Molecular and functional characterization of a SCD 1b from European sea bass (Dicentrarchus labrax L.).

Fatty acid desaturation is a highly complex and regulated process involving different molecular and genetic actors. Ultimally, the fatty acid desaturase enzymes are responsible for the introduction of double bonds at different positions of specific substrates, resulting in a wide variety of mono- and poly-unsaturated fatty acids. This substrate-specificity makes it possible to meet all the functional needs of the different tissues against a wide variety of internal and external conditions, giving rise to a varied profile of expression and functionality of the different desaturases in the body. Being our main interest to study and characterize at the molecular level the fatty acid desaturation process in fishes, we have focused our effort on characterizing SCD 1b from European sea bass (Dicentrarchus labrax, L.). In this work, we have characterized a tearoyl-CoA Desaturase cDNA that codes a protein of 334 amino acids, which shares the greatest homology to marine fish SCD 1b. Northern blot analysis showed two transcripts of 3.5kb and 1.4kb. Two putative cis-acting conserved motifs are localized in the cDNA 5'-end: a polypyrimidine CT dinucleotide repeat tract and two non-palindromic putative NRL-response elements (NREs). The deduced protein presents two Delta9 FADs like domain, three His-rich motifs, a total of nine His residues acting as di‑iron coordination ligands. The SCD 1b 3D protein modelling shows a structure made up primarily of alpha-helices, four of which could be transmembrane helices. The catalytic region is oriented to the cytosolic side of the Endoplasmic Reticulum membrane, where the 9-histidine residues are arranged coordinated to two non-heme Fe2+ ions. A new His-containing motif NX3H-like includes an Asn residue that participates in the coordination of Fe2+1 through a water molecule. The protein has a large pocket with a large opening to the outside. It includes a tunnel in which the substrate-binding site is located. The external shape is reminiscent of a boathook. It shows group specificity, although a greater preference for 18C substrates. The length of the tunnel, delimited by seven amino acids that forms a pocket at the end of the tunnel, the possibility that the substrates adopt different conformations inside the tunnel as well as and the movement of acyl chain inside the tunnel, could explain the high preference for 18C fatty acids and the group specificity of the enzyme. The cDNA encodes a functional SCD enzyme, whose subcellular localization is the Endoplasmic Reticulum, which complements the ole1Delta gene-disrupted gene in DTY-11A Saccharomyces cerevisiae strain and produces an increment of palmitoleic and oleic acids. The scd 1b gene is expressed in all tested tissues, showing the liver and adipose tissue a higher level of expression against the brain, heart, gonad and intestine. Scd 1b expression was always bigger than those of the Delta6 fad gene, being especially significant in adipose tissue and liver. From our data, we conclude that, in contrast to the functional significance of SCD 1b in adipose tissue, liver and heart, Delta6 FAD seems to play a more determining role in the biosynthesis of unsaturated fatty acids in the intestine, brain and gonad in fish

Nitric oxide synthase-dependent immune response against gram negative bacteria in a crustacean, Litopenaeus vannamei

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In previous studies, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide in vitro e in vivo. Hyperimmune serum was obtained from rabbits immunized with a P. argus eNOS fragment of 31 kDa produced in Escherichia coli, which specifically detected the recombinant polypeptide and the endogenous NOS from lobster hemocytes by western blotting and immunofluorescence. In the present work, we demonstrate that the hyperimmune serum obtained against P. argus NOS also recognizes Litopenaeus vannamei NOS in hemocytes by western blotting and immunofluorescence. Our data also show that while the hemolymph of L. vannamei has a strong antibacterial activity against the Gram negative bacteria Aeromonas hydrophila, the administration of the anti NOS serum reduce the natural bacterial clearance. These results strongly suggest that NOS is required for the shrimp immune defense toward Gram negative bacteria. Therefore, the monitoring of induction of NOS could be an important tool for testing immunity in shrimp farming

Cloning, tissue expression pattern and daily rhythms of Period1, Period2, and Clock transcripts in the XatWsh Senegalese sole, Solea senegalensis

An extensive network of endogenous oscillators governs vertebrate circadian rhythmicity. At the molecular level, they are composed of a set of clock genes that participate in transcriptional–translational feedback loops to control their own expression and that of downstream output genes. These clocks are synchronized with the environment, although entrainment by external periodic cues remains little explored in Wsh. In this work, partial cDNA sequences of clock genes representing both positive (Clock) and negative (Period1, Period2) elements of the molecular feedback loops were obtained from the nocturnal XatWsh Senegalese sole, a relevant species for aquaculture and chronobiology. All of the above genes exhibited high identities with their respective teleost clock genes, and Per– Arnt–Sim or basic helix–loop–helix binding domains were recognized in their primary structure. They showed a widespread distribution through the animal body and some of them displayed daily mRNA rhythms in central (retina, optic tectum, diencephalon, and cerebellum) and peripheral (liver) tissues. These rhythms were most robust in retina and liver, exhibiting marked Period1 and Clock daily oscillations in transcript levels as revealed by ANOVA and cosinor analysis. Interestingly, expression proWles were inverted in retina and optic tectum compared to liver. Such diVerences suggest the existence of tissue-dependent zeitgebers for clock gene expression in this species (i.e., light for retina and optic tectum and feeding time for liver). This study provides novel insight into the location of the molecular clocks (central vs. peripheral) and their diVerent phasing and synchronization pathways, which contributes to better understand the teleost circadian systems and its plasticity

