Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.     

Genetic Structure of Wild Bonobo Populations: Diversity of Mitochondrial DNA and Geographical Distribution

Bonobos (Pan paniscus) inhabit regions south of the Congo River including all areas between its southerly tributaries. To investigate the genetic diversity and evolutionary relationship among bonobo populations, we sequenced mitochondrial DNA from 376 fecal samples collected in seven study populations located within the eastern and western limits of the species’ range. In 136 effective samples from different individuals (range: 7–37 per population), we distinguished 54 haplotypes in six clades (A1, A2, B1, B2, C, D), which included a newly identified clade (D). MtDNA haplotypes were regionally clustered; 83 percent of haplotypes were locality-specific. The distribution of haplotypes across populations and the genetic diversity within populations thus showed highly geographical patterns. Using population distance measures, seven populations were categorized in three clusters: the east, central, and west cohorts. Although further elucidation of historical changes in the geological setting is required, the geographical patterns of genetic diversity seem to be shaped by paleoenvironmental changes during the Pleistocene. The present day riverine barriers appeared to have a weak effect on gene flow among populations, except for the Lomami River, which separates the TL2 population from the others. The central cohort preserves a high genetic diversity, and two unique clades of haplotypes were found in the Wamba/Iyondji populations in the central cohort and in the TL2 population in the eastern cohort respectively. This knowledge may contribute to the planning of bonobo conservation.     

Home range of newborn blacktip reef sharks (Carcharhinus melanopterus), as estimated using mark-recapture and acoustic telemetry

Coral Reefs - Sharks play important functional roles in coral reef ecosystems. Studying reef shark populations’ spatial ecology also contributes important data for effective conservation...     

Ectomycorrhizal fungal diversity in Quercus ilex Mediterranean woodlands: variation among sites and over soil depth profiles in hyphal exploration types, species richness and community composition

Understanding the factors underlying the distribution of biodiversity is a challenging issue in ecology. Here, we examined the distribution patterns of ectomycorrhizal fungal diversity across the soil profile in three Quercus ilex forests. Contact exploration type strongly dominated at all sites, but was most prevalent in the upper, organic-rich soil layers. At each site, three quarters of the ectomycorrhizal tips and 59 % of taxa were restricted to the ten first centimeters of the soil profile. The relative abundance of the dominant family Russulaceae increased with increasing soil depth. Species composition varied significantly among sites, with most species being rare. Species that occurred in only one of the three sites accounted for 78.9 % of all species, and 57.3 % of species were represented by a single ECM root tip. Our results suggest that (i) rare species at both local and regional scales contribute to the highly diverse fungal assemblages in Mediterranean forests and (ii) multi-sites studies including the whole soil profile are needed to provide comprehensive overviews of the taxonomic and functional diversities of ectomycorrhizal communities.     

Synthesis and FTIR Conformational Studies of Peptides From the Basic Region of c-Jun: a Critical Analysis on the Basis of CD and NMR Data

Abstract

The peptide (35 residues) corresponding to the basic subdomain (bSD) of c-Jun (residues 252–281) and its fragments NP (N-terminal peptide, 1–19) and CP (C-terminal peptide, 1635) were synthesized in stepwise solid-phase using the tert-butyloxycarbonyl/benzyl strategy. In a previous paper, we have shown that during its binding to the DNA site CRE (cAMP- responsive element) the bSD structure was converted into α-helix from an initial random coil conformation [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 231, 370–380 (1995)]. Our results suggested both a high flexibility and a helical potential in bSD, these two properties seeming crucial for the accommodation of the basic subdomain of c-Jun to its specific DNA targets. In this work, we assessed the conformational variability of bSD through the study of the secondary structures of its NP and CP fragments in trifluoroethanol (TFE)/²H₂O mixtures, using Fourier transform infrared (FTIR) spectroscopy. The IR results were critically analyzed in light of our previously reported circular dichroism (CD) and NMR data [Krebs, D., Dahmani, B., Monnot, M., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 235, 699–712 (1996)]. Upon addition of TFE, the relative areas of the seven components of the amide I band (1700–1620 cm^?1) reflected the conversion of a large amount of random coil conformation into α-helix for the two fragments and bSD. This effect was accompanied by more subtle variations of the less populated structures, in agreement with the results of CD and NMR experiments. The IR results stipulated the conservation of the parent bSD secondary structures in both fragments; however, NP and CP peptides did not display similar random-to-α-helix stabilization pattern upon additions of TFE to aqueous solutions. The profile from CD signal at 222 nm was found sigmoidal for NP and almost linear for CP, while that corresponding to the parent peptide bSD was just in between those of its fragments. Thus, the present study confirms the high flexibility and helix propensity of the c-Jun basic subdomain and suggests that the N- and C-terminal parts of the peptide do not follow the same random-to-helix conversion profile during their complexation with DNA.     

