Mostrando recursos 61 - 80 de 506

  1. Dietary lysine imbalance affects muscle proteome in zebrafish (Danio rerio): a comparative 2D-DIGE study

    Vareilles, Mahaut; Conceição, L. E. C.; Gómez-Requeni, Pedro; Kousoulaki, Katerina; Richard, N.; Rodrigues, Pedro; Fladmark, K. E.; Rønnestad, I.
    Lysine (Lys) is an indispensable amino acid (AA) and generally the first limiting AA in vegetable protein sources in fish feeds. Inadequate dietary Lys availability may limit protein synthesis, accretion and growth of fish. This experiment aimed to further elucidate the role of Lys imbalance on growth by examining the myotomal muscle proteome of juvenile zebrafish (Danio rerio). Quadruplicate groups of 8 fish were fed either a low-Lys [Lys(−), 1.34 gkg−1], medium/control (Lys, 2.47 gkg−1) or high-Lys [Lys(+), 4.63 gkg−1] diet. Fish growth was monitored from 33 to 49 days postfertilization (dpf) and trunk myotomal muscle proteome of Lys(−) and Lys(+) treatments...

  2. Structure and photochemistry of a novel tetrazole-saccharyl conjugate isolated in solid argon

    Ismael, A.; Borba, A.; Duarte, L.; Giuliano, B. M.; Gómez-Zavaglia, A.; Cristiano, Maria Lurdes Santos
    A combined matrix isolation FTIR and theoretical DFT/B3LYP/6-311++G(3df,3pd) study of the novel synthesised tetrazole-saccharyl conjugate 2-[1-(1H-tetrazol-5-yl)ethyl]-1,2-benzisothiazol-3(2H)-one 1,1-dioxide [1-TE-BZT] was performed. In the gas phase, at room temperature, the compound exists as a mixture of six isomeric forms (four conformers of 1H tautomer and two conformers of 2H tautomer). According to theoretical calculations, conformers 1H were the most stable and the relative energies among the three most stable forms are lower than 4 kJ mol 1. These conformers benefit from stabilising intramolecular hydrogen bonds-like interactions involving the 1H of the tetrazole ring and the carbonyl oxygen of the saccharyl moiety. The photochemistry...

  3. Localization of sites of expression of the BAT-1 gene enconding a RNA Helicase

    Leite, Ricardo B.; Gavaia, Paulo J.; Cancela, Leonor
    BAT-1 is a member of the DEAD-box family of ATP-dependent helicases involved in a number of cellular functions including initiation of translation, RNA splicing, and ribosome assembly.

  4. Inclusion of protein hydrolysates in the diet of white seabream (Diplodus sargus) larvae: impacts on skeleton quality and larvae proteome expression

    Richard, N.; De Vareilles, M.; Gavaia, Paulo J.; Silva, Tomé S.; Cordeiro, O.; Yúfera, M.; Rodrigues, Pedro; Conceição, L. E. C.
    In order to investigate the effects of dietary protein hydrolysate on larval skeleton quality and larvae proteome expression, triplicate groups of white seabream (Diplodus sargus) larvae were co-fed from first-feeding onwards with live feed and three microencapsulated diets ir differing only on the molecular weight of their protein hydrolysate fraction.

  5. Expression of osteonectin correlates with levels of fin regeneration in zebrafish (Danio rerio)

    Brito, A. B.; Cancela, Leonor; Gavaia, Paulo J.
    Mammals have the ability to regenerate some tissues such as blood and liver, but the majority of organs fail to regenerate. In contrast, zebrafish is capable of regenerating complex organs/tissues such as optic nerve, scales, heart and fins, and is presently one of the most used metazoan in regeneration research.

  6. Determinism and causative factors for morphological anomalies in reared European fishes

    Boglione, C.; Fontagné, Stéphanie; Gavaia, Paulo J.; Gisbert, Enric; Kjorsvik, E.; Koumoundouros, Giorgos; Moren, M.; Moutou, K. A.; Witten, Paul
    The presence of sublethal morphological deformities represents one of the main bottleneck of the industrial finfish hatchery production, resulting in major economic loss due to reduced growth and marketing ability of the final product, that has to be transformed (filets) or sold for fish flour. Furthermore, the elimination of deformed fishes from the productive cycle needs for periodic selections at present carried out by manual sorting. This represents an additional economic cost, and a stress for fishes.

