Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

Plant cell growth and stress signaling require Ca²âº influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²âº-permeable conductance that permits Ca²âº influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²âº-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca²âº- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²âº in response to OH•. An OH•-activated Ca²âº conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²âº-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²âº in plants.

Available, accessible, aware, appropriate, and acceptable: a strategy to improve participation of teenagers and young adults in cancer trials.

Under-representation of teenagers and young adults in clinical trials for cancer is acknowledged internationally and might account for the lower survival gains noted for this group. Little research has focused on strategies to increase participation of teenagers and young adults in clinical trials. We applied a conceptual framework for barriers to recruitment of under-represented populations to data for cancer clinical trials in teenagers and young adults. We did a systematic analysis of data for clinical trial enrolment in Great Britain over 6 years (2005-10), and reviewed the published work for the origins and scientific rationale of age eligibility criteria in clinical trials for cancer. Our Review revealed little scientific evidence for use of age eligibility criteria in cancer clinical trials. Participation in cancer trials fell as age increased. Between 2005 and 2010, participation rates increased for children and young people aged 0-24 years. The highest increase in participation was for teenagers aged 15-19 years, with smaller improvements in rates for 20-24 year olds. Improvements were related to five key criteria, the five As: available, accessible, aware, appropriate, and acceptable. In studies for which age eligibility criteria were appropriate for inclusion of teenagers or young adults or amended during the study period, participation rates for 15-19 year olds were similar to those for 10-14 year olds. We propose a conceptual model for a strategic approach to improve recruitment of teenagers and younger adults to clinical trials for cancer, with use of the five As, which is applicable worldwide for investigators, regulatory authorities, representatives in industry, policy makers, funders, and health-care professionals.

Purification of annexin V and its use in the detection of apoptotic cells.

Cell death by apoptosis has been studied for many years using fluorescently labeled annexin V. Annexin V shows high affinity for the phosphatidylserine that becomes enriched in the outer leaflet of the plasma membrane during apoptosis, but not necrosis, allowing differentiation between the two types of cell death. In this chapter we detail two methods for the purification of annexin V. The first is an untagged recombinant protein using a three step Fast Protein Liquid Chromatography (FPLC) method, and the second using a single step purification protocol via a glutathione S-transferase (GST) tag. Labeling of the resulting annexin V with a fluorescent dye to allow visualization of the protein is also explained. Finally, two methods are described in which a fluorescently labeled derivative of annexin V is used to detect apoptosis, namely the in vitro method of fluorescence-activated cell sorting (FACS) where fluorescent annexin V is used to differentiate apoptotic and necrotic cells within a population; and detection of apoptosing retinal cells (DARC) allowing the identification of apoptotic cells in the retina in vivo.

Heme-independent soluble and membrane-associated peroxidase activity of a Zea mays annexin preparation.

Annexins are cytosolic proteins capable of reversible, Ca(2+)-dependent membrane binding or insertion. Animal annexins form and regulate Ca(2+)-permeable ion channels and may therefore participate in signaling. Zea mays (maize) annexins (ZmANN33 and ZmANN35) have recently been shown to form a Ca(2+)-permeable conductance in planar lipid bilayers and also exhibit in vitro peroxidase activity. Peroxidases form a superfamily of intra- or extracellular heme-containing enzymes that use H(2)O(2) as the electron acceptor in a number of oxidative reactions. Maize annexin peroxidase activity appears independent of heme and persists after membrane association, the latter suggesting a role in reactive oxygen species signaling.

Zea mays annexins modulate cytosolic free Ca2+ and generate a Ca2+-permeable conductance.

Regulation of reactive oxygen species and cytosolic free calcium ([Ca(2+)](cyt)) is central to plant function. Annexins are small proteins capable of Ca(2+)-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca(2+)](cyt) when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca(2+)-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca(2+) influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca(2+)-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd(3+) and had a Ca(2+)-to-K(+) permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca(2+) influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca(2+)-permeable transport pathways, regulate [Ca(2+)](cyt), and may function as peroxidases in vitro.

Towards engineering increased pantothenate (vitamin B(5)) levels in plants.

Pantothenate (vitamin B(5)) is the precursor of the 4'-phosphopantetheine moiety of coenzyme A and acyl-carrier protein. It is made by plants and microorganisms de novo, but is a dietary requirement for animals. The pantothenate biosynthetic pathway is well-established in bacteria, comprising four enzymic reactions catalysed by ketopantoate hydroxymethyltransferase (KPHMT), L: -aspartate-alpha-decarboxylase (ADC), pantothenate synthetase (PS) and ketopantoate reductase (KPR) encoded by panB, panD, panC and panE genes, respectively. In higher plants, the genes encoding the first (KPHMT) and last (PS) enzymes have been identified and characterised in several plant species. Commercially, pantothenate is chemically synthesised and used in vitamin supplements, feed additives and cosmetics. Biotransformation is an attractive alternative production system that would circumvent the expensive procedures of separating racemic intermediates. We explored the possibility of manipulating pantothenate biosynthesis in plants. Transgenic oilseed rape (Brassica napus) lines were generated in which the E. coli KPHMT and PS genes were expressed under a strong constitutive CaMV35SS promoter. No significant change of pantothenate levels in PS transgenic lines was observed. In contrast plants expressing KPHMT had elevated pantothenate levels in leaves, flowers siliques and seed in the range of 1.5-2.5 fold increase compared to the wild type plant. Seeds contained the highest vitamin content, indicating that they might be the ideal target for production purposes.