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Τo exploit the solid residue of olive mill wastewaters (OMWS) towards the production of an olive paste-type product, natural fermentation of OMWS in NaCl (6% w/v) was implemented for 14 days at 20 °C. The microbiological and physicochemical characteristics of OMWS were examined during fermentation. Fermentation of OMWS was shown to be relied on yeasts while absence of enterobacteria, pseudomonads and staphylococci as well as LAB was observed. Molecular identification of yeast included RFLP analysis of the 5.8S-ITS region. For some selected isolates, sequence analysis of the 5.8S-ITS region was performed. The results were compared to sequences held in public databases (NCBI) and data of papers published previously. These techniques allowed to identify the isolates as Saccharomyces cerevisiae (99% homology) and Pichia membranefasciens (100% homology). Beta-glucosidase was the most important biochemical activity observed, mainly responsible for removing the bitter taste. Paste preparations of fermented OMWS did not differ in sensory characteristics than the controls.

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.

The correlation between the natural levels of GST and the tolerance to the insecticide decamethrin (dMT), as well as the interaction between the molecules of affinity purified enzyme and the insecticide were investigated in order to collect further information on the obscure role of the Glutathione S-transferase system (GST) as a mechanism of defence against pyrethroids. The studies were carried out, comparatively, on the larvae and pupae developmental stages of the coleopteran Tenebrio molitor, which exhibit varying natural levels of GST activity. No stage dependent susceptibility of the insect against pyrethroid insecticides was found during the first 24 h, however 48 h after treatment, the KD50 dose increased significantly due to the recovery of some individuals from the larvae stage. Simultaneous injection of decamethrin with compounds which inhibit GST activity in vitro, resulted in an increased tolerance, which was more pronounced in the pupae stage. Inhibition studies combined with competitive fluorescence spectroscopy and high pressure liquid chromatography (HPLC) showed that the insecticide binds probably to the active site of the enzyme inhibiting its activity towards CDNB in a competitive manner, but is not conjugated with GSH. According to this, GST offers a passive protection towards pyrethroid insecticides by binding to their molecule in a sequestering mechanism.

The correlation between the natural levels of glutathione S-transferase (GST) and the tolerance to the organophosphorus insecticides parathion-methyl and paraoxon-methyl, as well as the interaction of affinity-purified enzyme and the insecticides were investigated in order to collect further information on the role of the glutathione S-transferase system as a mechanism of defence against insecticides in insects. The studies were carried out on the larvae and pupae of the coleopteran Tenebrio molitor L, which exhibit varying natural levels of GST activity. Stage-dependent susceptibility of the insect against insecticides was observed during the first 24 h. However, 48 h after treatment, the KD50 value increased significantly due to the recovery of some individuals. Simultaneous injection of insecticide with compounds which inhibit GST activity in vitro caused an alteration in susceptibility of insects 24 or 48 h post-treatment, depending on stage and insecticide used. Inhibition studies combined with competitive fluorescence spectroscopy revealed that the insecticides probably bind to the active site of the enzyme, thus inhibiting its activity towards 1-chloro-2,4-dinitrobenzene in a competitive manner. High-performance liquid chromatography and gas chromatography revealed that T molitor GST catalyses the conjugation of the insecticides studied to a reduced form of glutathione (GSH). From the above experimental results, it is considered that GST offers a protection against the organophosphorus insecticides studied by active site binding and subsequent conjugation with GSH.

