Neurotranmission systems as targets for toxicants: a review

Neurotransmitters are chemicals that transmit impulses from one nerve to another or from nerves to effector organs. Numerous neurotransmitters have been described in mammals, amongst them acetylcholine, amino acids, amines, peptides and gases. Toxicants may interact with various parts of neurotransmission systems, including synthetic and degradative enzymes, presynaptic vesicles and the specialized receptors that characterize neurotransmission systems. Important toxicants acting on the cholinergic system include the anticholinesterases (organophosphates and carbamates) and substances that act on receptors such as nicotine and the neonicotinoid insecticides, including imidacloprid. An important substance acting on the glutamatergic system is domoic acid, responsible for amnesic shellfish poisoning. 4-Aminobutyric acid (GABA) and glycine are inhibitory neurotransmitters and their antagonists, fipronil (an insecticide) and strychnine respectively, are excitatory. Abnormalities of dopamine neurotransmission occur in Parkinson’s disease, and a number of substances that interfere with this system produce Parkinsonian symptoms and clinical signs, including notably 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is the precursor of 1-methyl-4-phenylpyridinium. Fewer substances are known that interfere with adrenergic, histaminergic or seroninergic neurotransmission, but there are some examples. Among peptide neurotransmission systems, agonists of opioids are the only well-known toxic compounds.     

The importance of topography and climate on short-term revegetation of coal wastes in Spain

The initial colonization phase is the crucial start point for succession and therefore for restoration. However, little is known about abiotic factors that influence the early stages of revegetation dynamics on restored coal mines, particularly in a Mediterranean climate. This information is crucial for improving our ability to reclaim land despoiled by mining. Here, we characterized the short-term plant community development in the first 3 years after hydroseeding on a topographically diverse reclaimed open-pit coal mines in Spain. Topography influenced both community composition and diversity producing different trajectories between the three different aspects (north-facing, south-facing, flat). Hydroseeded species provided most of the initial vegetation cover, which brought about most of the compositional differences between these aspects. We also found that the changing climate through the growing season also influenced floristic composition and diversity. Summer drought reduced the cover of hydroseeded perennial species and allowed native species to colonize. These results emphasize the need to take topography into account when developing reclamation management plans in Mediterranean ecosystems, and an acknowledgement that climate might drive the succession in the desired direction, hence increasing success in restoring mining impacts.     

Establishment and characterization of equine autonomic ganglion cell lines to enable direct testing of candidate toxins involved in equine dysautonomia (grass sickness)

To enable direct testing of a range of potential toxins or pathogens that might be involved in grass sickness, equine thoracic sympathetic chain ganglion cell lines were established from primary cell cultures by retroviral-mediated transduction of the temperature-sensitive mutant of the establishment oncogene encoding SV40 large T antigen. Morphological and behavioral features, temperature dependence, and immunocytochemical characteristics of the cell lines were investigated. The majority of cells were noradrenergic neurons in which dopamine-β-hydroxylase, the enzyme that catalyzes norepinephrine synthesis, and neuropeptide Y coexisted. Cells treated with plasma from grass sickness cases that had previously been shown to induce autonomic nervous system damage when injected into normal horses showed significantly decreased mitochondrial function after 1 day. After 3 days exposure most cells showed severe degeneration in contrast to those treated with normal plasma. Liver and lung cell lines were also susceptible to plasma, suggesting that the toxin is not specifically neurotoxic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vegetation succession on reclaimed coal wastes in Spain: the influence of soil and environmental factors

Question: How is vegetation succession on coal mine wastes under a Mediterranean climate affected by the restoration method used (topsoil addition or not)? How are plant successional processes influenced by local landscape and soil factors? Location: Reclaimed coal mines in the north of Palencia province, northern Spain (42°47′‐42°50′ N, 4°32′‐4°53′ W). Methods: In Jun–Jul 2008, vascular plant species cover was monitored in 31 coal mines. The mines, which had been restored using two restoration methods (topsoil addition or not), comprised a chronosequence of different ages from 1 to 40 yr since restoration started. Soil and environmental factors at each mine were monitored and related to species cover using a combination of ordination methods and Huisman–Olff–Fresco modeling. Results: Plant succession was affected by restoration method . Where topsoil was added, succession was influenced by age since restoration and soil pH. Where no topsoil was added, soil factors seem to arrest succession. Vegetation composition on topsoiled sites showed a gradient with age, from the youngest, with early colonizing species, to oldest, with an increase in woody species. Vegetation on non‐topsoiled sites comprised mainly early‐successional species. Response to age and pH of 37 species found on topsoiled mines is described. Conclusions: Restoration of coal mines under this Mediterranean climate can be relatively fast if topsoil is added, with a native shrub community developing after 15 yr. However, if topsoil is not used, it takes more than 40 yr. For topsoiled mines, the species found in the different successional stages were identified, and their tolerance to soil pH was derived. This information will assist future restoration projects in the area.     

