Rho GTPase signaling in Dictyostelium discoideum: insights from the genome

Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches participating in the regulation of many cellular processes. We present an inventory of proteins involved in Rho-regulated signaling pathways in Dictyostelium discoideum that have been identified in the completed genome sequence. In Dictyostelium the Rho family is encoded by 18 genes and one pseudogene. Some of the Rho GTPases (Rac1a/b/c, RacF1/F2 and RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily. The Cdc42 and Rho subfamilies, characteristic of metazoa and fungi, are absent. The activities of these GTPases are regulated by two members of the RhoGDI family, by eight members of the Dock180/zizimin family and by a surprisingly large number of proteins carrying RhoGEF (42 genes) or RhoGAP (43 genes) domains or both (three genes). Most of these show domain compositions not found in other organisms, although some have clear homologs in metazoa and/or fungi. Among the (in many cases putative) effectors found in Dictyostelium are the CRIB domain proteins (WASP and two related proteins, eight PAK kinases and a novel gelsolin-related protein), components of the Scar/WAVE complex, 10 formins, four IQGAPs, two members of the PCH family, numerous lipid kinases and phospholipases, and components of the NADPH oxidase and the exocyst complexes. In general, the repertoire of Rho signaling components of Dictyostelium is similar to that of metazoa and fungi.     

Activity of grape extracts from Greek varieties of Vitis vinifera against mutagenicity induced by bleomycin and hydrogen peroxide in Salmonella typhimurium strain TA102

Several in vivo and in vitro studies have shown that grape extracts could prevent certain steps in carcinogenesis and a few mechanisms have been proposed for this activity. In this study, the potential antimutagenic activity of methanolic and aqueous extracts from two Greek grape varieties of Vitis vinifera against DNA damage induced by reactive oxygen species (ROS) was assessed as a potential novel chemopreventive mechanism, using Salmonella typhimurium strain TA102. The two grape varieties were Assyrtiko (white grapes) and Mandilaria (red grapes), while the oxidant mutagens used were bleomycin (BLM) and hydrogen peroxide (H(2)O(2)). Since it has been considered that polyphenols present in grapes are their most potent biologically active compounds, we also tested the effects of polyphenol-rich fractions as well as some of the more common grape polyphenols on the activity of the two test mutagens. These polyphenols were quercetin, (+)-catechin, (-)-epicatechin, trans-resveratrol, gallic acid and protocatechuic acid. Almost all extracts showed inhibitory activity against both mutagens. On the other hand, polyphenol-rich fractions as well as individual polyphenols at concentrations found in the extracts either did not diminish or did enhance the activity of the mutagens. These results suggest that the protection of DNA from mutations induced by ROS may be one of the mechanisms accounting for the chemopreventive activity of grape extracts. However, it seems that this protective activity may not be attributed to polyphenols but rather to a synergism of many compounds in the grapes.     

Dendritic cell activation and function in response to Schistosoma mansoni

Dendritic cells (DC) are uniquely specialised for both antigen acquisition and presentation, linking innate and adaptive immunity. Their central role in the activation of na?ve T cells gives DC a strategic position in the control of immune responses. While the mechanisms by which viral, bacterial or protozoal pathogens interact with and activate DC are increasingly understood, much less is known about how these cells react to more complex organisms such as schistosomes. Recent studies have examined the impact on DC of antigens from different life cycle stages of Schistosoma mansoni and have revealed a DC phenotype quite distinct to that of conventional activation. Schistosome antigens elicit little of the cytokine secretion and costimulation that are abundantly triggered in DC by unicellular, proinflammatory pathogens and indeed may even actively inhibit such events. The DC response is not a null one, however, since S. mansoni-exposed DC still act as potent antigen presenting cells capable of generating a powerful Th2 immune response. Understanding the interaction between schistosomes and DC is therefore not only addressing fundamental questions of DC biology and immunity to multicellular parasites but also opens the way to therapeutic manipulation of the immune system.     

Selective photostimulation of genetically chARGed neurons.

