Getting to the root of neuronal signalling in plant-parasitic nematodes using RNA interference

A variety of genes expressed in preparasitic second-stage juveniles (J2) of plant-parasitic nematodes appear to be vulnerable to RNA interference (RNAi) in vitro by coupling double-stranded (ds)RNA soaking with the artificial stimulation of pharyngeal pumping. Also, there is mounting evidence that the in planta generation of nematode-specific double-stranded RNAs (dsRNAs) has real utility in the control of these pests. Although neuronally-expressed genes in Caenorhabditis elegans are commonly refractory to RNAi, we have discovered that neuronally-expressed genes in plant-parasitic nematodes are highly susceptible to RNAi and that silencing can be induced by simple soaking procedures without the need for pharyngeal stimulation. Since most front-line anthelmintics that are used for the control of nematode parasites of animals and humans act to disrupt neuromuscular coordination, we argue that intercellular signalling processes associated with neurons have much appeal as targets for transgenic plant-based control strategies for plant-parasitic nematodes. FMRFamide-like peptides (FLPs) are a large family of neuropeptides which are intimately associated with neuromuscular regulation, and our studies on flp gene function in plant-parasitic nematodes have revealed that their expression is central to coordinated locomotory activities. We propose that the high level of conservation in nervous systems across nematodes coupled with the RNAi-susceptibility of neuronally-expressed genes in plant-parasitic nematodes provides a valuable research tool which could be used to interrogate neuronal signalling processes in nematodes.

A set of primers for plastid indels and nuclear microsatellites in the invasive plant Heracleum mantegazzianum (Apiaceae) and their transferability to Heracleum sphondylium

This study reports the isolation and polymorphism characterization of four plastid indels and six nuclear microsatellite loci in the invasive plant Heracleum mantegazzianum. These markers were tested in 27 individuals from two distant H. mantegazzianum populations. Plastid indels revealed the presence of five chlorotypes while five nuclear microsatellite loci rendered polymorphism. Applications of these markers include population genetics and phylogeography of H. mantegazzianum. A very good transferability of markers to Heracleum sphondylium was demonstrated.

Development of microsatellites for the invasive riparian plant Impatiens glandulifera (Himalayan balsam) using intersimple sequence repeat cloning

Impatiens glandulifera (Himalayan balsam) is an invasive riparian plant species that can outcompete native perennials. Population genetic data on dispersal may aid in the management of invasive species, so we have developed microsatellite markers for this significant invader using an intersimple sequence repeat (ISSR)-based cloning method. Eight polymorphic markers displayed between two and five alleles, with overall levels of observed and expected heterozygosities ranging from 0.0500 to 0.7500 and from 0.1449 to 0.7692, respectively.

Determination of absolute configuration of conformationally flexible cis-dihydrodiol metabolites: Effect of diene substitution pattern on the circular dichroism spectra and optical rotations

Absolute configurations of a number of cis-dihydrodiols (cis-1,2-dihydroxy-3,5-cyclohexadienes), synthetically useful products of TDO-catalyzed dihydroxylations of 1,2- and 1,3-disubstituted benzene derivatives, have been determined by a comparison of calculated and experimental CD spectra and optical rotations and by methods involving X-ray crystallography, H-1 NMR spectra of diastereoisomeric derivatives, and by stereochemical correlations. The computations disclosed a significant effect of the substituents on conformational equilibria of cis-dihydrodiols and chiroptical properties of individual conformers. The assigned absolute configurations of cis-dihydrodiols have allowed the validity of a simple predictive model for TDO-catalyzed arene dihydroxylations to be extended.

Enzyme-catalysed synthesis and absolute configuration assignments of cis-dihydrodiol metabolites from 1,4-disubstituted benzenes

A series of ten cis-dihydro-diol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and H-1 NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

Absolute configuration of conformationally flexible cis-dihydrodiol metabolites by the method of confrontation of experimental and calculated electronic CD spectra and optical rotations

We have determined the absolute configurations of conformationally flexible cis-dihydrodiol metabolites (cis-1,2-dihydroxy-3,5-cyclohexadienes), bearing different substituents (e.g., Br, F, CF3, CN, Me) in 3- and 5-positions, by the method of confrontation of experimental and calculated electronic CD spectra and optical rotations. Convergent results were obtained by both methods in eight out of ten cases. For the difficult cases, where either conformer population and/or chiroptical properties (calculated rotational strengths of the long-wavelength Cotton effect or optical rotations) of contributing conformers remain inconclusive, the absolute configuration could still be correctly assigned based on one of the biased properties (either ECD or optical rotation). This approach appears well-suited for a broad spectrum of conformationally flexible chiral molecules.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45 min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95 pmol/8 x 101 RBCs and 2, 5, 2, 3, 20 ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200 mu l of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients. (C) 2008 Elsevier B.V. All rights reserved.

Relative activity of triclabendazole metabolites against the liver fluke, Fasciola hepatica

A study has been carried Out to determine the relative activity Of triclabendazole (TCBZ) and its sulphoxide (TCBZ(center dot)SO) and sulphone (TCBZ(center dot)SO(2)) metabolites against the adult stage of the liver fluke, Fasciola hepatica. Flukes were incubated for 24 h in vitro in 15 mu g/ml of each of the compounds and prepared for scanning and transmission electron microscopy.

Variability in thermal and UV-B energy fluxes through time and their influence on plant diversity and speciation

Throughout Earth's history there have been temporal and spatial variations in the amount of visible and ultraviolet radiation received by ecosystems. This paper examines if temporal changes in these forms of energy receipt could have influenced the tempo and mode of plant diversity and speciation, focusing in particular upon Cenozoic time-scales. Evidence for changing patterns of plant diversity and speciation apparent in various fossil records and molecular phylogenies are considered alongside calculated changes in thermal and solar ultraviolet energy (specifically UV-B) over the past 50 Myr. We suggest that changes in thermal energy influx (amount and variability) affected the tempo of evolution through its influence upon community dynamics (e.g. population size, diversity, turnover, extinctions). It was not only the amount of thermal energy but also variability in its flux that may have influenced these processes, and ultimately the rate of diversification. We suggest that variations in UV-B would have influenced the mode and tempo of speciation through changes to genome stability during intervals of elevated UV-B. We argue, therefore, that although variability in thermal energy and UV-B fluxes through time may lead to the same end-point (changing the rate of diversification), the processes responsible are very different and both need to be considered when linking evolutionary processes to energy flux.

Cut down by some cowardly miscreants’: Plant maiming, or the Malicious Cutting of Plants as an Act of Protest in Eighteenth and Nineteenth Century Rural England

Since the publication of Hobsbawm and Rudé's Captain Swing our understanding of the role(s) of covert protests in Hanoverian rural England has advanced considerably. Whilst we now know much about the dramatic practices of incendiarism and animal maiming and the voices of resistance in seemingly straightforward acquisitive acts, one major gap remains. Despite the fact that almost thirty years have passed since E. P. Thompson brought to our attention that under the notorious ‘Black Act’ the malicious cutting of trees was a capital offence, no subsequent research has been published. This paper seeks to address this major lacuna by systematically analysing the practices and patterns of malicious attacks on plants (‘plant maiming’) in the context of late eighteenth- and early nineteenth-century southern England. It is shown that not only did plant maiming take many different forms, attacking every conceivable type of flora, but also that it was universally understood and practised. In some communities plant maiming was the protestors' weapon of choice. As a social practice it therefore embodied wider community beliefs regarding the defence of plebeian livelihoods and identities.