Cholinesterase activity and effects of its inhibition by neurotoxic drugs in Dictyostelium discoideum

Recently, we found acetylcholinesterase (AChE) activity in the ciliate protozoan Paramecium primaurelia. As in the slime mould Dictyostelium discoideum the presence of a serine esterase was found with strong sequence identity to Torpedo AChE, we extended to D. discoideum the investigation on the characterization and possible functions of cholinesterases (ChEs). In amoeboid cells, histochemical, biochemical, and electrophoresis analyses evidenced both a ChE activity able to hydrolyze the substrate PrTChI, and AChE (E.C. 3.1.1.7.) activity similar to Electrophorus electricus AChE. Conversely, butyrylcholinesterase activity was nearly absent, according to our previous results on P. primaurelia. Moreover, the possibility to utilize D. discoideum in a bioassay for the pre-chemical screening both of moist environments and fresh waters, in relation to the occurrence of the neurotoxic organophosphate drugs, such as "basudin", inhibiting ChE activity, was investigated. Exposure to basudin inhibited propionylcholinesterase (PrChE) activity in a dose-dependent manner in the range 10(-1)-10(-7) M (60% at 10(-4) M), without any significant effect on AChE activity. PrChE activity was inhibited slightly by 10(-5) M eserine, and reduced significantly both by 10(-5) M iso-OMPA and BW284C51, classically used to discriminate the different ChE molecular forms. The effects on cell morphology, cell density, and differentiation were evaluated in cultures exposed to PrTChI 10(-5) M or basudin 10(-4) M for a three-day period. The PrTChI-exposed sample exhibited cell morphology, cell density, ability to aggregate, and to form fruiting bodies similar to the control; whereas, the basudin-exposed sample showed anomalies in cell morphology and lower cell density than the control, together with inability to aggregate.     

Bioherbicidal activity of a germacranolide sesquiterpene dilactone from Ambrosia artemisiifolia L.

Ambrosia artemisiifolia L. (common ragweed) is an invasive plant whose allelopathic properties have been suggested by its field behaviour and demonstrated through phytotoxicity bioassays. However, the nature of the molecules responsible for the allelopathic activity of common ragweed has not been explored. The main objective of this study was to identify the phytotoxic molecules produced by A. artemisiifolia. A preliminary investigation has indicated that a methanol extract of A. artemisiifolia completely inhibited the germination of cress and radish. Semi-preparative fractionation of the methanol extract allowed separating of phytotoxic fraction which contained a single compound. The structure of this compound was elucidated by liquid chromatography–mass spectrometry (LC-MS)/MS, high-resolution mass spectral, nuclear magnetic resonance, and Fourier transform infrared spectra as sesquiterpene lactone isabelin (C₁₅H₁₆O₄). The effect of pure isabelin was tested on four different weed species, confirming the inhibitory activity of molecule. The results indicate directions for the future studies about herbicidal specific activity of isabelin, as pure molecule or in the crude extract, as a potential candidate for biological weed control.     

Single and combined effects of heavy metals and hormones on lysosomes of haemolymph cells from the mussel Mytilus galloprovincialis

Effects of heavy metals on lysosomes were studied in living cells from the mussel (Mytilus galloprovincialis Lam.). Haemolymph cells were obtained from the mussel adductor muscle, stained with neutral red (NR), and analysed by digital imaging to evaluate NR retention times within lysosomes. Exposure to Hg²⁺, Cd²⁺ and Cu²⁺ induced a reduction of NR retention time, indicating lysosomal membrane destabilisation. The intensity of these effects was correlated with the metal affinity for sulfhydryls. In contrast, Zn²⁺ showed no effect on lysosomes. Moreover, 200 μM Zn²⁺ protected lysosomes against the effects of Cd²⁺ and Cu²⁺, but not against Hg²⁺. Cell loading with the fluorescent pH probe Lyso Sensor followed by digital imaging showed a rise of lysosomal pH induced by Cd²⁺ and Hg²⁺, while Zn²⁺ prevented the effect of Cd²⁺ and also partially that of Hg²⁺. The different protective effect of Zn²⁺ against Hg²⁺ suggests a dual action of Hg²⁺ on lysosomes, possibly involving both membrane destabilisation and proton pump inhibition. Cell exposure to 17 β-estradiol also caused a reduction of NR retention time, which was synergistic to that of Hg²⁺. This suggests a common pathway between metals and hormone, possibly involving Ca²⁺ signaling. Received: 17 November 1999 / Accepted: 29 June 2000     

