Multi-laboratory Validation of a New Marine Biodegradation Screening Test for Chemical Persistence Assessment.

Current biodegradation screening tests are not specifically designed for persistence assessment of chemicals, often show high inter- and intra-test variability, and often give false negative biodegradation results. Based on previous studies and recommendations, an international ring test involving 13 laboratories validated a new test method for marine biodegradation with a focus on improving the reliability of screening to determine the environmental degradation potential of chemicals. The new method incorporated increased bacterial cell concentrations to better represent the microbial diversity; a chemical is likely to be exposed in the sampled environments and ran beyond 60 days, which is the half-life threshold for chemical persistence in the marine environment. The new test provided a more reliable and less variable characterization of the biodegradation behavior of five reference chemicals (sodium benzoate, triethanolamine, 4-nitrophenol, anionic polyacrylamide, and pentachlorophenol), with respect to REACH and OSPAR persistence thresholds, than the current OECD 306 test. The proposed new method provides a cost-effective screening test for non-persistence that could streamline chemical regulation and reduce the cost and animal welfare implications of further higher tier testing.

Proteomic sample preparation by microdialysis: easy, speedy, and nonselective.

Microdialysis tools have been developed for parallelized medium exchange designated for sample volumes from 10 to 100 µl, compatible with the microplate format, and guaranteeing maximum recoveries without selectivity. These tools are applicable to both protein and peptide analysis. Moreover, they may be used for binding studies as well as for reconcentration and as unique sample containers for complex operating sequences allowing contemporaneous processing and high throughput.

Functional characterization of a class III acid endochitinase from the traps of the carnivorous pitcher plant genus, Nepenthes.

Carnivory in plants is an adaptation strategy to nutrient-poor environments and soils. Carnivorous plants obtain some additional mineral nutrients by trapping and digesting prey; the genus Nepenthes is helped by its specialized pitcher traps. To make the nutrients available, the caught prey needs to be digested, a process that requires the concerted activity of several hydrolytic enzymes. To identify and investigate the various enzymes involved in this process, fluid from Nepenthes traps has been analysed in detail. In this study, a novel type of Nepenthes endochitinase was identified in the digestion fluid of closed pitchers. The encoding endochitinase genes have been cloned from eight different Nepenthes species. Among these, the deduced amino acid sequence similarity was at least 94.9%. The corresponding cDNA from N. rafflesiana was heterologously expressed, and the purified protein, NrChit1, was biochemically characterized. The enzyme, classified as a class III acid endochitinase belonging to family 18 of the glycoside hydrolases, is secreted into the pitcher fluid very probably due to the presence of an N-terminal signal peptide. Transcriptome analyses using real-time PCR indicated that the presence of prey in the pitcher up-regulates the endochitinase gene not only in the glands, which are responsible for enzyme secretion, but at an even higher level, in the glands' surrounding tissue. These results suggest that in the pitchers' tissues, the endochitinase as well as other proteins from the pitcher fluid might fulfil a different, primary function as pathogenesis-related proteins.

Alamethicin-induced electrical long distance signaling in plants.

Systemic signals induced by wounding and/or pathogen or herbivore attack may be realized by either chemical or mechanical signals. In plants a variety of electrical phenomena have been described and may be considered as signal-transducing events; such as variation potentials (VPs) and action potentials (APs) which propagate over long distances and hence are able to carry information from organ to organ. In addition, we recently described a new type of electrical long-distance signal that propagates systemically, i.e. from leaf to leaf, the 'system potential' (SP). This was possible only by establishing a non-invasive method with micro-electrodes positioned in sub-stomatal cavities of open stomata and recording apoplastic responses. Using this technical approach, we investigated the function of the peptaibole alamethicin (ALA), a channel-forming peptide from Trichoderma viride, which is widely used as agent to induce various physiological and defence responses in eukaryotic cells including plants. Although the ability of ALA to initiate changes in membrane potentials in plants has always been postulated it has never been demonstrated. Here we show that both local and long-distance electrical signals, namely depolarization, can be induced by ALA treatment.

System potentials, a novel electrical long-distance apoplastic signal in plants, induced by wounding.

Systemic signaling was investigated in both a dicot (Vicia faba) and a monocot (Hordeum vulgare) plant. Stimuli were applied to one leaf (S-leaf), and apoplastic responses were monitored on a distant leaf (target; T-leaf) with microelectrodes positioned in substomatal cavities of open stomata. Leaves that had been injured by cutting and to which a variety of cations were subsequently added caused voltage transients at the T-leaf, which are neither action potentials nor variation potentials: with respect to the cell interior, the initial polarity of these voltage transients is hyperpolarizing; they do not obey the all-or-none rule but depend on both the concentration and the type of substance added and propagate at 5 to 10 cm min(-1). This response is thought to be due to the stimulation of the plasma membrane H(+)-ATPase, a notion supported by the action of fusicoccin, which also causes such voltage transients to appear on the T-leaf, whereas orthovanadate prevents their propagation. Moreover, apoplastic ion flux analysis reveals that, in contrast to action or variation potentials, all of the investigated ion movements (Ca(2+), K(+), H(+), and Cl(-)) occur after the voltage change begins. We suggest that these wound-induced "system potentials" represent a new type of electrical long-distance signaling in higher plants.

The coupling ion in the methanoarchaeal ATP synthases: H(+) vs. Na(+) in the A(1)A(o) ATP synthase from the archaeon Methanosarcina mazei Gö1.

To establish a system to analyze ATP synthesis by the archaeal A(1)A(o) ATP synthase and to address the nature of the coupling ion, the operon encoding the A(1)A(o) ATP synthase from the mesophile Methanosarcina mazei Gö1 was cloned in an expression vector and it was expressed in the F(1)F(o) ATP synthase-negative mutant Escherichia coli DK8. Western blot analyses revealed that each of the subunits was produced, and the subunits assembled to a functional, membrane-embedded ATP synthase/ATPase. ATP hydrolysis was inhibited by dicyclohexylcarbodiimide but also by tributyltin, which turned out to be the most efficient inhibitor of the A(o) domain of A(1)A(o) ATP synthase known to date. ATP hydrolysis was not dependent on the Na(+) concentration of the medium, and inhibition of the enzyme by dicyclohexylcarbodiimide could not be relieved by Na(+). The enzyme present in the cytoplasmic membrane of E. coli catalyzed ATP synthesis driven by an artificial DeltapH but not by DeltapNa or DeltamuNa(+).

Oral secretions from herbivorous lepidopteran larvae exhibit ion channel-forming activities.

Feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Various OS-derived molecules must be involved in subsequent processes including the induction of plant defence reactions. Using the planar lipid bilayer membrane technique, isolated OS were analyzed with respect to their membrane activities. Transmembrane ion fluxes were generated by OS of eight different lepidopteran larvae, all of which form comparable ion channels in artificial membranes. Currents were characterized by long lasting open times and conductivities from 250pS up to 1100pS. Channels formed by Spodoptera exigua secretions showed a preference for cations over anions. OS also induced a transient increase of the cytosolic calcium concentration in soybean cells, determined by the aequorin technique. Known compounds of the OS, fatty-acid-glutamine conjugates, also interfered with the membrane but were unable to form stable channels. Since ion fluxes and depolarization are early responses upon insect feeding, OS-derived components may be involved in the elicitation process by direct interaction with the plant membranes.