Characterization of the endogenous GIT1-betaPIX complex, and identification of its association to membranes

G protein-coupled receptor kinase interactors (GITs) are adaptor proteins with ADP-ribosylating factor--GTPase-activating protein (ARF-GAP) activity that form complexes with the p21-activated kinase-interacting exchange factor (PIX) guanine nucleotide exchanging factors for Rac and Cdc42. In this study we have characterized the endogenous GIT1/p95-APP1/Cat1 (GIT1)- PIX complexes in neuronal and non-neuronal cells. In COS7 cells, immunocytochemical analysis shows the localization of endogenous GIT1 in the perinuclear region of the cell, as well as at the cell periphery, where GIT1 co-localizes with filamentous actin. The perinuclear localization of endogenous GIT1 was confirmed in avian fibroblasts. In COS7 cells, immunoprecipitation and microsequencing experiments with either anti-GIT1 or anti-betaPIX antibodies unequivocally show that betaPIX is uniquely associated with GIT1 in lysates from these cells, while GIT2/PKL/p95-APP2/Cat2 (GIT2) is undetectable in the endogenous complexes. Moreover, this analysis demonstrates that betaPIX is the limiting factor for the formation of the endogenous complexes, since a small fraction of GIT1 can be co-immunoprecipitated with most betaPIX from these cells. Saponin treatment of unfixed cells indicates that betaPIX-bound GIT1 is preferentially retained in the saponin-resistant fraction when compared to betaPIX-free GIT1. Moreover, analysis by tissue fractionation shows that a significant fraction of the endogenous GIT1-betaPIX complex is firmly associated to membranes from brain homogenates. Our findings show the specific localization of the complex at intracellular membranes, and indicate a correlation between the association of GIT1 to betaPIX, and the localization of the endogenous complex at membranes.     

Population structure of ice-breeding seals

The development of population genetic structure in ice-breeding seal species is likely to be shaped by a combination of breeding habitat and life-history characteristics. Species that return to breed on predictable fast-ice locations are more likely to exhibit natal fidelity than pack-ice-breeding species, which in turn facilitates the development of genetic differentiation between subpopulations. Other aspects of life history such as geographically distinct vocalizations, female gregariousness, and the potential for polygynous breeding may also facilitate population structure. Based on these factors, we predicted that fast-ice-breeding seal species (the Weddell and ringed seal) would show elevated genetic differentiation compared to pack-ice-breeding species (the leopard, Ross, crabeater and bearded seals). We tested this prediction using microsatellite analysis to examine population structure of these six ice-breeding species. Our results did not support this prediction. While none of the Antarctic pack-ice species showed statistically significant population structure, the bearded seal of the Arctic pack ice showed strong differentiation between subpopulations. Again in contrast, the fast-ice-breeding Weddell seal of the Antarctic showed clear evidence for genetic differentiation while the ringed seal, breeding in similar habitat in the Arctic, did not. These results suggest that the development of population structure in ice-breeding phocid seals is a more complex outcome of the interplay of phylogenetic and ecological factors than can be predicted on the basis of breeding substrate and life-history characteristics.     

Linoleic acid increases chorda tympani nerve responses to and behavioral preferences for monosodium glutamate by male and female rats

Previous studies suggest that the chorda tympani nerve (CT) is important in transmitting fat taste information to the central nervous system. However, the contribution of the CT in this process may depend upon the presence of other taste stimuli and/or differ in males and females. Accordingly, the present study investigated the role of the CT in free fatty acid taste processing by examining electrophysiological activity of the CT in response to the free fatty acid linoleic acid (LA), as well as by measuring behavioral responses to LA-taste mixtures. We recorded whole nerve responses from the CT in response to lingual application of LA with or without monosodium glutamate (MSG) in anesthetized male and female rats. In addition, we examined preferences for MSG + LA taste mixtures in behavioral tests. Although lingual application of LA alone did not produce CT whole nerve responses, coapplication of LA and MSG elicited greater CT responses than did MSG alone. These findings were paralleled by greater preferences for MSG + LA taste mixtures than for MSG alone. In both cases, the effect was particularly pronounced in male rats. Thus LA enhances CT activity and behavioral responses to LA + MSG taste mixtures, although there are sex differences in the effects. These results suggest that CT input is important in mediating behavioral responses to fat taste, but the effects depend upon other taste stimuli and differ in males and females.     

The multiple phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two distinct protein lineages

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.     

Leg-length inequality is not associated with greater trochanteric pain syndrome

Introduction  

Greater trochanteric pain syndrome (GTPS) is a common condition, the pathogenesis of which is incompletely understood. Although leg-length inequality has been suggested as a potential risk factor for GTPS, this widely held assumption has not been tested.     