Characterization of post-consumer polyolefin wastes by hyperspectral imaging for quality control in recycling processes

In this paper new analytical inspection strategies, based on hyperspectral imaging (HSI) in the VIS–NIR and NIR wavelength ranges (400–1000 and 1000–1700 nm, respectively), have been investigated and set up in order to define quality control logics that could be applied at industrial plant level for polyolefins recycling. The research was developed inside the European FP7 Project W2Plastics “Magnetic Sorting and Ultrasound Sensor Technologies for Production of High Purity Secondary Polyolefins from Waste”. The main aim of the project is the separation of pure polyethylene and polypropylene adopting an innovative process, the magnetic density separation (MDS). Spectra of plastic particles and contaminants resulting from post-consumer complex wastes and of virgin polyolefins have been acquired by HSI and by Raman spectroscopy. The classification results obtained applying principal component analysis (PCA) on HSI data have been compared with those obtained by Raman spectroscopy, in order to validate the proposed innovative methodology. Results showed that HSI sensing techniques allow to identify both polyolefins and contaminants. Results also demonstrated that HSI has a great potentiality as a tool for quality control of feed (identification of contaminants in the plastic waste) and of the two different pure polypropylene and polyethylene flow streams resulting from the MDS-based recycling process.     

Heterogeneity of soil surface ammonium concentration and other characteristics, related to plant specific variability in a Mediterranean-type ecosystem

Heterogeneity and dynamics of eight soil surface characteristics essential for plants—ammonium and nitrate concentrations, water content, temperature, pH, organic matter, nitrification and ammonification rates—were studied in a Mediterranean-type ecosystem on four occasions over a year. Soil properties varied seasonally and were influenced by plant species. Nitrate and ammonium were present in the soil at similar concentrations throughout the year. The positive correlation between them at the time of greatest plant development indicates that ammonium is a readily available nitrogen source in Mediterranean-type ecosystems. The results presented here suggest that plant cover significantly affects soil surface characteristics.     

Leaching of S-metolachlor,terbuthylazine, desethyl-terbuthylazine,mesotrione, flufenacet,isoxaflutole, and diketonitrile in field lysimeters as affected by the time elapsed between spraying and first leaching event

The effect of elapsed time between spraying and first leaching event on the leaching behavior of five herbicides (terbuthylazine, S-metolachlor, mesotrione, flufenacet, and isoxaflutole) and two metabolites (desethyl-terbuthylazine and diketonitrile) was evaluated in a 2011–2012 study in northwest Italy. A battery of 12 lysimeters (8.4 m² long with a depth of 1.8 m) were used in the study, each filled with silty-loam soil and treated during pre-emergence with the selected herbicides by applying a mixture of commercial products Lumax (4 L ha^?1) and Merlin Gold (1 L ha^?1). During treatment periods, no gravity water was present in lysimeters. Irrigation events capable of producing leaching (40 mm) were conducted on independent groups of three lysimeters on 1 day after treatment (1 DAT), 7 DAT, 14 DAT, and 28 DAT. The series was then repeated 14 days later. Leachate samples were collected a few days after irrigation; compounds were extracted by solid phase extraction and analyzed by high-performance liquid chromatography and gas chromatography–mass spectrometry. Under study conditions, terbuthylazine and S-metolachlor showed the highest leaching potentials. Specifically, S-metolachlor concentrations were always found above 0.25 µg L^?1. Desethyl-terbuthylazine was often detected in leached waters, in most cases at concentrations above 0.1 µg L^?1. Flufenacet leached only when irrigation occurred close to the time of herbicide spraying. Isoxaflutole and mesotrione were not measured (<0.1 µg L^?1), while diketonitrile was detected in concentrations above 0.1 µg L^?1 on 1 DAT in 2011 only.     