To permit direct functional analyses of neural circuits, we have developed a method for stimulating groups of genetically designated neurons optically. Coexpression of the Drosophila photoreceptor genes encoding arrestin-2, rhodopsin (formed by liganding opsin with retinal), and the alpha subunit of the cognate heterotrimeric G protein--an explosive combination we term "chARGe"--sensitizes generalist vertebrate neurons to light. Illumination of a mixed population of neurons elicits action potentials selectively and cell-autonomously in its genetically chARGed members. In contrast to bath-applied photostimulants or caged neurotransmitters, which act indiscriminately throughout the illuminated volume, chARGe localizes the responsiveness to light. Distributed activity may thus be fed directly into a circumscribed population of neurons in intact tissue, irrespective of the spatial arrangement of its elements.     

Pathogenicity and proteomic signatures of autoantibodies to Ro and La

Ro/SSA and La/SSB comprise a linked set of autoantigens that are clinically important members of the extractable nuclear antigen family and key translational biomarkers for lupus and primary Sj?gren's syndrome. Autoantibodies directed against the Ro60 and La polypeptide components of the Ro/La ribonucleoprotein complex, and the structurally unrelated Ro52 protein, mediate tissue damage in the neonatal lupus syndrome, a model of passively acquired autoimmunity in humans in which the most serious manifestation is congenital heart block (CHB). Recent studies have concentrated on two distinct pathogenic mechanisms by which maternal anti-Ro/La autoantibodies can cause CHB: by forming immune complexes with apoptotic cells in developing fetal heart; and/or by acting as functional autoantibodies that cross-react with and inhibit calcium channels. Although the precise role of the individual autoantibodies is yet to be settled, maternal anti-Ro60 and anti-Ro52 remain the most likely culprits. This article will discuss the molecular pathways that culminate in the development of CHB, including the recent discovery of β2 glycoprotein I as a protective factor, and present a proteomic approach based on direct mass spectrometric sequencing, which may give a more representative snapshot of the idiotype repertoire of these autoantibodies than genomic-based technologies.     

The radiosensitizing potential of glutaraldehyde on MCF7 breast cancer cells as quantified by means of the G2-chromosomal radiosensitivity assay

Glutaraldehyde (GA) is a high production volume chemical that is very reactive with a wide spectrum of medical, scientific and industrial applications. Concerning the genotoxic and carcinogenic effect of GA, controversial results have been reported, while in humans no studies with positive carcinogenic results for GA have been published. However, our previous study concerning the combined effects of exposure to both GA and ionising radiation (IR) in peripheral blood lymphocytes of healthy donors has shown that non-genotoxic doses of the chemical induces a statistically significant increase in chromosomal radiosensitivity. The lack of information concerning the radiosensitizing potential of GA on cancerous cells triggered us to test the radiosensitizing effect of GA on breast cancer cells (MCF7). For this purpose the G2-chromosomal radiosensitivity assay (G2-assay) was used. The assay involves G2-phase irradiation and quantitation of the chromosomal fragility in the subsequent metaphase. The experimental data show that 48 h exposure to GA, at doses that are not clastogenic to MCF7 breast cancer cells enhances G2-chromosomal radiosensitivity of this cell line. In an effort to evaluate whether the observed increase in GAs-induced G2-chromosomal radiosensitization is linked to GA-induced alterations in the cell cycle and feedback control mechanism, Mitotic Index analysis was performed. The results have shown that such a mechanism cannot be directly related to the observed GA-induced increase in G2-chromosomal radiosensitivity. Since increased G2-chromosomal radiosensitivity has been linked with cancer proneness, the radiosensitizing effect of GA at non-clastogenic doses highlights its potential carcinogenic profile.     

