Cold atmospheric pressure plasma jet interactions with plasmid DNA

The effect of a cold (<40 °C) radio frequency-driven atmospheric pressure plasma jet on plasmid DNA has been investigated. Gel electrophoresis was used to analyze the DNA forms post-treatment. The experimental data are fitted to a rate equation model that allows for quantitative determination of the rates of single and double strand break formation. The formation of double strand breaks correlates well with the atomic oxygen density. Taken with other measurements, this indicates that neutral components in the jet are effective in inducing double strand breaks.

Altered interactions within FY/AtCPSF complexes required for Arabidopsis FCA-mediated chromatin silencing.

The role of RNA metabolism in chromatin silencing is now widely recognized. We have studied the Arabidopsis RNA-binding protein FCA that down-regulates an endogenous floral repressor gene through a chromatin mechanism involving histone demethylase activity. This mechanism needs FCA to interact with an RNA 3' processing/polyadenylation factor (FY/Pfs2p), but the subsequent events leading to chromatin changes are unknown. Here, we show that this FCA-FY interaction is required for general chromatin silencing roles where hairpin transgenes induce DNA methylation of an endogenous gene. We also show 2 conserved RNA processing factors, AtCPSF100 and AtCPSF160, but not FCA, are stably associated with FY in vivo and form a range of different-sized complexes. A hypomorphic fy allele producing a shorter protein, able to provide some FY functions but unable to interact with FCA, reduces abundance of some of the larger MW complexes. Suppressor mutants, which specifically disrupt the FY motif through which FCA interacts, also lacked these larger complexes. Our data support a model whereby FCA, perhaps after recognition of a specific RNA feature, transiently interacts with FY, an integral component of the canonical RNA 3' processing machinery, changing the interactions of the different RNA processing components. These altered interactions would appear to be a necessary step in this RNA-mediated chromatin silencing.

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

Interactions of 12-lipoxygenase with phospholipase A(2) isoforms following platelet activation through the glycoprotein VI collagen receptor

Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethyl phosphocholine, but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A(2) (PLA(2)) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA(2), (sPLA(2)). Finally, venoms containing sPLA(2) acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA(2) and that 12-LOX functionally associates with both PLA(2) isoforms. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Characterization and parametrization in terms of atomic number of x-ray emission from K-shell filling during ion-surface interactions

When highly charged ions are incident on a surface, part of their potential energy is emitted as characteristic radiation. The energies and yields of these characteristic x rays have been measured for a series of elements at the Tokyo electron-beam ion trap. These data have been used to develop a simple model of the relaxation of the hollow atoms which are formed as the ion approaches the surface, as well as a set of semiempirical scaling laws, which allow for the ready calculation of the K-shell x-ray spectrum which would be produced by an arbitrary slow bare or hydrogenlike ion on a surface. These semiempirical scaling laws can be used to assess the merit of highly charged ion fluorescence x-ray generation in a wide range of applications.

Novel interactions between UFH and TFPI in children

The impact of age upon therapeutic response to unfractionated heparin (UFH) in children is proposed to reflect quantitative and potentially qualitative differences in coagulation proteins across childhood. This study explores the UFH-dependent tissue factor pathway inhibitor (TFPI) release in children compared to previously published data in adults. Children <16 years of age undergoing cardiac angiography formed the population for this prospective cohort study. TFPI release was measured prior to (baseline) and at 15, 30, 45 and 120 min post-UFH dose. This study demonstrated that, whilst the immediate release of TFPI post-UFH was similar in children compared to adults, TFPI release in children remained increased and consistent for a significantly longer period post-UFH administration compared to adults. Plasma TFPI levels in children did not demonstrate an UFH concentration –dependent reduction, as has been previously reported in adults. The prolonged TFPI-mediated anticoagulant levels observed in children administered UFH may contribute to the increased rate of major bleeding reported in children compared to adults. Furthermore, we postulate that this sustained UFH-dependent increase in TFPI levels in children may influence the binding of UFH to competitive plasma proteins, such as those involved in the immunological response to UFH associated with heparin-induced thrombocytopenia.