An application of multivariate analysis to acid rain data in Northern Italy to discriminate natural and man-made compounds

This paper presents the preliminary results of a study, the aim of which was to analyse the pluviometric and chemical rain data, recorded by a wet only network located in Northern Italy.More in detail, precipitations were collected on a weekly basis and chemical analysis was performed on pH, electric conductivity and Ca, Mg, Na, K, NH₄, NO₃, SO₄, Cl concentrations.The Principal Components Analysis pointed out that the first three components are enough to explain more than 90% of the variability of the parameters. Moreover each component may have a different physical interpretation, that is the first one is mainly related to the precipitation amount, while the second to the man made and natural sources and the last one to the sea/soil contribution.     

Fate of domoic acid ingested by the copepod <Emphasis Type="Italic">Acartia clausi</Emphasis>

Two important issues in the studies of harmful algae include ecological role of the toxic compounds and their fate through the food web. The aims of this study were to determine whether the production of domoic acid is a strategy evolved to avoid predation and the role of copepods in the fate of this toxic compound through the food web. The copepod Acartia clausi was fed with single and mixed cultures of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia delicatissima. Ingestion rate as a function of diatom abundance was the same for the toxic and non-toxic Pseudo-nitzschia species, indicating no selective feeding behaviour against P. multiseries. The toxins ingested by the copepods did not affect mortality, feeding behaviour, egg production and egg hatching of the copepods. Copepods assimilated the 4.8% of the total domoic acid ingested. Although the amount of toxins daily detoxificated by the copepods was 63.6%, the copepods accumulated domoic acid in their tissues. We conclude that domoic acid is not toxic for copepods and, probably for this reason, this toxin does not act as feeding deterrent for copepods. However, even though the production of domoic acid has apparently not evolved to deter predation, copepods may play an important role on the fate of this toxic compound through the marine food web.     

Metallothioneins and lysosomes in metal toxicity and accumulation in marine mussels: the effect of cadmium in the presence and absence of phenanthrene

It has been confirmed that metallothioneins play an important role in the accumulation of cadmium (Cd) in the digestive gland cells of mussels (Mytilus galloprovincialis Lam.). The content of Cd in the tissue of mussels exposed for 9 d to the metal (estimated dosage of 180 g Cd mussel^-1 d^-1) was 66.2 ppm. This value is about the same as the metal content found in the digestive gland of Cd-exposed mussels kept in clean water for a recovery period of 28 d. At the end of the recovery period, however, the Cd bound to thionein had increased by approximately 250%. Our data demonstrate that the stability of lysosomes, a biological parameter adopted as a cellular stress index, is extremely low in mussels exposed to Cd for 9 d, but returns to control values in the digestive gland cells of mussels allowed to recover for 28 d in uncontaminated sea water. At this point most of the Cd present in the cytosol is bound to thionein. These data demonstrate the importance of metallothionein induction in the reduction of the cytotoxic effects exerted by high levels of Cd accumulation. The results of tests designed to clarify the reasons for the long biological half-life of Cd demonstrated that, in the digestive gland of mussels, the lysosomes are not able to eliminate Cd either bound to insoluble thionein polymers or to lipid peroxidation products such as lysosomal lipofuscin, both of which are apparently involved in the elimination of copper. The absence of these two mechanisms of metal sequestration and elimination via excretion of residual bodies (tertiary lysosomes) is in agreement with the persistence of cadmium in the digestive gland of mussels. Finally, the results also demonstrate that simultaneous exposure of mussels to Cd and phenanthrene, an established lysosomal membrane destabilizer, did not significantly alter the accumulation of Cd or the kinetics of the metal in mussels.