  7. Identification of an osteopontin-like protein in fish associated with mineral formation

    Fonseca, V. G.; Laizé, Vincent; Valente, M. S.; Cancela, Leonor
    Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones...

  8. Establishment of primary cell cultures from fish calcified tissues

    Marques, C. L.; Rafael, M. S.; Cancela, Leonor; Laizé, Vincent
    Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed...

  9. Serum-specific stimulation of proliferation and mineralization of fish bone-derived cells

    Rosa, J.; Tiago, Daniel M.; Dias, J.; Cancela, Leonor; Laizé, Vincent
    Teleost fish have recently been implemented as suitable model organisms to study vertebrate development, in particular skeletogenesis. In vitro cell systems derived from fish bone have been successfully established, although their development has been hampered by the limited availability of fish serum to supplement culture medium. Commercially available sera are mostly of mammalian origin and thus not necessarily adequate to fish cell growth. The main objective of this work was to compare proliferative and mineralogenic potential of bovine and fish sera using fish bone-derived cell lines VSa13 and VSa16. Fish serum was shown to (i) strongly stimulate cell proliferation in...

  10. Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: Evidence for more than one osteonectin in Salmonidae

    Laizé, Vincent; Pombinho, A. R.; Cancela, Leonor
    Osteonectin is a matricellular protein involved in various cellular mechanisms but its exact function remains unclear despite numerous studies. We present here the cloning of Sparus aurata partial osteonectin cDNA and the reconstruction of 15 other sequences from both vertebrates and invertebrates, almost doubling the set of available sequences (a total of 35 sequences is now available). Taking advantage of the resulting large amount of data, we have created multiple sequence alignments and identified osteonectin putative conserved features (intra- and inter-disulfide bonds, collagen- and calcium-binding domains and phosphorylation sites) likely to be important for protein structure and function. This work...

  11. Identification of Sparus aurata bone morphogenetic protein 2: Molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates

    Rafael, M. S.; Laizé, Vincent; Cancela, Leonor
    Bone morphogenetic protein 2 (BMP-2) is a secreted signaling molecule that acts as an inducer of bone formation and a regulator of embryonic development. The objectives of this work were as follows: (1) to clone the full-length cDNA of BMP-2 in a marine fish model, (2) analyze its gene expression during development, in adult tissues and in cell lines, and (3) identify protein conserved features of vertebrate BMP-2. Using a combination of RT- and 5′-RACE-PCR, a 1653-bp fragment corresponding to Sparus aurata BMP-2 cDNA (SaBMP-2) was amplified. Levels of SaBMP-2 gene expression were estimated using quantitative real-time PCR and shown...

  12. Vanadate proliferative and anti-mineralogenic effects are mediated by MAPK and PI 3K/Ras/Erk pathways in a fish chondrocyte cell line

    Tiago, Daniel M.; Cancela, Leonor; Aureliano, M.; Laizé, Vincent
    We recently reported proliferative and anti-mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF-1, while it inhibits chondrocyte differentiation/mineralization through a putative PI-3K/Ras/Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and...

  13. Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperature

    Ferreira, MC De Jesus; Bao, X.; Laizé, Vincent; Hohmann, S.
    Using transposon mutagenesis in the haploid Saccharomyces cerevisiae strain W303-1A we have identified genes required for growth in high salt medium, survival of a hypo-osmotic shock and growth at 15 degrees C. Screening 25,000 transposon insertions revealed a total of 61 insertions that caused salt-sensitivity; and those insertions affected 31 genes. Only 12 of those genes were previously known to be required for salt-tolerance. Among the 61 insertions, three caused general osmo-sensitivity. We identified one single insertion mutant in the already-known gene, FPS1, required for survival of hypo-osmotic shock. A total of 31 insertions caused failure to grow at low...