Optimization of a fluorescent-based phosphor imaging dot blot DNA hybridization assay to assess E. coli virulence gene profiles

To increase the efficiency and consistency in screening Escherichia coli for virulence genes, a Phosphor Imager was adopted for signal detection in Dot Blot DNA hybridization replacing X-ray film read by eye. We assessed not only the reliability of the instrument-based procedure, but the impact of going from an outcome measured by visualization on a semi-quantitative scale to a digitized readout on an interval scale. We analyzed technical and biological variability of the assay and the factors contributing to the variability. In spite of high variability both within and between membranes in the Phosphor Imager readings, we were able to define classification rules for gene presence that were remarkably consistent. Using the X-ray film signal detection procedure with Southern confirmation as a gold standard, we obtained a sensitivity and specificity of 87-99% for a rule requiring no retesting for all but one gene probe.     

Bioluminescence detection of ATP release mechanisms in epithelia

Autocrine and paracrine release of and extracellular signalingby ATP is a ubiquitous cell biological and physiological process. Despite this knowledge, the mechanisms and physiological roles ofcellular ATP release are unknown. We tested the hypothesis thatepithelia release ATP under basal and stimulated conditions by using anewly designed and highly sensitive assay for bioluminescence detectionof ATP released from polarized epithelial monolayers. Thisbioluminescence assay measures ATP released from cystic fibrosis (CF)and non-CF human epithelial monolayers in a reduced serum mediumthrough catalysis of the luciferase-luciferin reaction, yielding aphoton of light collected by a luminometer. This novel assay measuresATP released into the apical or basolateral medium surroundingepithelia. Of relevance to CF, CF epithelia fail to release ATP acrossthe apical membrane under basal conditions. Moreover, hypotonicity isan extracellular signal that stimulates ATP release into bothcompartments of non-CF epithelia in a reversible manner; the responseto hypotonicity is also lost in CF epithelia. The bioluminescencedetection assay for ATP released from epithelia and other cells will beuseful in the study of extracellular nucleotide signaling inphysiological and pathophysiological paradigms. Taken together, theseresults suggest that extracellular ATP may be a constant regulator ofepithelial cell function under basal conditions and an autocrineregulator of cell volume under hypotonic conditions, two functions thatmay be lost in CF and contribute to CF pathophysiology.

     

Determining the important environmental variables controlling plant species community composition in mesotrophic grasslands in Great Britain

Question: What is the relative importance of local site effects and selected important environmental variables in determining plant species composition? How do species respond to these environmental variables? Location: Ten mesotrophic grassland sites of high conservation value in southern England. Methods: Species cover was assessed in between 10 and 25 randomly selected 1‐m² quadrats at each site. At each quadrat degree of waterlogging (W), soil Olsen extractable phosphorus (P) and soil pH were measured. Variation partitioning was used to separate site and soil effects, and HOF (Huisman, Olff & Fresco) modelling was used to produce response curves for the major species on soil gradients, based on coenoclines derived from partial canonical correspondence analysis (pCCA). Results: Variation partitioning identified Site as the most important environmental variable (34.6%). Only 18.7% was accounted for by the three soil variables together; W (degree of waterlogging), P and pH accounted for 11.1%, 5.7% and 4.3%, respectively in raw form with 2.4% shared. However, when Site and the other soil variables were removed the variation explained reduced to 2.3% for W, 1.1% for P and 1.0% for pH. The species responses to each of these soil environmental factors could be separated into four types on each gradient. Most species were abundant at low W, low soil P and intermediate pH. Conclusions: Site‐based factors were more important than the three soil variables, which were assumed to be directly or indirectly associated with productivity. This implies that each site has unique properties that are more important than the soil variables. The three soil factors were, however, significant and the groups of the most common species, based on significant response curves, can be used as a first approximation of indicators of environmental conditions in British mesotrophic grasslands for conservation. However, W accounted for most variation, and the current reliance on soil available P and soil pH for assessing conservation/restoration potential should be viewed with caution.     

Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.