Effects of the litter layer of Pteridium aquilinum on seed banks under experimental restoration

Questions: Does the litter layer of Pteridium aquilinum (bracken) act as a barrier to certain species in the seed bank? Does bracken control/restoration treatment affect seed transfer through the litter layer? Location: Five experiments at three sites across the UK covering two major vegetation types; acid‐grassland and heath‐land. Methods: At each experiment a range of bracken control and vegetation restoration treatments were applied for about ten years. The seed bank was sampled in both the bracken litter and the soil. The cover (%) of each species in the vegetation and the bracken litter abundance (cover and depth) was also estimated. Results: The bracken litter layer acts as an inert barrier as it contained a large proportion of seeds available in the litter‐soil profile (38%– 67% of the total). Bracken litter depth and cover also influenced significantly the seed bank composition in both the bracken litter and the soil. These effects were site‐specific, and species‐specific. The application of treatments changed significantly the balance between seed inputs and outputs in the bracken litter layer for some species. This was either a positive or negative response relative to the untreated control plots. Conclusion: For heathland and acid‐grassland restoration, the bracken litter layer may be an important seed source, but it must be disturbed particularly before seed addition.     

Coyotes (Canis latrans) are definitive hosts of Neospora caninum

Four captive-raised coyote pups consumed tissues from Neospora caninum-infected calves. Faeces were examined from 4 days before to 28 days after infection. One pup shed N. caninum-like oocysts, which tested positive for N. caninum and negative for Hammondia heydorni using PCR tests. Coyotes are the second discovered definitive host of N. caninum, after dogs. In North America, the expanding coyote ranges and population increase the probability of contact with domestic livestock. To reduce the risk of transmission of N. caninum to intensively farmed cattle, we recommend protection of feedstuffs using canid-proof fences, and careful disposal of dead stock.     

Inhibiting aggregation of alpha-synuclein with human single chain antibody fragments

The alpha-synuclein protein has been strongly correlated with Parkinson's disease (PD) and is a major component of the hallmark Lewy body aggregates associated with PD. Two different mutations in the alpha-synuclein gene as well as increased gene dosage of wild-type alpha-synuclein all associate with early onset cases of PD; and transgenic animal models overexpressing alpha-synuclein develop PD symptoms. Alpha-synuclein, a natively unfolded protein, can adopt a number of different folded conformations including a beta-sheet form that facilitates formation of numerous aggregated morphologies, including long fibrils, spherical and linear protofibrils, and smaller aggregates or oligomers. The roles of the various morphologies of alpha-synuclein in the progression of PD are not known, and different species have been shown to be toxic. Here we show that single chain antibody fragments (scFv's) isolated from na?ve phage display antibody libraries can be used to control the aggregation of alpha-synuclein. We isolated an scFv with nanomolar affinity for monomeric alpha-synuclein (K(D) = 2.5 x 10(-8) M). When co-incubated with monomeric alpha-synuclein, the scFv decreased not only the rate of aggregation of alpha-synuclein, but also inhibited the formation of oligomeric and protofibrillar structures. The scFv binds the carboxyl terminal region of alpha-synuclein, suggesting that perturbation of this region can influence folding and aggregation of alpha-synuclein in vitro along with the previously identified hydrophobic core region of alpha-synuclein (residues 61-95, particularly residues 71-82). Since the scFv has been isolated from an antibody library based on human gene sequences, such scFv's can have potential therapeutic value in controlling aggregation of alpha-synuclein in vivo when expressed intracellularly as intrabodies in dopaminergic neurons.     

Sequence differentiation associated with an inversion on the neo-X chromosome of Drosophila americana

Sex chromosomes originate from pairs of autosomes that acquire controlling genes in the sex-determining cascade. Universal mechanisms apparently influence the evolution of sex chromosomes, because this chromosomal pair is characteristically heteromorphic in a broad range of organisms. To examine the pattern of initial differentiation between sex chromosomes, sequence analyses were performed on a pair of newly formed sex chromosomes in Drosophila americana. This species has neo-sex chromosomes as a result of a centromeric fusion between the X chromosome and an autosome. Sequences were analyzed from the Alcohol dehydrogenase (Adh), big brain (bib), and timeless (tim) gene regions, which represent separate positions along this pair of neo-sex chromosomes. In the northwestern range of the species, the bib and Adh regions exhibit significant sequence differentiation for neo-X chromosomes relative to neo-Y chromosomes from the same geographic region and other chromosomal populations of D. americana. Furthermore, a nucleotide site defining a common haplotype in bib is shown to be associated with a paracentric inversion [In(4)ab] on the neo-X chromosome, and this inversion suppresses recombination between neo-X and neo-Y chromosomes. These observations are consistent with the inversion acting as a recombination modifier that suppresses exchange between these neo-sex chromosomes, as predicted by models of sex chromosome evolution.     

Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis

This paper describes the use of fluorescence two-dimensional differential in-gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK(1)R-/-) and wild type (NK(1)R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK(1)R-/- and NK(1)R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two-dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In-gel Analysis software. A Cy2-labeled sample pool was co-electrophoresed with all Cy3- and Cy5-labeled sample pairs as an internal standard providing a link for inter-gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK(1)R-/- mice compared to NK(1)R+/+ controls. Matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.     

Techniques for gene transfer into neurons

To illuminate the function of the thousands of genes that make up the complexity of the nervous system, it is critical to be able to introduce and express DNA in neurons. Over the past two decades, many gene transfer methods have been developed, including viral vectors, liposomes and electroporation. Although the perfect gene transfer technique for every application has not yet been developed, recent technical advances have facilitated the ease of neuronal gene transfer and have increased the accessibility of these techniques to all laboratories. In order to select a transfection method for any particular experiment, the specific advantages and disadvantages of each technique must be considered.     

Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.     

Posters. P17 Accuracy of fine needle aspiration cytology in diagnosis of thyroid swellings

Introduction Fine needle aspiration cytology is regarded as the gold standard investigation in diagnosis of thyroid swellings. Published data suggest an overall accuracy rate of 75% 1 in the detection of thyroid malignancy. The aim of this study was to determine the accuracy of FNA cytology in detection of thyroid malignancy in our surgical unit. Methods Between 1989–2002, 144 patients who underwent thyroid resection by single consultant surgeon and who had pre‐operative FNA were enrolled in this retrospective study. The pre‐operative FNA results were compared with definitive histological diagnosis following thyroid resection. Fine needle aspiration cytology was performed using aspirate and non‐aspirate techniques on each thyroid swelling. The cytological sample was assessed by a single cytopathologist and was classified as inadequate, non‐neoplastic, neoplastic, suspicious or indeterminate. The histology was classified as non‐neoplastic (benign) and neoplastic (malignant). Results Fine needle aspiration cytology analysis revealed 94 (13.88%) non‐neoplastic, six (65.27%) neoplastic and 20 (4.16%) suspicious aspirates. Twenty (13.88%) samples were inadequate and four (2.77%) samples were indeterminate. Histological analysis showed 118 (81.94%) benign, 26 (18.05%) malignant specimens. Fine needle aspiration cytology had a sensitivity, specificity and accuracy rate of 52.6%, 86.6% and 79.1%, respectively for diagnosing thyroid malignancy. Conclusion The results are comparable with the current published data and demonstrate that FNA cytology in our hands is accurate investigation for pre‐operative diagnosis for the detection of thyroid malignancy.