  14. Existence of a tightly regulated water channel in saccharomyces cerevisiae

    Meyrial, V.; Laizé, Vincent; Gobin, R.; Ripoche, P.; Hohmann, S.; Tacnet, F.
    The Saccharomyces cerevisiae strain Σ1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the...

  15. Ser3p (Yer081wp) and Ser33p (Yil074cp) are phosphoglycerate dehydrogenases in Saccharomyces cerevisiae

    Albers, E.; Laizé, Vincent; Blomberg, A.; Hohmann, S.; Gustafsson, L.
    Texto integral disponível através do editor

  16. Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

    Pombinho, A. R.; Laizé, Vincent; Molha, D. M.; Marques, S.; Cancela, Leonor
    A growing interest in the understanding of the ontogeny and mineralization of fish skeleton has emerged from the recent implementation of fish as a vertebrate model, particularly for skeletal development. Whereas several in vivo studies dealing with the regulation of bone formation in fish have been published, in vitro studies have been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the development and the characterization of two new cell lines, designated VSa13 and VSa16, derived from the vertebra of the gilthead sea bream. Both cell types exhibit a spindle-like phenotype and slow growth when cultured...

  17. Rapid identification of differentially expressed genes by in situ screening of bacteria

    Fonseca, V. G.; Lago-Lestón, A.; Laizé, Vincent; Cancela, Leonor
    The identification of differentially expressed genes is a key step in the understanding of specific molecular mechanisms. Various methods have been developed to search for differences in expression but most of them are time or money consuming. We present here an alternative technique that connects standard suppression subtractive hybridization with in situ screening of bacteria to isolate and identify differentially expressed transcripts. The in situ differential screening is based on the transfer of bacteria directly from cultures onto nylon membranes with no need of phenol/chloroform extraction, colony lifting, or polymerase chain reaction amplification. This improved method was successfully applied and...

  18. Identification of an osteocalcin isoform in fish with a large acidic prodomain

    Laizé, Vincent; Viegas, C. S. B.; Price, P. A.; Cancela, Leonor
    Osteocalcin is a small, secreted bone protein whose gene consists of four exons. In the course of analyzing the structure of fish osteocalcin genes, we recently found that the spotted green pufferfish has two possible exon 2 structures, one of 15 bp and the other of 324 bp. Subsequent analysis of the pufferfish cDNA showed that only the transcript with a large exon 2 exists. Exon 2 codes for the osteocalcin propeptide, and exon 2 of pufferfish osteocalcin is ∼3.4-fold larger than exon 2 previously found in other vertebrate species. We have termed this new pufferfish osteocalcin isoform OC2. Additional...

  19. Desenvolvimento de sistemas celulares de peixe adequados ao estudo da mineralização in vitro

    Marques, C. L.; Rafael, M. S.; Tiago, Daniel M.; Cancela, Leonor; Laizé, Vincent
    Os peixes foram recentemente reconhecidos como organismos modelo apropriados para o estudo da biologia de vertebrados, particularmente de processos relacionados com a mineralização tecidular e o desenvolvimento do esqueleto. Apesar de existirem alguns estudos in vivo, a identificação de mecanismos associados à calcificação em peixes tem sido prejudicada pelo facto de não existirem sistemas celulares para estudos in vitro. Este artigo descreve um protocolo simples e de baixo custo adequado ao desenvolvimento de culturas celulares mineralogénicas, derivadas de tecidos calcificados de peixes.

  20. Evolution of matrix and bone gamma-carboxyglutamic acid proteins in vertebrates

    Laizé, Vincent; Martel, Paulo; Viegas, C. S. D.; Price, P. A.; Cancela, Leonor
    The evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating the evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. The purpose of this study was to collect and compare sequences of matrix and bone γ-carboxyglutamic acid proteins (MGP and BGP, respectively) to identify common features and determine the evolutionary relationship between MGP and BGP. Thirteen cDNAs and genes were cloned using standard methods or reconstructed through the use of comparative genomics and data mining. These sequences were compared with available annotated sequences (a total of 48 complete or nearly complete...

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