Accumulation of 15-deoxy-delta(12,14)-prostaglandin J2 adduct formation with Keap1 over time: effects on potency for intracellular antioxidant defence induction

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelchlike erythroid cell-derived protein with 'capn'collar homologyassociated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very lowconcentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97-99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.     

Interactions of Colletotrichum musae and Lasiodiplodia theobromae and their biocontrol by Pantoea agglomerans and Flavobacterium sp. in expression of crown rot of “Embul” banana

Previous research has shown that two local isolates of bacteria (Pantoea agglomerans and Flavobacterium sp.) are capable of biocontrol of the two main pathogens (Colletotrichum musae and Lasiodiplodia theobromae) known to cause crown rot on “Embul” (Musa, AAB) banana. In this investigation an attempt was made to elucidate the comparative virulence of these pathogens and to determine the underlying biocontrol mechanisms. L. theobromae was more virulent, causing faster spread of the disease, whereas C. musae was more resistant to the bacterial antagonists. Viable cells of the antagonists were more effective at suppressing conidial germination than cell-free culture media. It seemed that antifungal compounds acting on conidial germination may be heat stable and those acting on mycelia may include heat labile compounds also. Considering the specialized roles observed for each pathogen in terms of causing the disease, and in modes of control by antagonists, future field investigations on biocontrol should consider the roles played by the pathogens and the antagonists.

A hydrogen peroxide electrode assay to measure thiol peroxidase activity for organoselenium and organotellurium drugs

Molecular mimics of the enzyme glutathione peroxidase (GPx) are increasingly being evaluated as redox active drugs. Their molecular mechanism of action parallels that of the native enzyme; however, a major distinction is that GPx mimics can use alternative thiol substrates to glutathione. This generic thiol peroxidase activity implies that it is necessary to assess a GPx mimic's recognition of a range of cellular thiols in order to determine its potential therapeutic effects. We report an electrochemical assay that, by measuring the rate of decrease of the peroxide substrate, allows the activity of GPx mimics to be directly compared against an array of thiols. The derived pseudo zero-order rate constants, k(obs), for representative GPx mimics range between 0 and 6.6min(-1) and can vary by more than an order of magnitude depending on the thiol electron donor. An additional advantage of the assay is that it enables synergistic interactions between GPx mimics and cellular proteins to be evaluated. Here we report that glutathione disulfide reductase, which is commonly used to evaluate GPx mimic activity, recognizes the GPx mimic ebselen as a substrate, increasing its apparent k(obs). Therefore, reports relying on glutathione disulfide reductase to evaluate GPx mimic activity may exaggerate drug antioxidant action.     

Mining the archival formalin-fixed paraffin-embedded tissue proteome: opportunities and challenges

The significant potential of tissue-based proteomic biomarker studies can be restricted by difficulties in accessing samples in optimal fresh-frozen form. While archival formalin-fixed tissue collections with attached clinical and outcome data represent a valuable alternate resource, the use of formalin as a fixative which induces protein cross-linking, has generally been assumed to render them unsuitable for proteomic studies. However, this view has been challenged recently with the publication of several papers accomplishing variable degrees of heat-induced reversal of cross-links. Although still in its infancy and requiring the quantitative optimisation of several critical parameters, formalin-fixed tissue proteomics holds promise as a powerful tool for biomarker-driven translational research. Here, we critically review the current status of research in the field, highlighting challenges which need to be addressed for robust quantitative application of protocols to ensure confident high impact inferences can be made.     

Targeted sequencing supports morphology and embryo features in resolving the classification of Cyperaceae tribe Fuireneae s.l.

Molecular phylogenetic studies based on Sanger sequences have shown that Cyperaceae tribe Fuireneae s.l. is paraphyletic. However, taxonomic sampling in these studies has been poor, topologies have been inconsistent, and support for the backbone of trees has been weak. Moreover, uncertainty still surrounds the morphological limits of Schoenoplectiella, a genus of mainly small, amphicarpic annuals that was recently segregated from Schoenoplectus. Consequently, despite ample evidence from molecular analyses that Fuireneae s.l. might consist of two to four tribal lineages, no taxonomic changes have yet been made. Here, we use the Angiosperms353 enrichment panel for targeted sequencing to (i) clarify the relationships of Fuireneae s.l. with the related tribes Abildgaardieae, Eleocharideae, and Cypereae; (ii) define the limits of Fuireneae s.s., and (iii) test the monophyly of Fuireneae s.l. genera with emphasis on Schoenoplectus and Schoenoplectiella. Using more than a third of Fuireneae s.l. diversity, our phylogenomic analyses strongly support six genera and four major Fuireneae s.l. clades that we recognize as tribes: Bolboschoeneae stat.nov., Fuireneae s.s., Schoenoplecteae, and Pseudoschoeneae tr. nov. These results are consistent with morphological, micromorphological (nutlet epidermal cell shape), and embryo differences detected for each tribe. At the generic level, most sub-Saharan African perennials currently treated in Schoenoplectus are transferred to Schoenoplectiella. Our targeted sequencing results show that these species are nested in Schoenoplectiella, and their treatment here is consistent with micromorphological and embryo characters shared by all Schoenoplectiella species. Keys to recognized tribes and genera are provided.