Lipoxygenase from cucumber fruit: Localization and properties

The subcellular localization of lipoxygenase (LOX) from cucumber fruit has been studied. Two methods have been employed to obtain organelles; (1) maceration of the tissue, followed by separation on a linear sucrose gradient and (2) release from protoplasts by osmotic shock, followed by a discontinuous Ficoll gradient. It was possible to obtain high LOX activity in the intact protoplasts from both peel and flesh tissue. However, fewer intact vacuoles were obtained following osmotic rupture than from macerated tissue. Both methods produced more particulate LOX activity from the peel than from flesh tissue, and both showed that this activity was associated with the vacuoles. The cucumber LOX enzyme was similar to the potato and tomato enzymes, both in pH characteristics and substrate specificity.     

Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.     

Does Mutational Robustness Inhibit Extinction by Lethal Mutagenesis in Viral Populations?

Lethal mutagenesis is a promising new antiviral therapy that kills a virus by raising its mutation rate. One potential shortcoming of lethal mutagenesis is that viruses may resist the treatment by evolving genomes with increased robustness to mutations. Here, we investigate to what extent mutational robustness can inhibit extinction by lethal mutagenesis in viruses, using both simple toy models and more biophysically realistic models based on RNA secondary-structure folding. We show that although the evolution of greater robustness may be promoted by increasing the mutation rate of a viral population, such evolution is unlikely to greatly increase the mutation rate required for certain extinction. Using an analytic multi-type branching process model, we investigate whether the evolution of robustness can be relevant on the time scales on which extinction takes place. We find that the evolution of robustness matters only when initial viral population sizes are small and deleterious mutation rates are only slightly above the level at which extinction can occur. The stochastic calculations are in good agreement with simulations of self-replicating RNA sequences that have to fold into a specific secondary structure to reproduce. We conclude that the evolution of mutational robustness is in most cases unlikely to prevent the extinction of viruses by lethal mutagenesis.     

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A₂ receptor

In humans, thromboxane (TX) A₂ signals through the TPα and TPβ isoforms of its G-protein coupled TXA₂ receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA₂ mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.     

Evaluation of attractant flavours for use in oral vaccine baits for badgers (<Emphasis Type="Italic">Meles meles</Emphasis>)

European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis infection (tuberculosis) in Ireland and the UK and are implicated in the transmission of infection to livestock. Vaccination of badgers with the human BCG vaccine (Bacille Calmette Guerin) is considered as an important strategy to reduce the burden of disease in this species, and a pragmatic approach is likely to involve oral vaccination. In this study, we evaluated nine different flavours for use as attractants in a prototype oral vaccine bait for European badgers (M. meles): aniseed, apple, cocoa powder, carob powder, curry, fish, garlic, peanut and strawberry. The bait matrix was composed of a natural lipid formulation, developed as a vehicle for oral vaccination against tuberculosis in wildlife. A ‘food for work’ paradigm was employed during the trials to ensure the animals were actively seeking the baits. The trials showed carob and cocoa powders were equally attractive and more attractive than any of the other candidates. Carob and cocoa show potential as bait attractants for badgers and might form part of a novel vaccine delivery system.     

Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK1: role in endothelial cell migration and angiogenesis

Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein the high-density lipoprotein (HDL) scavenger receptor class B, type 1 (SR-B1) adapter protein PDZ domain-containing protein 1 (PDZK1) was identified as an interactant of the human prostacyclin receptor (hIP) involving a Class I PDZ ligand at its carboxyl terminus and PDZ domains 1, 3, and 4 of PDZK1. Although the interaction is constitutive, it may be dynamically regulated following cicaprost activation of the hIP through a mechanism involving cAMP-dependent protein kinase (PK)A-phosphorylation of PDZK1 at Ser-505. Although PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface, enhancing ligand binding and cicaprost-induced cAMP generation. Consistent with its role in re-endothelialization and angiogenesis, cicaprost activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects completely abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR-B1, small interfering RNA (siRNA)-targeted disruption of PDZK1 abolished cicaprost-mediated endothelial responses but did not affect VEGF responses. Considering the essential role played by prostacyclin throughout the cardiovascular system, identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players, and potentially HDL/SR-B1, within the vascular endothelium.     

Investigating the effects of physiological bile acids on GLP-1 secretion and glucose tolerance in normal and GLP-1R(-/-) mice

Physiological secretion of bile acids has previously been linked to the regulation of blood glucose. GLP-1 is an intestinal peptide hormone with important glucose-lowering actions, such as stimulation of insulin secretion and inhibition of glucagon secretion. In this investigation, we assessed the ability of several bile acid compounds to secrete GLP-1 in vitro in STC-1 cells. Bile acids stimulated GLP-1 secretion from 3.3- to 6.2-fold but some were associated with cytolytic effects. Glycocholic and taurocholic acids were selected for in vivo studies in normal and GLP-1R(-/-) mice. Oral glucose tolerance tests revealed that glycocholic acid did not affect glucose excursions. However, taurocholic acid reduced glucose excursions by 40% in normal mice and by 27% in GLP-1R(-/-) mice, and plasma GLP-1 concentrations were significantly elevated 30 min post-gavage. Additional studies used incretin receptor antagonists to probe involvement of GLP-1 and GIP in taurocholic acid-induced glucose lowering. The findings suggest that bile acids partially aid glucose regulation by physiologically enhancing nutrient-induced GLP-1 secretion. However, GLP-1 secretion appears to be only part of the glucose-lowering mechanism and our studies indicate that the other major incretin GIP is not involved.