Mink with Divergent Activity Levels have Divergent Reproductive Strategies

Stable individual differences in activity levels within populations have been linked to differences in reproductive rate or parental care in several species, including American mink (Neovison vison). Fur‐farmed mink are good models for studying such effects because they yield large sample sizes and readily allow investigations into maternal behaviour, reproductive success, offspring performance and the relationships between these factors. On farms, very inactive individuals generally have smaller litters, and this held true in our study populations. We tested two competing hypotheses to explain this: (1) inactive individuals are failing to cope with a challenging environment and experiencing chronic stress and/or depression‐like ‘apathy’; this predicts female‐skewed litters, poorer maternal care, higher infant mortality and poorer infant growth and (2) inactive individuals do not have reduced fitness but instead employ an alternative adaptive reproductive strategy, trading off offspring quantity for quality; this predicts enhanced maternal care, reduced infant mortality and enhanced infant growth. Inactive females’ kits, especially their sons, grew faster than active females’, even after statistically controlling for litter size; and by 21 d, inactive and active dams’ litters no longer differed in total biomass, despite the former’s smaller litter sizes. In kit retrieval tests, inactive females were faster than active dams to reach their sons (as well as more likely to contact their sons than their daughters: a bias towards male kits not evident in the active dams). Furthermore, kit growth rates and dam latencies to touch them co‐varied, suggesting the existence of consistent differences in maternal style across inactive and active dams. Hypothesis 2 was thus supported: inactive females favour offspring quality over quantity, investing more resources in fewer kits, particularly males. This potentially boosts their sons’ adult fitness. More broadly for laboratory‐based studies, possible ‘captivity effects’ on the fitness correlates of activity and other personality traits are discussed.     

Differences in kainate receptor involvement in hippocampal mossy fibre long-term potentiation depending on slice orientation

Long-term potentiation (LTP) is a well-established experimental model used to investigate the synaptic basis of learning and memory. LTP at mossy fibre - CA3 synapses in the hippocampus is unusual because it is normally N-methyl-d-aspartate (NMDA) receptor-independent. Instead it seems that the trigger for mossy fibre LTP involves kainate receptors (KARs). Although it is generally accepted that pre-synaptic KARs play an essential role in frequency facilitation and LTP, their subunit composition remains a matter of significant controversy. We have reported previously that both frequency facilitation and LTP can be blocked by selective antagonism of GluK1 (formerly GluR5/Glu(K5))-containing KARs, but other groups have failed to reproduce this effect. Moreover, data from receptor knockout and mRNA expression studies argue against a major role of GluK1, supporting a more central role for GluK2 (formerly GluR6/Glu(K6)). A potential reason underlying the controversy in the pharmacological experiments may reside in differences in the preparations used. Here we show differences in pharmacological sensitivity of synaptic plasticity at mossy fibre - CA3 synapses depend critically on slice orientation. In transverse slices, LTP of fEPSPs was invariably resistant to GluK1-selective antagonists whereas in parasagittal slices LTP was consistently blocked by GluK1-selective antagonists. In addition, there were pronounced differences in the magnitude of frequency facilitation and the sensitivity to the mGlu2/3 receptor agonist DCG-IV. Using anterograde labelling of granule cells we show that slices of both orientations possess intact mossy fibres and both large and small presynaptic boutons. Transverse slices have denser fibre tracts but a smaller proportion of giant mossy fibre boutons. These results further demonstrate a considerable heterogeneity in the functional properties of the mossy fibre projection.     

A predictive model of avian natal dispersal distance provides prior information for investigating response to landscape change

1. Informative Bayesian priors can improve the precision of estimates in ecological studies or estimate parameters for which little or no information is available. While Bayesian analyses are becoming more popular in ecology, the use of strongly informative priors remains rare, perhaps because examples of informative priors are not readily available in the published literature. 2. Dispersal distance is an important ecological parameter, but is difficult to measure and estimates are scarce. General models that provide informative prior estimates of dispersal distances will therefore be valuable. 3. Using a world-wide data set on birds, we develop a predictive model of median natal dispersal distance that includes body mass, wingspan, sex and feeding guild. This model predicts median dispersal distance well when using the fitted data and an independent test data set, explaining up to 53% of the variation. 4. Using this model, we predict a priori estimates of median dispersal distance for 57 woodland-dependent bird species in northern Victoria, Australia. These estimates are then used to investigate the relationship between dispersal ability and vulnerability to landscape-scale changes in habitat cover and fragmentation. 5. We find evidence that woodland bird species with poor predicted dispersal ability are more vulnerable to habitat fragmentation than those species with longer predicted dispersal distances, thus improving the understanding of this important phenomenon. 6. The value of constructing informative priors from existing information is also demonstrated. When used as informative priors for four example species, predicted dispersal distances reduced the 95% credible intervals of posterior estimates of dispersal distance by 8-19%. Further, should we have wished to collect information on avian dispersal distances and relate it to species' responses to habitat loss and fragmentation, data from 221 individuals across 57 species would have been required to obtain estimates with the same precision as those provided by the general model.