An N-terminal, 830 residues intrinsically disordered region of the cytoskeleton-regulatory protein supervillin contains Myosin II- and F-actin-binding sites

Supervillin, the largest member of the villin/gelsolin family, is a cytoskeleton regulating, peripheral membrane protein. Supervillin increases cell motility and promotes invasive activity in tumors. Major cytoskeletal interactors, including filamentous actin and myosin II, bind within the unique supervillin amino terminus, amino acids 1–830. The structural features of this key region of the supervillin polypeptide are unknown. Here, we utilize circular dichroism and bioinformatics sequence analysis to demonstrate that the N-terminal part of supervillin forms an extended intrinsically disordered region (IDR). Our combined data indicate that the N-terminus of human and bovine supervillin sequences (positions 1–830) represents an IDR, which is the largest IDR known to date in the villin/gelsolin family. Moreover, this result suggests a potentially novel mechanism of regulation of myosin II and F-actin via the intrinsically disordered N-terminal region of hub protein supervillin.     

Revising the Absolute Configurations of Coatlines via Density Functional Theory Calculations of Electronic Circular Dichroism Spectra

Coatline A ( 1 ) and α‐epi‐coatline A ( 4 ) co‐occur in the trunk extract of Andira coriacea. Inspection of their chiroptical properties led to intriguing results. After a careful examination of the experimental data used for the previously reported absolute configuration of these compounds, some uncertainties were identified. A combined theoretical approach including conformational analyses and calculation of electronic circular dichroism (ECD) spectra, in addition with experimental data obtained for schoepfin A ( 5 ) and the new schoepfin D ( 6 ) isolated from Senna quinquangulata, allowed the revision of the absolute configuration of coatlines A ( 1 ) and B ( 2 ). Chirality 25:180–184, 2013. © 2012 Wiley Periodicals, Inc.     

Genetic Analysis of the Biosynthesis of 2-Methoxy-3-Isobutylpyrazine,a Major Grape-Derived Aroma Compound Impacting Wine Quality