Understanding the control of piscine fatty acid metabolism is important for determining the nutritional requirements of fish, and hence for the production of optimal aquaculture diets. The regulation and expression of carnitine palmitoyltransferase 1 (CPT1; EC No 2.3.1.21) are critical processes in the control of fatty acid metabolism, and here we report a cDNA from gilthead sea bream (Sparus aurata) which encodes a protein with high identity to vertebrate CPT1. This sea bream CPT1 mRNA is predominantly expressed in skeletal and cardiac muscle, with little expression in other tissues. Phylogenetic analysis of other vertebrate CPT1 sequences show that fish genomes contain a single gene related to mammalian CPT1B, and a further two multi-gene families related to mammalian CPT1A. Genes related to mammalian CPT1C are absent in fish. Therefore, based on both functional and evolutionary orthology to mammalian CPT1B, the sea bream CPT1 reported here is a CPT1B isoform. Sea bream CPT1B mRNA expression progressively decreases in heart and muscle up to 12 h after last feeding, but returns to initial, non-fasted levels after 72 h. In contrast, in liver non-fasted expression is low, but strongly increases at 24 and 72 h after last feeding. In white muscle and liver, CPT1B mRNA expression is highly correlated with the expression of peroxisomal proliferator-activated receptor β (PPARβ). Thus fatty acid metabolism by CPT1B and its control by PPARs are similar in fish and mammals, but multiple genes for CPT1A-like proteins in fish also suggest different and more complex pathways of lipid utilisation than in mammals.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily that functions as critical regulators of lipid and energy homeostasis. Although intensively studied in mammals, their basic biological functions are still poorly understood. The objective of this work was to characterize PPARbeta subtypes in a fish, the Atlantic salmon (Salmo salar), in order to address PPAR function and the regulation of lipid homeostasis in lower vertebrates. The screening of an Atlantic salmon genomic library revealed the presence of four genes for PPARbeta subtypes. Based on comparisons of exons and exon-flanking regions, these genes were assigned into two families, ssPPARbeta1 and ssPPARbeta2, each family containing two isotypes: ssPPARbeta1A and beta1B and ssPPARbeta2A and beta2B. Two full-length cDNAs for ssPPARbeta1A and ssPPPARbeta2A were isolated. Transcripts for ssPPARbeta1A and ssPPARbeta2A have distinct tissue expression profiles, with ssPPARbeta1A predominating in liver and ssPPARbeta2A predominating in gill. Expression levels of mRNA of either isotypes were up to tenfold lower in kidney, heart, spleen, muscle, and brain. In cellular transfection assays, ssPPARbeta1A is activated by monounsaturated fatty acids, 2-bromopalmitate, and mammalian PPARbeta-specific ligand GW501516. In contrast, PPARbeta2A was not activated by any of the compounds tested. Furthermore, ssPPARbeta2A repressed both the basal reporter gene activity and the GW501516-induced activity of ssPPARbeta1A. The results indicate unexpected levels of variety and complexity in PPAR subtype and mechanism of action in lower vertebrates.

The cloning and characterization of cDNAs and genes encoding three peroxisome proliferator-activated receptor (PPAR) isotypes from two species of marine fish, the plaice (Pleuronectes platessa) and the gilthead sea bream (Sparus aurata), are reported for the first time. Although differences in the genomic organization of the fish PPAR genes compared with their mammalian counterparts are evident, sequence alignments and phylogenetic comparisons show the fish genes to be homologs of mammalian PPARα, PPARβ/δ, and PPARγ. Like their mammalian homologs, fish PPARs bind to a variety of natural PPAR response elements (PPREs) present in the promoters of mammalian or piscine genes. In contrast, the mRNA expression pattern of PPARs in the two fish species differs from that observed in other vertebrates. Thus, PPARγ is expressed more widely in fish tissues than in mammals, whereas PPARα and β are expressed similarly in profile to mammals. Furthermore, nutritional status strongly influences the expression of all three PPAR isotypes in liver, whereas it has no effect on PPAR expression in intestinal and adipose tissues. Fish PPARα and β exhibit an activation profile similar to that of the mammalian PPAR in response to a variety of activators/ligands, whereas PPARγ is not activated by mammalian PPARγ-specific ligands. Amino acid residues shown to be critical for ligand binding in mammalian PPARs are not conserved in fish PPARγ and therefore, together with the distinct tissue expression profile of this receptor, suggest potential differences in the function of PPARγ in fish compared with mammals.