Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts, We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha -particles). The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 mum and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell, Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish, This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved. (C) 2001 Cancer Research Campaign.

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5-3' exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R(2) > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC-MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.

Naturalising the Highlands: geographies of mountain fieldwork in late-Victorian Scotland

Through a detailed examination of two late-Victorian field clubs dedicated to the exploration of alpine botany in the Scottish Highlands, this paper contributes to work on the historical geographies of civic science. By focusing on the scientific and social character of mountain fieldwork it analyses the reciprocal relations between the spaces, practices and science of Highland botanising and wider concerns with sociability, character and civic virtue. In so doing it investigates the transposition of a variety of discursive resources from evolutionism to tourism into the language and practices of botanical science. This focus enables the paper to complicate more general accounts of natural history in the Victorian period and to consider a number of methodological issues relevant to reconstructing the historical geographies of science.

Patterns of cytoplasmic variation in Arabidopsis thaliana (Brassicaceae) revealed by polymorphic chloroplast microsatellites

Despite being the model organism for plant molecular genetic studies, little is known about the origins and evolutionary history of extant natural populations of Arabidopsis thaliana. We have analysed phylogenetic relationships between worldwide populations of Arabidopsis using polymorphic chloroplast microsatellites. These highly variable markers have revealed previously undetected levels of cytoplasmic variation and confirm previous hypotheses of a recent and rapid expansion of the species from its centre of origin. Furthermore, the results seem to verify previous nuclear analyses that call into question the true origin of several individual Arabidopsis ecotypes.

Algal epiphytes of Zostera marina: Variation in assemblage structure from individual leaves to regional scale

Data from a hierarchical study of four Zostera marina beds in Wales were used to identify the spatial scales of variation in epiphyte assemblages. There were significant within and among bed differences in assemblage structure. The differences in assemblage structure with spatial scale generally persisted when species identifications were aggregated into functional groups. There was also significant within and among bed variability in Zostera density and average length. Local variations in Zostera canopy variables at the quadrat scale (total leaf length, average leaf length and leaf density per quadrat) were not related to epiphyte species richness nor to the structure of the assemblage. In contrast, individual leaf length was significantly related to species richness in two of the beds and the structure of epiphyte assemblages was always related to individual leaf lengths. The absence of links between quadrat scale measurements of canopy variables and assemblage structure may reflect the high turnover of individual Zostera leaves. Experimental work is required to discriminate further between the potential causes of epiphyte assemblage variation within and between beds. No bed represented a refuge where a rare species was abundant. If a species was uncommon at the bed scale, it was also uncommon in beds where it occurred. The heterogeneous assemblages found in this study suggest that a precautionary approach to conservation is advisable.

cpDNA-RFLP in Ceramium (Rhodophyta): Intraspecific polymorphism and species-level phylogeny

Restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNA is a powerful tool for the study of microevolutionary processes in land plants, yet has not previously been applied to seaweed populations. We used cpDNA-RFLP, detected on Southern blots using labeled total plastid DNA, to search for intraspecific and intrapopulational cpDNA RFLP polymorphism in two species of the common red algal genus Ceramium in Ireland and Britain. In C. botryocarpum one polymorphism was detected in one individual among 18 from two populations. Twenty-six individuals of C. virgatum from five populations at three locations exhibited a total of four haplotypes. One was frequent (80.8% of individuals); the others were rare (7.7, 7.7 and 4.2%) and were private to particular populations. Polymorphism was observed in two populations. The corrected mean was 2.26 +/- 0.36 haplotypes per population, which was within the typical range determined for higher plants using similar techniques. The spatial distribution of haplotypes was heterogeneous, with highly significant population differentiation (P = 0.00018; Fisher's exact test). Intraspecific polymorphism in C. virgatum had no impact on species-level phylogenetic reconstruction. This is the first unequivocal report of both intraspecific and intrapopulational cpDNA-RFLP polymorphism in algae.

THE DISTRIBUTION AND UTILIZATION OF ZOSTERA SPECIES IN STRANGFORD LOUGH, NORTHERN-IRELAND

Strangford Lough is a sheltered marine inlet on the east coast of Co. Down, Northern Ireland. The distribution of Zostera in the Lough and its exploitation by wildfowl was investigated. There was an estimated 1100 tonnes (fresh weight) of Zostera in the Lough in October 1991 covering some 6.3 km2 of the northern mudflats and representing some 12% of the total intertidal area of the Lough. This was regarded as peak biomass. By January 1992, Zostera was reduced following grazing by wildfowl and weathering to 20% of the initial biomass as measured in the exclusion experiment. The above-ground and below-ground biomass of Zostera were not equally affected with reduction to 7% and 26%, respectively.

Detection of short lived radioisotopes as a fast diagnostic for intense laser-solid interactions

As a diagnostic of high-intensity laser interactions (> 10(19) W cm(-2)), the detection of radioactive isotopes is regularly used for the characterization of proton, neutron, ion, and photon beams. This involves sample removal from the interaction chamber and time consuming post shot analysis using NaI coincidence counting or Ge detectors. This letter describes the use of in situ detectors to measure laser-driven (p,n) reactions in Al-27 as an almost real-time diagnostic for proton acceleration. The produced Si-27 isotope decays with a 4.16 s half-life by the predominantly beta+ emission, producing a strong 511 keV annihilation peak. (c) 2006 American Institute of Physics.

Laser generation of proton beams for the production of short-lived positron emitting radioisotopes

Protons of energies up to 37 MeV have been generated when ultra-intense lasers (up to 10(20) W cm(-2)) interact with hydrogen containing solid targets. These protons can be used to induce nuclear reactions in secondary targets to produce P-emitting nuclei of relevance to the nuclear medicine community, namely C-11 and N-13 via (p, n) and (p, alpha) reactions. Activities of the order of 200 kBq have been measured from a single laser pulse interacting with a thin solid target. The possibility of using ultra-intense lasers to produce commercial amounts of short-lived positron emitting sources for positron emission tomography (PET) is discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

Rice-arsenate interactions in hydroponics:whole genome transcriptional analysis

Rice (Oryza sativa) varieties that are arsenate-tolerant (Bala) and -sensitive (Azucena) were used to conduct a transcriptome analysis of the response of rice seedlings to sodium arsenate (AsV) in hydroponic solution. RNA extracted from the roots of three replicate experiments of plants grown for 1 week in phosphate-free nutrient with or without 13.3 muM AsV was used to challenge the Affymetrix (52K) GeneChip Rice Genome array. A total of 576 probe sets were significantly up-regulated at least 2-fold in both varieties, whereas 622 were down-regulated. Ontological classification is presented. As expected, a large number of transcription factors, stress proteins, and transporters demonstrated differential expression. Striking is the lack of response of classic oxidative stress-responsive genes or phytochelatin synthases/synthatases. However, the large number of responses from genes involved in glutathione synthesis, metabolism, and transport suggests that glutathione conjugation and arsenate methylation may be important biochemical responses to arsenate challenge. In this report, no attempt is made to dissect differences in the response of the tolerant and sensitive variety, but analysis in a companion article will link gene expression to the known tolerance loci available in the BalaxAzucena mapping population.