Contrasting patterns of genetic differentiation in Macaronesian lineages of Ilex (Aquifoliaceae)

Islands offer an interesting framework in which to study the effect of geographical isolation on population genetic differentiation. For plant species with high dispersal abilities, however, oceanic barriers may not represent a factor promoting strong population structure. In this work, we analysed seven nuclear microsatellite loci in Ilex (Aquifoliaceae), a bird‐dispersed plant group, to infer patterns of genetic differentiation among Macaronesian taxa: I. canariensis, I. perado ssp. lopezlilloi, I. perado ssp. platyphylla (Canary Islands) and I. perado ssp. azorica (Azores). In agreement with current taxonomic classification, our results revealed a high genetic differentiation between Ilex lineages (I. canariensis and the I. perado complex), and also supported previous hypotheses that these are the result of independent dispersal events to the islands. In contrast, genetic differentiation between I. perado ssp. azorica and the two subspecies from the Canaries was high, suggesting that taxonomic revision may be necessary. Levels of genetic variation at microsatellite loci in ssp. azorica were, in addition, the lowest reported among Macaronesian bird‐dispersed taxa. Lastly, low genetic differentiation was observed between subspecies occurring on the same island (sspp. platyphylla and lopezlilloi). In summary, our results revealed contrasting patterns between Macaronesian Ilex lineages: I. canariensis displayed moderate population structure across islands, whereas the I. perado complex showed strong differentiation among populations sampled on different islands. Thus, the Macaronesian Ilex taxa show that long‐distance dispersal syndromes (ornithochory) do not always ensure genetic connectivity across large areas in island systems. Plant groups that successfully colonized the islands on multiple occasions may have found barriers to gene flow within certain lineages. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 258–268.     

Functional and Structural Effects of Amyloid-β Aggregate on Xenopus laevis Oocytes

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of “spontaneous” blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca²⁺ was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca²⁺-dependent Cl⁻ currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T_out) and the serum-activated, oscillatory Cl⁻ currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca²⁺-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.     

Tomato T2 ribonuclease LE is involved in the response to pathogens

T2 ribonucleases (RNases) are RNA-degrading enzymes that function in various cellular processes, mostly via RNA metabolism. T2 RNase-encoding genes have been identified in various organisms, from bacteria to mammals, and are most diverse in plants. The existence of T2 RNase genes in almost every organism suggests an important biological function that has been conserved through evolution. In plants, T2 RNases are suggested to be involved in phosphate scavenging and recycling, and are implicated in defence responses to pathogens. We investigated the function of the tomato T2 RNase LE, known to be induced by phosphate deficiency and wounding. The possible involvement of LE in pathogen responses was examined. Expression analysis showed LE induction during fungal infection and by stimuli known to be associated with pathogen inoculation, including oxalic acid and hydrogen peroxide. Analysis of LE-suppressed transgenic tomato lines revealed higher susceptibility to oxalic acid, a cell death-inducing factor, compared to the wild type. This elevated sensitivity of LE-suppressed lines was evidenced by visual signs of necrosis, and increased ion leakage and reactive oxygen species levels, indicating acceleration of cell death. Challenge of the LE-suppressed lines with the necrotrophic pathogen Botrytis cinerea resulted in accelerated development of disease symptoms compared to the wild type, associated with suppressed expression of pathogenesis-related marker genes. The results suggest a role for plant endogenous T2 RNases in antifungal activity.     

Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.     

