Ryanodine receptor calcium release channels in trophoblasts and their role in cell migration.

Trophoblasts are specialized epithelial cells of the placenta that are involved in invasion, communication and the exchange of materials between the mother and fetus. Cytoplasmic Ca2+ ([Ca2+]c) plays critical roles in regulating such processes in other cell types, but relatively little is known about the mechanisms that control this second messenger in trophoblasts. In the current study, the presence of RyRs and their accessory proteins in placental tissues and in the BeWo choriocarcinoma, a model trophoblast cell-line, were examined using immunohistochemistry and Western immunoblotting. Contributions of RyRs to Ca2+ signalling and to random migration in BeWo cells were investigated using fura-2 fluorescent and brightfield videomicroscopy. The effect of RyR inhibition on reorganization of the F-actin cytoskeleton elicited by the hormone angiotensin II, was determined using phalloidin-labelling and confocal microscopy. RyR1 and RyR3 proteins were detected in trophoblasts of human first trimester and term placental villi, along with the accessory proteins triadin and calsequestrin. Similarly, RyR1, RyR3, triadin and calsequestrin were detected in BeWo cells. In this cell-line, activation of RyRs with micromolar ryanodine increased [Ca2+]c, whereas pharmacological inhibition of these channels reduced Ca2+ transients elicited by the peptide hormones angiotensin II, arginine vasopressin and endothelin 1. Angiotensin II increased the velocity, total distance and Euclidean distance of random migration by BeWo cells and these effects were significantly reduced by tetracaine and by inhibitory concentrations of ryanodine. RyRs contribute to reorganization of the F-actin cytoskeleton elicited by angiotensin II, since inhibition of these channels restores the parallelness of these structures to control levels. These findings demonstrate that trophoblasts contain a suite of proteins similar to those in other cell types possessing highly developed Ca2+ signal transduction systems, such as skeletal muscle. They also indicate that these channels regulate the migration of trophoblast cells, a process that plays a key role in development of the placenta.

Inhibitory Properties of Aldehydes and Related Compounds against Phytophthora infestans-Identification of a New Lead.

The pathogen Phytophthora infestans is responsible for catastrophic crop damage on a global scale which totals billions of euros annually. The discovery of new inhibitors of this organism is of paramount agricultural importance and of critical relevance to food security. Current strategies for crop treatment are inadequate with the emergence of resistant strains and problematic toxicity. Natural products such as cinnamaldehyde have been reported to have fungicidal properties and are the seed for many new discovery research programmes. We report a probe of the cinnamaldehyde framework to investigate the aldehyde subunit and its role in a subset of aromatic aldehydes in order to identify new lead compounds to act against P. infestans. An ellipticine derivative which incorporates an aldehyde (9-formyl-6-methyl ellipticine, 34) has been identified with exceptional activity versus P. infestans with limited toxicity and potential for use as a fungicide.

Synthesis and Evaluation of Novel Ellipticines and Derivatives as Inhibitors of Phytophthora infestans.

The pathogen Phytophthora infestans is responsible for worldwide catastrophic crop damage and discovery of new inhibitors of this organism is of paramount agricultural and industrial importance. Current strategies for crop treatment are inadequate with limitations of efficacy and market alternatives. Ellipticines have recently been reported to have fungicidal properties and have been assessed against P. infestans growth with promising results. We hereby report a probe of the ellipticine framework to investigate the alkyl subunit and screen a set ellipticines and derivatives to identify new lead compounds to act against P. infestans. A series of ellipticinium salt derivatives have been identified with exceptional growth inhibitory activity and apparent lack of toxicity towards a human cell-line surpassing the effect of known and marketed fungicides. This report identifies the potential of this natural product derivative as a novel fungicide.

Polycystic Kidney Disease Ryanodine Receptor Domain (PKDRR) Proteins in Oomycetes.

In eukaryotes, two sources of Ca2+ are accessed to allow rapid changes in the cytosolic levels of this second messenger: the extracellular medium and intracellular Ca2+ stores, such as the endoplasmic reticulum. One class of channel that permits Ca2+ entry is the transient receptor potential (TRP) superfamily, including the polycystic kidney disease (PKD) proteins, or polycystins. Channels that release Ca2+ from intracellular stores include the inositol 1,4,5-trisphosphate/ryanodine receptor (ITPR/RyR) superfamily. Here, we characterise a family of proteins that are only encoded by oomycete genomes, that we have named PKDRR, since they share domains with both PKD and RyR channels. We provide evidence that these proteins belong to the TRP superfamily and are distinct from the ITPR/RyR superfamily in terms of their evolutionary relationships, protein domain architectures and predicted ion channel structures. We also demonstrate that a hypothetical PKDRR protein from Phytophthora infestans is produced by this organism, is located in the cell-surface membrane and forms multimeric protein complexes. Efforts to functionally characterise this protein in a heterologous expression system were unsuccessful but support a cell-surface localisation. These PKDRR proteins represent potential targets for the development of new "fungicides", since they are of a distinctive structure that is only found in oomycetes and not in any other cellular organisms.

Calcium Signaling in Oomycetes: An Evolutionary Perspective.

Oomycetes are a family of eukaryotic microbes that superficially resemble fungi, but which are phylogenetically distinct from them. These organisms cause major global economic losses to agriculture and fisheries, with representative pathogens being Phytophthora infestans, the cause of late potato blight and Saprolegnia diclina, the instigator of "cotton molds" in fish. As in all eukaryotes, cytoplasmic Ca(2+) is a key second messenger in oomycetes, regulating life-cycle transitions, controlling motility and chemotaxis and, in excess, leading to cell-death. Despite this, little is known about the molecular mechanisms regulating cytoplasmic Ca(2+) concentrations in these organisms. Consequently, this review analyzed the presence of candidate calcium channels encoded within the nine oomycete genomes that are currently available. This revealed key differences between oomycetes and other eukaryotes, in particular the expansion and loss of different channel families, and the presence of a phylum-specific group of proteins, termed the polycystic kidney disease tandem ryanodine receptor domain (PKDRR) channels.