Northwest passages: conservation genetics of Arctic Island wolves

Wolves in the Canadian Arctic Archipelago face several challenges to persistence: a harsh habitat, an unstable prey base, and potentially significant anthropogenic influences. These external factors, if combined with genetic constraints common to island populations, could be particularly difficult to withstand. To determine the genetic status of Arctic Island wolves, we used 14 microsatellite loci to estimate population variation and the extent of inter-island and island-mainland gene flow. All island populations were significantly less variable than mainland wolves; although inbreeding is currently insignificant, the two least variable populations, Banks and the High Arctic (Ellesmere and Devon Islands), showed genetic signatures of recent population declines. Recovery after a bottleneck appears to result, in large part, via recolonization from other islands. These extinction-recolonization dynamics, and the degree of similarity among island wolves revealed by Bayesian clustering, suggest that Arctic Island wolves function as a metapopulation. Persistence of the metapopulation may be supported by periodic migration from mainland populations, occurring primarily through two corridors: Baffin Island in the Eastern Arctic, and Victoria Island in the Western Arctic. This gene flow could be compromised or eliminated by loss—due to climatic warming or increased human activity—of sea ice in the Northwest Passage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plant-nematode interactions: environmental signals detected by the nematode's chemosensory organs control changes in the surface cuticle and behaviour

Plant parasitic nematodes have developed the capacity to sense and respond to chemical signals of host origin and the ability to orientate towards plant roots enhances the nematode's chance of survival. Root exudates contain a range of compounds which mediate belowground interactions with pathogenic and beneficial soil organisms. Chemical components of root exudates may deter one organism while attracting another and these compounds alter nematode behaviour and can either attract nematodes to the roots or result in repellence, motility inhibition or even death. In vitro, plant signals present in root exudates, trigger a rapid alteration of the surface cuticle of Meloidogyne incognita and the same changes were also induced by indole-acetic acid (IAA). IAA binds to the chemosensory organs of M. incognito and it is possible that IAA acts as a signal that orientates the nematode on the root surface in the rhizosphere and/or inside the root tissue and thereby promotes nematode infection.     

WAVECLOCK: wavelet analysis of circadian oscillation

Oscillations in mRNA and protein of circadian clock components can be continuously monitored in vitro using synchronized cell lines. These rhythms can be highly variable due to culture conditions and are non-stationary due to baseline trends, damping and drift in period length. We present a technique for characterizing the modal frequencies of oscillation using continuous wavelet decomposition to non-parametrically model changes in amplitude and period while removing baseline effects and noise. AVAILABILITY: The method has been implemented as the package waveclock for the free statistical software program R and is available for download from http://cran.r-project.org/     

The cost of males in Daphnia pulex

Sex is paradoxical, because asexuals should replace their sexual ancestors by avoiding the demographic cost of producing males (hereafter referred to as the cost‐of‐males). Despite the large body of theoretical and empirical work dealing with the paradox of sex, the cost‐of‐males assumption has been rarely tested. In the present study, we tested the cost‐of‐males assumption in the cladoceran Daphnia pulex. Populations of this species consist of both cyclically parthenogenetic (i.e. sexuals) and obligately parthenogenetic (i.e. asexuals) lineages. In addition, some of the asexual lineages produce only female offspring, whereas others produce functional males, which can mate with sexual females. We compared the reproductive investment of sexuals, male‐producing asexuals, and non‐male‐producing asexuals when raised separately under various environmental conditions. We also determined the outcome of competition between pair‐wise combinations of these reproductive modes. The cost of males was evident when sexual and asexual females were raised separately: sexuals produced fewer female offspring. However, there was no cost of males when reproductive modes were raised in pairs, as sexuals won the competition with asexuals. Our results directly relate to the field conditions experienced by D. pulex. Sexuals might suffer the cost of males at the beginning of the season, when resource competition is low; but when conditions deteriorate as the population approaches carrying capacity, sexuals seem to be better competitors in spite of male production.     

Development of hydrogel-based keratoprostheses: a materials perspective

Research and development of artificial corneas (keratoprostheses) in recent years have evolved from the use of rigid hydrophobic materials such as plastics and rubbers to hydrophilic, water-swollen hydrogels engineered to support not only peripheral tissue integration but also glucose diffusion and surface epithelialization. The advent of the AlphaCor core-and-skirt hydrogel keratoprosthesis has paved the way for a host of new approaches based on hydrogels and other soft materials that encompass a variety of materials preparation strategies, from synthetic homopolymers and copolymers to collagen-based bio-copolymers and, finally, interpenetrating polymer networks. Each approach represents a unique strategy toward the same goal: to develop a new hydrogel that mimics the important properties of natural donor corneas. We provide a critical review of these approaches from a materials perspective and discuss recent experimental results. While formidable technical hurdles still need to be overcome, the rapid progress that has been made by investigators with these approaches is indicative that a synthetic donor cornea capable of surface epithelialization is now closer to becoming a clinical reality.