Search

The marine alga Isochrysis galbana was grown under the integrated influence of nitrogen starvation and sodium acetate, specifically to understand the associated remodelling of the fatty acid profiles. This is the first comparative proteomics study under four different set of conditions namely nutrient sufficient, with/without sodium acetate and N-deficient with/without sodium acetate. A total set of 546 unique peptides were detected. Due to the addition of sodium acetate, 56 proteins were down-regulated and 7 proteins showed up-regulation respectively in the nutrient complete and the nitrogen starvation media. Both cell growth and docosahexaenoic acid (DHA) accumulation in the nutrient replete cultures were favoured by the addition of reduced carbon source in the form of sodium acetate. The 14 days old sodium acetate supplemented nitrogen starved cells showed highest amounts of total fatty acids and an over accumulation of the oleic acid. Of the seven key genes of the lipid and fatty acid metabolic pathway that were studied, the IgTeCe, IgFad12, and IgFad4 showed over expressions under nitrogen starvation. The gene expressions of lipases and desaturases involved in the fatty acids metabolism were not found to be correlated with the changes observed in the fatty acid profiles under the experimental conditions. This suggested the presence of an alternative metabolic route and/or possibly the variants of fatty acid desaturase genes in I. galbana which was beyond the scope of this study. A translational or post-translational control can not either be ignored.

International audience; Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24 h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size.

The rise of human populations and the growth of cities contribute to the depletion of natural resources, increase their cost, and create potential climatic changes. To overcome difficulties in supplying populations and reducing the resource cost, a search for alternative pharmaceutical, nanotechnology, and energy sources has begun. Among the alternative sources, microalgae are the most promising because they use carbon dioxide (CO2) to produce biomass and/or valuable compounds. Once produced, the biomass is ordinarily harvested and processed (downstream program). Drying, grinding, and extraction steps are destructive to the microalgal biomass that then needs to be renewed. The extraction and purification processes generate organic wastes and require substantial energy inputs. Altogether, it is urgent to develop alternative downstream processes. Among the possibilities, milking invokes the concept that the extraction should not kill the algal cells. Therefore, it does not require growing the algae anew. In this review, we discuss research on milking of diatoms. The main themes are (a) development of alternative methods to extract and harvest high added value compounds; (b) design of photobioreactors; (c) biodiversity and (d) stress physiology, illustrated with original results dealing with oleaginous diatoms.

Lysophospholipids, such as lysophosphatidic acid or lysophosphatidylcholine, are important bioactive lipids, involved in various normal and pathological cellular processes. They also have industrial and pharmaceutical uses such as emulsifiers or components of drug delivery systems. Lipases, which natural substrates are long chain triacylglycerols, are important biocatalysts for organic synthesis mainly due to their broad substrate specificity and their ability to display high catalytic activity in organic media. This paper describes the various lipase-catalyzed reactions implemented for the production of lysophospholipids. They include hydrolysis or alcoholysis of phospholipids and acylation of the glycerophosphoryl moiety. Special emphasis is made on our work dealing with the production of lysophospholipids rich in dososahexaenoic acid, an important dietary polyunsaturated fatty acid via the hydrolysis of phospholipids extracted from the microalga Isochrysis galbana .

The authors determined levels of dopamine (DA) and its derivatives homovanillic acid (HVA), 3-4 dihydroxyphenylacetic acid (DOPAC), 3 methoxytyramine and norepinephrine + epinephrine (NE + E) in the urine, and DA, E and NE in the whole blood of 50 autistic children aged between 1 year 11 months and 16 years. An association was tested for between markers coding for the enzymes and D3 dopaminergic receptor genes implicated in the monoaminergic pathway and autism, using restriction fragment-length polymorphism. There were significant modifications of catecholamine metabolites, but no difference for allele frequencies of the genes coding for tyrosine hydroxylase, dopamine beta hydroxylase and DRD3 in this population compared with a healthy school population matched for chronological age. However, some of the data encourage a more complete study of chromosome 11.

Les lipides de microalgues marines sont riches en acide eicosapentaenoique (EPA, C20 :5) et en acide docosahexaenoique (DHA, C22 :6), deux acides gras hautement insatures de la serie ω3 (AGHI-ω3), et representent aujourd’hui une source alternative potentielle face aux huiles de poissons. Les microalgues synthetisent de l’EPA et\\ou du DHA dans des proportions relatives variables selon leur classe taxonomique. D’autre part, pour une meme espece, les teneurs en ces acides gras dependent fortement des conditions de culture. De part leur metabolisme photoautotrophe, la production de biomasse microalgale a grande echelle necessite la conception de bioreacteurs specifiques, les photobioreacteurs, qui representent encore aujourd’hui un defi technologique. La poursuite de travaux de recherche, dans des domaines tres divers, reste necessaire pour confirmer la viabilite economique des procedes industriels de production d’EPA et de DHA par les microalgues.

