 mM suspension of oleic acid/oleate, in which oleic acid/oleate is predominantly present in the form of monomers and possibly non-vesicular aggregates. In both cases, changes in vesicle morphology were observed within tens of seconds after the transfer. After an initial increase of the vesicle cross-section, the vesicle started to evaginate, spawning dozens of satellite vesicles connected to the mother vesicle with narrow necks or tethers. In 60% of the cases of transfer into a 0.8 mM oleic acid suspension, the evagination process reversed and proceeded to the point where the membrane formed invaginations. In some of these cases, several consecutive transitions between invaginated and evaginated shapes were observed. In the remaining 40% of the cases of transfer into the 0.8 mM oleic acid suspension and in all cases of vesicle transfer into the 0.3 mM oleic acid suspension, no invaginations nor subsequent evaginations were observed. An interpretation of the observed vesicle shape transformation on the basis of the bilayer-couple model is proposed, which takes into account uptake of oleic acid/oleate molecules by the POPC vesicles, oleic acid flip-flop processes and transient pore formation.     

Photoreceptor responses of fruitflies with normal and reduced arrestin content studied by simultaneous measurements of visual pigment fluorescence and ERG

We have simultaneously measured the electroretinogram (ERG) and the metarhodopsin content via fluorescence in white-eyed, wild-type Drosophila and the arrestin2 hypomorphic mutant (w ⁻ ;arr2 ³ ) at a range of stimulus wavelengths and intensities. Photoreceptor response amplitude and termination (transition between full repolarization and prolonged depolarizing afterpotential, PDA) were related to visual pigment conversions and arrestin concentration. The data were implemented in a kinetic model of the rhodopsin–arrestin cycle, allowing us to estimate the active metarhodopsin concentration as a function of effective light intensity and arrestin concentration. Arrestin reduction in the mutant modestly increased the light sensitivity and decreased the photoreceptor dynamic range. Compared to the wild type, in the mutant the transition between full repolarization and PDA occurred at a lower metarhodopsin fraction and was more abrupt. We developed a steady-state stochastic model to interpret the dependence of the PDA on effective light intensity and arrestin content and to help deduce the arrestin to rhodopsin ratio from the sensitivity and PDA data. The feasibility of different experimental methods for the estimation of arrestin content from ERG and PDA is discussed.     

Reproductive phenology of the introduced kelp <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Phaeophyceae,Laminariales) in Port Phillip Bay (Victoria,Australia)

A thorough understanding of the reproductive phenology of introduced species is crucial for effective management and control. Undaria pinnatifida is an invasive macroalga from the Northwest Pacific which has been recently introduced into three countries in the Southern Hemisphere: Australia, New Zealand and Argentina. Reproductive phenological studies in Port Phillip Bay, Australia, were undertaken and compared with other populations in the Southern Hemisphere, especially with those from Tasmania which were suspected to be very different. The growth season began earlier in Port Phillip Bay than in Tasmanian populations, and abundance was higher. Growth rates were lower in Port Phillip Bay, but this might be due to the different morphology of both populations. The maximum spore release competency of U. pinnatifida in Port Phillip Bay was 12.1 × 10⁵ spores cm⁻² h⁻¹ which is 20 times the maximum obtained in Tasmania (0.6 × 10⁵ spores cm⁻² h⁻¹). For most of the growth season, spore release competency ranged between 2 and 3 × 10⁵ spores cm⁻² h⁻¹, 3–5 times more than in Tasmania. Undaria pinnatifida has not been established outside Port Phillip Bay in continental Australia, but a precautionary approach should be undertaken in order to avoid further spread. Monitoring for early detection and removal of immature sporophytes prior to spore release seem to be the best options. This monitoring should be continuous since new recruits may appear throughout the growth season (April–February) and it should be combined with informative programmes to reduce the chances of spread.     

