Fractionation of RNA polymerase subspecies from soybean hypocotyls by isoelectric focusing in Sephadex

RNA polymerase enzymes isolated from chromatin of 6-day-old soybean hypocotyls are resolved into two major and one minor species of activity by DEAE-Sephadex chromatography. A soluble form of the enzyme, isolated from the postchromatin supernatant fraction, shows a broad peak of activity when fractionated by this method. The elution characteristics and α-amanitin sensitivity data indicate the two major chromatin-bound activities to be Class I and III enzymes, while the minor chromatin-bound activity and the soluble enzyme are representative of the Class II enzymes. In contrast to these profiles, fractionation of these enzyme preparations by the new method of isoelectric focusing in Sephadex G-15 yields five distinct chromatin-bound and four soluble subspecies. The relationships of these observed activities to their parent DEAE classes are investigated, showing two subspecies within the Class I and III RNA polymerase enzymes, respectively, and four subspecies within the Class II enzymes.     

Protection of large unilamellar vesicles by trehalose during dehydration: retention of vesicle contents

The ability of trehalose and other sugars to maintain the integrity of large unilamellar vesicles subjected to dehydration and rehydration has been investigated. It is shown, employing freeze-fracture techniques, that large unilamellar vesicles prepared in the presence of trehalose at 125 mM or higher concentration do not exhibit significant structural changes during the dehydration-rehydration cycle. Further, up to 90% of entrapped 22Na or [3H]inulin is retained during this process. Other sugars also exhibited similar protective effects where trehalose was most effective, followed by sucrose, maltose, glucose and lactose. It is demonstrated that proton or Na+/K+ electrochemical gradients can be maintained during the dehydration-rehydration process, which can subsequently be used to drive the uptake of lipophilic cationic drugs such as adriamycin. The implications for long-term storage of liposomal systems for use in drug-delivery protocols are discussed.     

What hypothesis tests are not: a reply to Johnson

We are sorry that Johnson's pleasure at reading our recent paper(Colegrave and Ruxton, 2003) was so short-lived. Johnson (2005)is correct that the definition of the P value that we use inthe paper is incorrect, and we are grateful     

A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design

Cyclic nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes that regulate a variety of cellular processes. We describe a family of potent PDE4 inhibitors discovered using an efficient method for scaffold-based drug design. This method involves an iterative approach starting with low-affinity screening of compounds followed by high-throughput cocrystallography to reveal the molecular basis underlying the activity of the newly identified compounds. Through detailed structural analysis of the interaction of the initially discovered pyrazole carboxylic ester scaffold with PDE4D using X-ray crystallography, we identified three sites of chemical substitution and designed small selective libraries of scaffold derivatives with modifications at these sites. A 4,000-fold increase in the potency of this PDE4 inhibitor was achieved after only two rounds of chemical synthesis and the structural analysis of seven pyrazole derivatives bound to PDE4B or PDE4D, revealing the robustness of this approach for identifying new inhibitors that can be further developed into drug candidates.     

Microdialysis studies of extracellular reactive oxygen species in skeletal muscle: factors influencing the reduction of cytochrome c and hydroxylation of salicylate

Identification and quantification of specific reactive oxygen species (ROS) is essential to allow greater understanding into the role that ROS play in tissues and extracellular fluids. Previous studies have examined the reduction of cytochrome c and the hydroxylation of salicylate to detect superoxide and hydroxyl activity, respectively, although the specificity of these assays has been the subject of debate. This study aimed to identify the factors influencing hydroxylation of salicylate and reduction of cytochrome c in microdialysates from skeletal muscle extracellular fluid. Mice were anesthetized and treated with either polyethylene glycol-tagged superoxide dismutase (PEG-SOD), desferrioxamine mesylate (desferal) or N(G)-nitro-l-arginine methyl ester (l-NAME). A further cohort of untreated mice was also studied. Microdialysis probes were placed into the gastrocnemius muscle and perfused with salicylate or cytochrome c prior to, during, and after a period of demanding electrically stimulated contractions. Microdialysates were analysed for the reduction of cytochrome c and hydroxylation of salicylate. Contractile activity was found to increase both the reduction of cytochrome c and the hydroxylation of salicylate in the microdialysates. The reduction of cytochrome c was greater in mice treated with l-NAME compared with control untreated mice and was attenuated in mice treated with PEG-SOD. The hydroxylation of salicylate was attenuated in mice treated with desferal while there was no effect of l-NAME compared with untreated mice. Data support the hypothesis that superoxide and hydroxyl radical activity are the major contributors to the reduction of cytochrome c and hydroxylation of salicylate respectively in microdialysates from skeletal muscle extracellular fluid and indicate that these ROS are increased by contractile activity in skeletal muscle extracellular fluid.     

Inherited eye disease: cause and late effect

Molecular genetics has provided relatively few insights into late-onset eye disorders, but epidemiological data indicate that genetic factors are important in some late-onset eye disorders that cause major health burdens. Much clinical genetic research is based on the belief that developmental and late-onset disorders are not necessarily the result of defects in different genes, but are often caused by different mutations in the same collection of genes. Thus, mutations that either abolish or radically change gene function might cause early-onset disorders, whereas more-subtle changes in gene expression might underlie late-onset diseases. We present arguments and examples that indicate that this principle might be a fruitful guide to investigating the causes of late-onset eye disorders.