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.The pleasure experienced while enjoying a glass of wine is the result of sophisticated sensory, neurophysiological, and psychological processes triggered by wine aroma. Wine flavor is the result of a complex mixture of volatile compounds in the headspace of the glass that induces feelings of pleasure at the brain level (Shepherd, 2006). During the last 40 years, over 800 volatile molecules have been formally identified in wines, in concentrations ranging from hundreds of milligrams per liter down to a few picograms per liter (Ebeler and Thorngate, 2009; Styger et al., 2011). Among all of them, a relatively limited number of compounds, called varietal (or primary) aromas, play a crucial role in wine flavor and typicality. These aromas, which are related to the grape variety, belong to a limited number of chemical families, including monoterpenes, C13 norisoprenoids, volatile sulfur compounds, and methoxypyrazines (MPs; Ebeler and Thorngate, 2009). Quite frequently, they exist mostly in the grape (Vitis vinifera) berry as nonvolatile, odorless, “bound” forms that can be released by chemical and enzymatic reactions occurring during the winemaking and wine aging processes, thus enhancing wine’s varietal expression (Styger et al., 2011). Two classical examples are the glycoside precursors of the monoterpenols (Strauss et al., 1986) and the cysteinylated or glutathionylated precursors of the volatile thiols (Tominaga et al., 1998; Peña-Gallego et al., 2012). Noticeable exceptions are the MPs, which are found in grape berries exclusively as free, volatile molecules.MPs are strongly odorant volatile heterocycles, with vegetable-like fragrances, that are widely occurring in the plant kingdom (Maga, 1982). In grape, they can be detected in fruits, leaves, shoots, and roots (Dunlevy et al., 2010). They are found in different grape varieties and are particularly abundant in the so-called Bordeaux cultivars (i.e. cv Cabernet Franc, Cabernet Sauvignon [CS], Sauvignon Blanc, Merlot, and Carménère [Car]; Bayonove et al., 1975; Lacey et al., 1991; Roujou de Boubée et al., 2002; Belancic and Agosin, 2007), whereas they are rarely detected in other cultivars, such as cv Pinot Noir (PN), Chardonnay, or Petit Verdot (PV). This finding indicates a strong genotype dependency of MP biosynthesis (Koch et al., 2010). MPs are accumulated in berries until bunch closure or véraison, and then their level declines after véraison (Hashizume and Samuta, 1999; Ryona et al., 2008). MP concentration in wine is highly correlated with the grape berry content at harvest (Roujou de Boubée et al., 2002). Three MPs are found in grape berries: 2-methoxy-3-isobutylpyrazine (IBMP), which is the most abundant, and two others, 2-methoxy-3-isopropylpyrazine (IPMP) and 2-methoxy-3-sec-butylpyrazine (SBMP; Ebeler and Thorngate, 2009). Both IBMP and IPMP display very low sensory detection thresholds in the wine matrix, ranging from 1 to 16 ng L^–1.MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols (Dubourdieu et al., 2006; Lund et al., 2009). Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these extremely powerful odorants will reduce consumer acceptance (Parr et al., 2007). In red wine, MPs are considered as off-flavor, and red wines can be depreciated by concentrations above 10 ng L^–1 (Allen et al., 1991; Roujou de Boubée et al., 2000; Belancic and Agosin, 2007). Given the importance of MPs, either as typical varietal aromas or as detrimental off-flavors, deciphering the genetic and molecular determinism of their accumulation is of high interest for viticulture.In spite of this, until recently little was known about the MP biosynthesis pathway or the MP biosynthetic genes, either in grapevine or other plant species. Theoretical biosynthesis pathways have been proposed since the mid-1970s. They all start by the addition of an α-dicarbonyl on a branched amino acid (Leu for IBMP, Val for IPMP) to form a 2-hydroxy-3-alkylpyrazine, which is subsequently transformed into the corresponding MP, by a methoxylation reaction (Murray and Whitfield 1975; Gallois et al., 1988). While the initial addition step remains to be demonstrated in plants, an S-adenosyl-l-Met (SAM)-dependent O-methyltransferase (OMT), capable of converting 2-hydroxy-3-isobutylpyrazine (IBHP) into IBMP, has been detected in CS shoots, partially purified and sequenced (Hashizume et al., 2001a, 2001b; Fig. 1). Recently, Dunlevy et al. (2010) characterized two OMTs, VvOMT1 and VvOMT2, capable of methylating IBHP in vitro, albeit with high apparent K_m values. To investigate the genetic bases of MP biosynthesis in grape berries, we performed a quantitative trait loci (QTL) analysis, which has led to the identification of two previously uncharacterized OMTs termed VvOMT3 and VvOMT4. Functional characterization of these two OMTs showed that VvOMT3 was highly specific and efficient for IBHP methylation. Based on its differential expression in high-MP and low-MP grapevine varieties, we propose that VvOMT3 and, to a lesser extent, VvOMT4 are key genes for MP biosynthesis in grapevine berries.Open in a separate windowFigure 1.Putative biosynthesis pathway for IBMP adapted from Hashizume et al. (2001a). SAHcy, S-Adenosyl-l-homo-Cys.