An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato

Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases β-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes β-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene β-cyclase, an enzyme that converts lycopene to β-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that β-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.

Genomes OnLine Database (GOLD): a monitor of genome projects world-wide

GOLD is a comprehensive resource for accessing information related to completed and ongoing genome projects world-wide. The database currently provides information on 350 genome projects, of which 48 have been completely sequenced and their analysis published. GOLD was created in 1997 and since April 2000 it has been licensed to Integrated Genomics. The database is freely available through the URL: http://igweb.integratedgenomics.com/GOLD/.

Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization

In humans, SOX9 heterozygous mutations cause the severe skeletal dysmorphology syndrome campomelic dysplasia. Except for clinical descriptions, little is known about the pathogenesis of this disease. We have generated heterozygous Sox9 mutant mice that phenocopy most of the skeletal abnormalities of this syndrome. The Sox9+/− mice died perinatally with cleft palate, as well as hypoplasia and bending of many skeletal structures derived from cartilage precursors. In embryonic day (E)14.5 heterozygous embryos, bending of radius, ulna, and tibia cartilages was already prominent. In E12.5 heterozygotes, all skeletal elements visualized by using Alcian blue were smaller. In addition, the overall levels of Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type embryos. We propose that the skeletal abnormalities observed at later embryonic stages were caused by delayed or defective precartilaginous condensations. Furthermore, in E18.5 embryos and in newborn heterozygotes, premature mineralization occurred in many bones, including vertebrae and some craniofacial bones. Because Sox9 is not expressed in the mineralized portion of the growth plate, this premature mineralization is very likely the consequence of allele insufficiency existing in cells of the growth plate that express Sox9. Because the hypertrophic zone of the heterozygous Sox9 mutants was larger than that of wild-type mice, we propose that Sox9 also has a role in regulating the transition to hypertrophic chondrocytes in the growth plate. Despite the severe hypoplasia of cartilages, the overall organization and cellular composition of the growth plate were otherwise normal. Our results suggest the hypothesis that two critical steps of the chondrocyte differentiation pathway are sensitive to Sox9 dosage. First, an early step presumably at the stage of mesenchymal condensation of cartilage primordia, and second, a later step preceding the transition of chondrocytes into hypertrophic chondrocytes.

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

Chiral gold(I) vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor

The synthesis of a GSK 2nd generation inhibitor of the hepatitis C virus, by enantioselective 1,3-dipolar cycloaddition between a leucine derived iminoester and tert-butyl acrylate, was studied. The comparison between silver(I) and gold(I) catalysts in this reaction was established by working with chiral phosphoramidites or with chiral BINAP. The best reaction conditions were used for the total synthesis of the hepatitis C virus inhibitor by a four step procedure affording this product in 99% ee and in 63% overall yield. The origin of the enantioselectivity of the chiral gold(I) catalyst was justified according to DFT calculations, the stabilizing coulombic interaction between the nitrogen atom of the thiazole moiety and one of the gold atoms being crucial.

Binap-gold (I) versus Binap-silver trifluoroacetate complexes as catalysts in 1,3-dipolar cycloadditions of axomethine ylides

The 1,3-dipolar cycloaddition between azomethine ylides and alkenes is efficiently catalysed by [{(Sa)-Binap-Au(tfa)}2] (Binap=2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; tfa=trifluoroacetyl). Maleimides, 1,2-bis(phenylsulfonyl)ethylene, chalcone and nitrostyrene were suitable dipolarophiles even when using sterically hindered 1,3-dipole precursors. The results obtained in these transformations improve the analogous ones obtained in the same reactions catalysed by [Binap–Ag(tfa)]. In addition, computational studies have also been carried out to demonstrate both the high enantioselectivity exhibited by the chiral gold(I) complex, and the non-linear effect observed in this transformation.

Calibration of the length of a chain of single gold atoms

Using a scanning tunnelling microscope or mechanically controllable break junction it has been shown that it is possible to control the formation of a wire made of single gold atoms. In these experiments an interatomic distance between atoms in the chain of ∼3.6 Å was reported which is not consistent with recent theoretical calculations. Here, using precise calibration procedures for both techniques, we measure the length of the atomic chains. Based on the distance between the peaks observed in the chain length histogram we find the mean value of the interatomic distance before chain rupture to be 2.5±0.2 Å. This value agrees with the theoretical calculations for the bond length. The discrepancy with the previous experimental measurements was due to the presence of He gas, that was used to promote the thermal contact, and which affects the value of the work function that is commonly used to calibrate distances in scanning tunnelling microscopy and mechanically controllable break junctions at low temperatures.

Reutilización del Treebank de dependencias del euskera para la construcción del gold standard de la sintaxis superficial de la gramática de restricciones (CG)

El objetivo del trabajo consiste en reutilizar el Treebank de dependencias EPECDEP (BDT) para construir el gold standard de la sintaxis superficial del euskera. El paso básico consiste en el estudio comparativo de los dos formalismos aplicados sobre el mismo corpus: el formalismo de la Gramática de Restricciones (Constraint Grammar, CG) y la Gramática de Dependencias (Dependency Grammar, DP). Como resultado de dicho estudio hemos establecido los criterios lingüísticos necesarios para derivar la funciones sintácticas en estilo CG. Dichos criterios han sido implementados y evaluados, así en el 75% de los casos se derivan automáticamente las funciones sintácticas para construir el gold standard.