Morphological and physiological differentiation of seedlings between dry and wet habitats in a tropical dry forest

A common observation in tropical dry forests is the habitat preference of tree species along spatial soil water gradients. This pattern of habitat partitioning might be a result of species differentiation in their strategy for using water, along with competing functions such as maximizing water exploitation and tolerating soil water stress. We tested whether species from drier soil conditions exhibited a tolerance strategy compared with that of wet-habitat species. In a comparison of 12 morphophysiological traits in seedlings of 10 closely related dry and wet-habitat species pairs, we explored what trade-offs guide differentiation between habitats and species. Contrary to our expectations, dry-habitat species showed mostly traits associated with an exploitation strategy (higher carbon assimilation capacity, specific leaf area and leaf-specific conductivity and lower water-use efficiency). Strikingly, dry-habitat species tended to retain their leaves longer during drought. Additionally, we detected multiple strategies to live within each habitat, in part due to variation of strategies among lineages, as well as functional differentiation along the water storage capacity-stem density (xylem safety) trade-off. Our results suggest that fundamental trade-offs guide functional niche differentiation among tree species expressed both within and between soil water habitats in a tropical dry forest.     

Periconceptional alcohol consumption-induced changes in embryonic prostaglandin E levels in mouse organogenesis: modulation by nitric oxide

The mechanisms of the teratogenic effects of maternal alcohol consumption remain unclear. The aim of the present work was to study the organogenic PGE(2) levels and the modulation of PGE(2) levels by NO after periconceptional alcohol ingestion. Female mice were intoxicated with a 10% ethanol in drinking water before pregnancy and up to day 10 of gestation. The PGE(2) released from organogenic embryos was measured by radio immunoassay following incubation with or without the addition of either a NO donor or a NO synthase (NOS) inhibitor. In the ethanol-treated females, we found increased percentages of retarded embryos, associated with a significantly elevated resorption rate (p<0.05), very high quantities of morphologically abnormal E.10 embryos (p<0.001) and significantly increased PGE(2) release, as compared to the embryo parameters of control females. While in the control-derived E.10 embryos the NO donor produced significantly increased PGE(2) release, in the ethanol-derived embryos decreased quantities of PGE(2) were observed. L-NMMA inhibited PGE(2) release in both control and ethanol-derived embryos at different concentrations, whereas it decreased PGE(2) content in controls but not in ethanol-derived embryos. The periconceptional alcohol ingestion produced excessive PGE(2) release, decreased PGE(2) content and disruption of the regulatory NO-PGE(2) pathways. These PGs alterations may be related to delayed organogenesis and abnormal neural tube development after chronic periconceptional consumption of alcohol.     

Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe

A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS/PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4-nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mM was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (K(i) = 0.14 microM), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDI-MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight beta-sheets surrounded by alpha-helical segments in the form of a saddle.     

Diet reveals links between morphology and foraging in a cryptic temperate reef fish

Predators select prey so as to maximize energy and minimize manipulation time. In order to reduce prey detection and handling time, individuals must actively select their foraging space (microhabitat) and populations exhibit morphologies that are best suited for capturing locally available prey. We explored how variation in diet correlates with habitat type, and how these factors influence key morphological structures (mouth gape, eye diameter, fin length, fin area, and pectoral fin ratio) in a common microcarnivorous cryptic reef fish species, the triplefin Helcogrammoides cunninghami. In a mensurative experiment carried out at six kelp‐dominated sites, we observed considerable differences in diet along 400 km of the Chilean coast coincident with variation in habitat availability and prey distributions. Triplefins preferred a single prey type (bivalves or barnacles) at northern sites, coincident with a low diversity of foraging habitats. In contrast, southern sites presented varied and heterogeneous habitats, where triplefin diets were more diverse and included amphipods, decapods, and cumaceans. Allometry‐corrected results indicated that some morphological structures were consistently correlated with different prey items. Specifically, large mouth gape was associated with the capture of highly mobile prey such as decapods, while small mouth gape was more associated with cumaceans and copepods. In contrast, triplefins that capture sessile prey such as hydroids tend to have larger eyes. Therefore, morphological structures co‐vary with habitat selection and prey usage in this species. Our study shows how an abundant generalist reef fish exhibits variable feeding morphologies in response to the distribution of potential habitats and prey throughout its range.     

Aquaporin inhibition changes protein phosphorylation pattern following sperm motility activation in fish

Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl₂, the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl₂(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.     

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes

We evaluated coenzyme Q₁₀ (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n = 39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann–Whitney-U test: p = 0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.