Carrot DNA‐methyltransferase is encoded by two classes of genes with differing patterns of expression

In the present study, the isolation and characterization of two distinct cDNAs that code for carrot DNA (cytosine-5)-methyltransferase (DNA-METase) are reported. The screening of a cDNA library with a carrot genomic DNA fragment, previously obtained by PCR using degenerate primers, has led to the isolation of clones that belong to two distinct classes of genes (Met1 and Met2) which differ in sequence and size. Met1–5 and Met2–21 derived amino acid sequences are more than 85% identical for most of the polypeptide and completely diverge at the N-terminus. The larger size of the Met2–21 cDNA is due to the presence of nearly perfect fivefold repeat of a 171 bp sequence present only once in the Met1–5 cDNA. Northern and in situ hybridization analyses with young carrot plants and somatic embryos indicate that both genes are maximally expressed in proliferating cells (suspension cells, meristems and leaf primordia), but differ quantitatively and spatially in their mode of expression. Polyclonal antibodies were raised in rabbit using fusion proteins corresponding to the regulatory and catalytic regions of the most highly expressed gene (Met1–5). In nuclear carrot extracts, both antibodies were found to recognize a band of about 200 kDa along with some additional bands of lower size. These results provide the first direct demonstration that DNA-METases of a higher eukaryote are encoded by a gene family.     

Nitric oxide promotes strong cytotoxicity of phenolic compounds against Escherichia coli: the influence of antioxidant defenses

The induction of mutagenic and cytotoxic effects by simple phenolics, including catechol (CAT), 3,4-dihydroxyphenylacetic acid (DOPAC), hydroquinone (HQ), and 2,5-dihydroxyphenylacetic (homogentisic) acid (HGA), appears to occur through an oxidative mechanism based on the ability of these compounds to undergo autoxidation, leading to quinone formation with the production of reactive oxygen species. This is supported by the detection of such adverse effects in plate assays using Escherichia coli tester strains deficient in the OxyR function, but not in OxyR(+) strains. The OxyR protein is a redox-sensitive regulator of genes encoding antioxidant enzymes including catalase and alkyl hydroperoxide reductase, which would eliminate hydrogen peroxide. Methyl-substituted phenolics such as 4-methylcatechol (MCAT) and methylhydroquinone (MHQ) produced, in addition to oxidative toxicity, marked cytotoxic effects against OxyR(+) cells, thus revealing a mechanism of toxicity not mediated by hydrogen peroxide that could involve quinones and quinone methides arising from MCAT and MHQ oxidation. Quinone compounds could also be responsible for the enhanced cytotoxicity of certain phenolics when combined with a nitric oxide (NO(*)) donor such as diethylamine/NO (DEA/NO). Phenolics scavenge NO(*) and, in turn, NO(*) oxidizes phenolics to form their quinone derivatives. In OxyR(+) cells, where the oxidative toxicity is inhibited, DEA/NO promoted exceptional increases in the cytotoxicity of CAT and 3,4-dihydroxycinnamic (caffeic) acid (CAF), which both exhibited very low oxidative cytotoxicity, as well as in that of MCAT, HQ, and MHQ. In contrast, DEA/NO failed to promote toxicity by DOPAC and HGA, probably due to their ability to undergo oxidative polymerization, leading to the formation of melanins. Spectroscopic studies demonstrated quinone generation from the oxidation of CAF, HQ, and MHQ by DEA/NO. The o-quinone derived from CAF was rather unstable and decomposed during its isolation. For the generation of toxic quinones, e.g., to be used as therapeutic agents producing antitumor or antibacterial effects, the isolation step could be avoided with the method proposed. It combines quinone precursors, i.e. phenolic compounds, with an oxidant such as NO(*).     

Hydrolases in supercritical CO<Subscript>2</Subscript> and their use in a high-pressure membrane reactor

The thermal stability and activity of enzymes in supercritical carbon dioxide (SC CO(2)) and near-critical propane were studied at a pressure of 300 bar in the temperature range 20-90 degrees C. Proteinase from Carica papaya was incubated in microaqueous SC CO(2) at atmospheric pressure in a nonaqueous system. Lipase stability in an aqueous medium at atmospheric pressure and in SC CO(2) as well as near-critical propane at 100 bar and 40 degrees C was studied. In order to investigate the impact of solvent on lipases, these were chosen from different sources: Pseudomonas fluorescences, Rhizpous javanicus, Rhizopus niveus and porcine pancreas. On the basis of our previous study on lipase activities in dense gases, a high-pressure continuous flat-shape membrane reactor was designed. The hydrolysis of sunflower oil in SC CO(2) was performed as a model reaction in this reactor. The reaction was catalyzed by the lipase preparation Lipolase 100T and was performed at 50 degrees C and 200 bar.