One-electron oxidation of "photo-Fenton" reagents and inhibition of lipid peroxidation

The "photo-Fenton" reagent, 2-mercaptopyridine N-oxide (MPO), which releases a hydroxyl radical on ultraviolet irradiation, has been found to act as an antioxidant. In the peroxidation of linoleate initiated by a water-soluble azo-initiator, MPO has about one-third the activity of the water-soluble vitamin E analogue Trolox C. In contrast, the oxygen-containing analogue, 2-hydroxypyridine N-oxide (HPO), does not have measurable antioxidant activity in this system. Both reagents react with hydroxyl radical with second order rate constants very close to the diffusion-controlled limit. With the less oxidising dithiocyanate radical anion, MPO reacts approximately 50 times more rapidly than HPO at pH>7. The more reducing properties of MPO result in its activity as an antioxidant and make it less suitable than HPO as a source of hydroxyl radicals for investigation of oxidative stress in biological systems.     

The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A2 at 1.5 A resolution provides an insight into the catalytic mechanism

The human group IIA secreted PLA(2) is a 14 kDa calcium-dependent extracellular enzyme that has been characterized as an acute phase protein with important antimicrobial activity and has been implicated in signal transduction. The selective binding of this enzyme to the phospholipid substrate interface plays a crucial role in its physiological function. To study interfacial binding in the absence of catalysis, one strategy is to produce structurally intact but catalytically inactive mutants. The active site mutants H48Q, H48N, and H48A had been prepared for the secreted PLA(2)s from bovine pancreas and bee venom and retained minimal catalytic activity while the H48Q mutant showed the maximum structural integrity. Preparation of the mutant H48Q of the human group IIA enzyme unexpectedly produced an enzyme that retained significant (2-4%) catalytic activity that was contrary to expectations in view of the accepted catalytic mechanism. In this paper it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions including assays in the presence of Co(2+) and Ni(2+) in place of Ca(2+). The crystallization of the H48Q mutant, yielding diffraction data to a resolution of 1.5 A, allowed a comparison with the corresponding recombinant wild-type enzyme (N1A) that was also crystallized. This comparison revealed that all of the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins, and the catalytic water molecule (w6) was clearly resolved in the H48Q mutant. We propose that a variation of the calcium-coordinated oxyanion ("two water") mechanism involving hydrogen bonding rather than the anticipated full proton transfer to the histidine will best explain the ability of an active site glutamine to allow significant catalytic activity.     

Field-based evaluation of biopesticide impacts on native biodiversity: malagasy coleoptera and anti-locust entomopathogenic fungi

A community of 225 species of Coleoptera was used as a surrogate to evaluate nontarget effects of entomopathogenic fungi under development as biopesticides for use against the Malagasy migratory locust Locusta migratoria capito Saussure (Orthoptera: Acrididae). Evaluation of a standard chemical treatment of fenitrothion + esfenvalerate, two indigenous isolates of Metarhiziumflavoviride Gams & Roszsypol (SP3 and SP9), and an indigenous isolate of Beauveria bassiana (Balsamo) Vuillemin (SP16) against an untreated control in a replicated field trial in southern Madagascar showed that one of the isolates of M. flavoviride (SP3) and fenitrothion + esfenvalerate had distinct effects on nontarget beetle communities that were similar to each other. The other two isolates had no detectable effects compared with the untreated control. Based on an evaluation of the species affected, the similar effects of SP3 and the chemical pesticide are hypothesized to be the result of a perturbation of predator-prey relationships, with a distinct tendency to be manifested via predators. The data indicate that use of SP9 and SP16 would have minimal detrimental effects on the biodiversity of nontarget beetles, but that SP3 needs further testing.     

MHCPred: bringing a quantitative dimension to the online prediction of MHC binding

The accurate prediction of T cell epitopes is one of the key aspirations of immunoinformatics. We have developed a partial least squares-based, robust multivariate statistical method for the quantitative prediction of peptide binding to major histocompatibility complexes (MHCs), the principal checkpoint on the antigen presentation pathway. As a service to the immunobiology community, we have made a Perl implementation of the method available as a World Wide Web server.     

Open pore block of connexin26 and connexin32 hemichannels by neutral, acidic and basic glycoconjugates

The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26-Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.     

Development of rhythmic melatonin secretion from the pineal gland of embryonic mummichog (Fundulus heteroclitus)

The pineal gland of vertebrates produces and secretes the hormone melatonin in response to changes in the light-dark cycle, with high production at night and low production during the day. Melatonin is thought to play an important role in synchronizing daily and/or seasonal physiological, behavioral, and developmental rhythms in vertebrates. In this study, the functional development of the pineal melatonin-generating system was examined in the mummichog, Fundulus heteroclitus, an euryhaline teleost. In this species, the pineal gland contains an endogenous oscillator, ultimately responsible for timing the melatonin rhythm. Oocytes from gravid females were collected and fertilized in vitro from sperm collected from mature males. Skull caps containing attached pineal glands were obtained from F. heteroclitus embryos at different embryonic stages and placed in static or perfusion culture under various photoperiodic regimes. Rhythmic melatonin secretion from pineal glands of embryonic F. heteroclitus embryos exposed to a 12L:12D cycle in static culture was observed at five days post-fertilization. The ontogeny of circadian-controlled melatonin production from F. heteroclitus pineal glands exposed to constant darkness for five days was also seen at day five post-fertilization. These data show that early development of the pineal melatonin-generating system in this teleost occurs prior to hatching. Pre-hatching development of the melatonin-generating system may confer some selective advantage in this species in its interactions with the environment.     

A high-throughput method for enzyme kinetic studies

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.     

Towards an e-biology of ageing: integrating theory and data

Ageing is a highly complex process; it involves interactions between numerous biochemical and cellular mechanisms that affect many tissues in an organism. Although work on the biology of ageing is now advancing quickly, this inherent complexity means that information remains highly fragmented. We describe how a new web-based modelling initiative is seeking to integrate data and hypotheses from diverse biological sources.     

A relational database for the discovery of genes encoding amino acid biosynthetic enzymes in pathogenic fungi

Fungal phytopathogens continue to cause major economic impact, either directly, through crop losses, or due to the costs of fungicide application. Attempts to understand these organisms are hampered by a lack of fungal genome sequence data. A need exists, however, to develop specific bioinformatics tools to collate and analyse the sequence data that currently is available. A web-accessible gene discovery database (http://cogeme.ex.ac.uk/biosynthesis.html) was developed as a demonstration tool for the analysis of metabolic and signal transduction pathways in pathogenic fungi using incomplete gene inventories. Using Bayesian probability to analyse the currently available gene information from pathogenic fungi, we provide evidence that the obligate pathogen Blumeria graminis possesses all amino acid biosynthetic pathways found in free-living fungi, such as Saccharomyces cerevisiae. Phylogenetic analysis was also used to deduce a gene history of succinate-semialdehyde dehydrogenase, an enzyme in the glutamate and lysine biosynthesis pathways. The database provides a tool and methodology to researchers to direct experimentation towards predicting pathway conservation in pathogenic microorganisms.