A functional light-entrainable molecular clock is revealed in gilthead seabream (Sparus aurata) from early developmental stages using an embryonic stem cell line

Like other vertebrates, fish exhibit physiological and behavioural rhythms that are synchronized by the cyclic variations in the external environment, being photoperiod, i.e., the alternation of light and dark during the 24-h cycle, the most predictable and powerful environmental cue. Many of these rhythms are endogenous and controlled by a molecular clock present in cells from early developmental stages. In a previous study, we have developed and characterised a new monoclonal embryonic stem cell line, SAEC-H7, derived from the morula stage embryos of a marine teleost, the gilthead seabream (Sparus aurata), a species with high commercial value for European aquaculture. In the present study, we aimed to identify the main components from the positive (clock and bmal1) and negative (cry1a, per1, per2 and per3) feedback loops of the molecular clock in this embryonic cell line and characterise putative daily variations and circadian rhythms in their expression under different photoperiod regimes, as well as the effects of nocturnal light pulses on their transcriptional activation. Our results revealed that SAEC-H7 cells contain a molecular clock that is entrained by the daily light-dark (LD) cycle. All clock genes analysed showed daily rhythms under LD conditions and were able to re-entrain to a 12-h phase shift (DL) in the photocycle, some of them (clock, bmal1, per1, per3) exhibiting circadian rhythms that persisted over a certain period under constant conditions, although with affected amplitudes and acrophases. Both cry1a and per2 genes showed a statistically significant increase in expression after a nocturnal light pulse, confirming the light-inducible character of these clock genes. The results obtained reveal that seabream embryonic cells are highly sensitive to light and already contain the phototransduction pathways that entrain the molecular clock and overt rhythms from the morula stage, making this embryonic cell line a useful tool for developmental and chronobiological studies in fish. Our results also reinforce the importance of maintaining natural cyclic light conditions in in vitro studies performed with fish cell lines as well as in embryonic and larval rearing protocols in aquaculture practices

The Biological Clock of Sole: From Early Stages to Adults

En el presente capítulo de libro se realiza una revisión de los estudios relacionados con las bases moleculares de los ritmos circadianos en peces, centrándose en el lenguado senegalés como modelo de estudio en cronobiología. Se centra en la clonación de los principales genes reloj, el análisis de las secuencias, la expresión en diversos tejidos centrales y periféricos así como durante la ontogenia del lenguado

Differential effects of transient constant light-dark conditions on daily rhythms of Period and Clock transcripts during Senegalese sole metamorphosis

En este estudio se investigó el funcionamiento del reloj biológico durante una etapa crucial en el desarrollo del lenguado: la metamorfosis. Además, se determinaron los efectos de condiciones transitorias de luz y oscuridad constantes. Todos los genes reloj estudiados mantuvieron los ritmos de expresión durante la metamorfosis, aunque con amplitudes menores que en otros periodos de desarrollo. Se observaron efectos prolongados de las condiciones continuas de iluminación u oscuridad en la expresión de dichos genes

The clock gene Period3 in the nocturnal flatfish Solea senegalensis: Molecular cloning, tissue expression and daily rhythms in central areas

En este trabajo se clonó el gen period3 en el lenguado senegalés, perteneciente a la maquinaria molecular del reloj biológico en peces. Se analizó la secuencia obtenida, incluyendo el árbol filogenético. Además, se estudió su expresión diaria en diversos tejidos tanto centrales como periféricos, revelando ritmos robustos de expresión en la mayoría de ellos, con acrofases al final de la noche-principio del día

The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

En este trabajo se determinó el momento de inicio del reloj endógeno en el lenguado senegalés durante los primeros días de desarrollo y cómo estaba influido por las condiciones de iluminación (ciclos de luz-oscuridad, luz continua y oscuridad continua). Se detectaron ritmos de expresión de genes reloj durante el primer día de desarrollo, con un ajuste gradual de las fases al fotoperiodo durante los días posteriores. Ritmos similares a los tejidos neurales adultos se alcanzaron durante el día 4 de desarrollo. Las condiciones de luz/oscuridad continua afectaron de forma muy patente la expresión y sincronización de los componentes del reloj biológico