Understanding the structure of the first atomic contact in gold

We have studied experimentally jump-to-contact (JC) and jump-out-of-contact (JOC) phenomena in gold electrodes. JC can be observed at first contact when two metals approach each other, while JOC occurs in the last contact before breaking. When the indentation depth between the electrodes is limited to a certain value of conductance, a highly reproducible behaviour in the evolution of the conductance can be obtained for hundreds of cycles of formation and rupture. Molecular dynamics simulations of this process show how the two metallic electrodes are shaped into tips of a well-defined crystallographic structure formed through a mechanical annealing mechanism. We report a detailed analysis of the atomic configurations obtained before contact and rupture of these stable structures and obtained their conductance using first-principles quantum transport calculations. These results help us understand the values of conductance obtained experimentally in the JC and JOC phenomena and improve our understanding of atomic-sized contacts and the evolution of their structural characteristics.

Synthetic scope and DFT analysis of the chiral binap-gold (I) complex-catalyzed 1,3-dipolar cycloaddition of azlactones with alkenes

The 1,3-dipolar cycloaddition between glycine-derived azlactones with maleimides is efficiently catalyzed by the dimeric chiral complex [(Sa)-Binap·AuTFA]2. The alanine-derived oxazolone only reacts with tert-butyl acrylate giving anomalous regiochemistry, which is explained and supported by Natural Resonance Theory and Nucleus Independent Chemical Shifts calculations. The origin of the high enantiodiscrimination observed with maleimides and tert-butyl acrylate is analyzed using DFT computed at M06/Lanl2dz//ONIOM(b3lyp/Lanl2dz:UFF) level. Several applications of these cycloadducts in the synthesis of new proline derivatives with a 2,5-trans-arrangement and in the preparation of complex fused polycyclic molecules are described.

Electrochemical analysis of the performance of carbon supported Pd nanoparticles for direct formic acid fuel cells: from gold supported electrodes to catalyst-coated membranes

In this work carbon supported Pd nanoparticles were prepared and used as electrocatalysts for formic acid electrooxidation fuel cells. The influence of some relevant parameters such as the nominal Pt loading, the Nafion/total solids ratio as well as the Pd loading towards formic acid electrooxidation was evaluated using gold supported catalytic layer electrodes which were prepared using a similar methodology to that employed in the preparation of conventional catalyst coated membranes (CCM). The results obtained show that, for constant Pd loading, the nominal Pd loading and the Nafion percentage on the catalytic layer do not play an important role on the resulting electrocatalytic properties. The main parameter affecting the electrocatalytic activity of the electrodes seems to be the Pd loading, although the resulting activity is not directly proportional to the increased Pd loading. Thus, whereas the Pd loading is multiplied by a factor of 10, the activity is only twice which evidences an important decrease in the Pd utilization. In fact, the results obtained suggest the active layer is the outer one being clearly independent of the catalytic layer thickness. Finally, catalyst coated membranes with Pd catalyst loadings of 0.1, 0.5 and 1.2 mg cm-2 were also tested in a breathing direct formic acid fuel cell.

Colloidal gold immobilized on mesoporous silica as a highly active and selective catalyst for styrene epoxidation with H2O2

Colloidal gold nanoparticles were synthesized by different procedures affording suspensions with two different mean sizes (2 and 5 nm). Au catalysts were prepared by sol immobilization onto several silica frameworks with different 2D and 3D mesoporosities. The catalysts were tested in styrene oxidation reactions showing excellent efficiency and selectivity. The effect of nanoparticle size and mesoporous framework on the physical and catalytic properties of the final materials was studied. The most selective catalyst was prepared from the 5 nm Au nanoparticles and the more interconnected silica framework (3D mesoporosity).

Modeling contact formation between atomic-sized gold tips via molecular dynamics

The formation and rupture of atomic-sized contacts is modelled by means of molecular dynamics simulations. Such nano-contacts are realized in scanning tunnelling microscope and mechanically controlled break junction experiments. These instruments routinely measure the conductance across the nano-sized electrodes as they are brought into contact and separated, permitting conductance traces to be recorded that are plots of conductance versus the distance between the electrodes. One interesting feature of the conductance traces is that for some metals and geometric configurations a jump in the value of the conductance is observed right before contact between the electrodes, a phenomenon known as jump-to-contact. This paper considers, from a computational point of view, the dynamics of contact between two gold nano-electrodes. Repeated indentation of the two surfaces on each other is performed in two crystallographic orientations of face-centred cubic gold, namely (001) and (111). Ultimately, the intention is to identify the structures at the atomic level at the moment of first contact between the surfaces, since the value of the conductance is related to the minimum cross-section in the contact region. Conductance values obtained in this way are determined using first principles electronic transport calculations, with atomic configurations taken from the molecular dynamics simulations serving as input structures.

Superior performance of gold supported on doped CeO2 catalysts for the preferential CO oxidation (PROX)

Herein, the preferential oxidation of CO in excess hydrogen (PROX reaction) was studied over Au catalysts supported on ceria and Y or Nb doped ceria. Both supports and catalysts have been extensively characterized by a number of advanced techniques; XRD, N2-adsortion, Raman spectroscopy, XPS, and H2-TPR. The catalytic results showed that when an ideal mixture of H2 and CO is used for the PROX reaction the gold supported on pure ceria behaves better than the others samples. However, when a typical reformate gas composition containing CO2 and H2O is used, the gold supported on Nb doped sample behaves better than gold supported in pure ceria. It is suggested that niobium hampers the strong adsorption of CO2 and H2O in the active sites, thus improving the catalytic performance in real reformate gas.