International audience; Marine microalgae are now well-known for their ability to produce omega-3 long chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Among these microalgae, Isochrysis galbana has received increasing interest especially because of its high DHA content and its common use in hatchery to feed fish larvae and clams. Moreover, lipolysis occurring from the biomass harvest stage suggests that I. galbana may contain lipolytic enzymes with potential interesting selectivities. For these reasons, the potential of this microalga for the production of valuable lipids and lipolytic enzymes was investigated. Lipid analysis revealed that DHA is mainly located at the sn-2 position of the phospholipids. Thus, I. galbana was considered as an interesting starting material for the lipase catalyzed production of 1-lyso-2-DHA-phospholipids which are considered as convenient vehicles for the conveyance of DHA to the brain. Lipids from I. galbana can also be used for the enzyme-catalyzed production of structured phospholipids containing one DHA and one medium chain fatty acid in order to combine interesting therapeutic and biological benefits. Starting from total RNA extract from I. galbana, coding sequences of putative lipolytic enzymes were obtained by RACE and Nested PCR. The heterologous expression of a sequence designated IgTeCe was implemented. An expression plasmid was constructed by ligating the coding sequence to a plasmid vector and then cloned and expressed in E. coli. Results showed the effective functionality of plasmid construction for the production of a recombinant protein with the expected molecular mass. Moreover, local alignment using BLASTP and biochemical evidences support the hypothesis that the expressed protein is a thioesterase. Keywords: microalgae / v-3 polyunsaturated fatty acids / phospholipids / lipolytic enzymes Résumé-Lipides et enzymes lipolytiques de la microalgue Isochrysis galbana. Les microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série v-3 comme l'acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérêt croissant du fait d'un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D'autre part, la lipolyse souvent observée dès l'étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu'I. galbana pourrait contenir des enzymes lipolytiques intéressantes en termes de sélectivité. Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d'intérêt et d'enzymes lipolytiques a été étudié. L'analyse des lipides d'I. galbana a tout d'abord révélé que le DHA était majoritairement greffé sur la position sn-2 des phospholipides. Dans ce contexte, les phospholipides d'I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2-DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. À partir des lipides d'I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants. À partir des ARN totaux d'I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L'expression hétérologue d'une séquence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que la; Les microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série ω-3 comme l’acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérèt croissant du fait d’un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D’autre part, la lipolyse souvent observée dès l’étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu’I. galbana pourrait contenir des enzymes lipolytiques intéressantes en terme de sélectivité.Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d’intérêt et d’enzymes lipolytiques a été étudié.L’analyse des lipides d’I. galbana a tout d’abord revélé que le DHA était majoritairement greffé sur la position sn-2 des phopholipides. Dans ce contexte, les phospholipids d’I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2–DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. A partir des lipides d’I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants.A partir des ARN totaux d’I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L’expression hétérologue d’une sequence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que la construction plasmidique permettait bien d’obtenir une protéine recombinante avec la masse moléculaire attendue. D’autre part, l’outil d’alignement local de séquences, BLASTP, ainsi que des données biochimiques ont permis de confirmer l’hypothèse que la protéine obtenue était une thioestérase.

Microalgae constitute a novel study area for characterising new lipolytic enzymes of biotechnological interest. A new gene from the microalga Isochrysis galbana has been isolated and a preliminary characterisation performed. The full-length cDNA contains 2,060 base pairs with an open reading frame of 1,374 nucleotides encoding a polypeptide of 457 amino acids with a predicted molecular mass of 49 kDa, and a theoretical pI of 5.65. The deduced protein includes highly conserved motifs found in alpha/beta fold hydrolases, and shares some similarities with putative or known lipases. Sequence comparison indicated that the catalytic triad corresponds to residues Ser(188), Asp(306) and His(391), with the nucleophilic residue Ser(188) positioned within the consensus G-X-S-188-X-G pentapeptide. Phylogenetic analyses established close relationships with fungal lipases and microalgal sequences.

This study examined the effects of dietary alpha-linolenic acid [18:3(n-3)] deficiency on dopaminergic and serotoninergic neurotransmission systems in 60-d-old male rats. Rats were fed semipurified diets containing either peanut oil [the (n-3)-deficient group] or peanut plus rapeseed oil (control group). We measured the densities of the serotonin-2 (5-HT2) receptors and the dopamine-2 (D2) receptors by autoradiography and membrane-binding assays in relation to the fatty acid composition and levels of endogenous monoamines in three cerebral regions: the frontal cortex, the striatum and the cerebellum. Long-term feeding of the (n-3)deficient diet induced a significantly higher 5-HT2 receptor density in the frontal cortex compared with the control rats without any difference in the endogenous serotonin concentrations. The results also showed some modification of dopaminergic neurotransmission specifically in the frontal cortex in the rats deficient in alpha-linolenic acid, with a significantly lower density of D2 receptors and a significantly lower concentration of endogenous dopamine than in control animals. Moreover, there were lower levels of (n-3) fatty acids in all the regions studied in the deficient rats, balanced by greater levels of (n-6) fatty acids. These results suggest that chronic consumption of an alpha-linolenic acid-deficient diet could induce modifications of the neurotransmission pathways; this might induce the behavioral disturbances previously described in this fatty acid-deficient animal model.

Levels of serotonin (5-HT), dopamine (DA), norepinephrine (NE) and epinephrine (E) were determined in the whole blood and urine of 23 children with autism and compared to those of normal children. Very significant group effects (low whole blood 5-HT, high urinary 5-HT and high NE+E in autism) and age effects (urinary 5-HT and DA decrease with age) were found. Moreover, the urinary DA and the whole blood E levels were correlated with clinical findings. The results suggest a maturation defect of noradrenergic systems, possibly disturbed dopaminergic and serotoninergic metabolism, and a functional imbalance among these neurotransmitters in autism.

We tested for an association between autism and genes coding for enzymes involved in monoaminergic metabolism and for a linked marker, c- Harvey-Ras-1 (HRAS 1), using restriction fragment length polymorphisms. We did not find evidence of an association between autism and genes coding for tyrosine hydroxylase, dopamine- β -hydroxylase (DBH), and tryptophan hydroxylase. However, we report a positive association between autism and the locus containing the gene for HRAS-1.

A simple method for the determination of N-acetyldopamine (NADA) (both free and conjugated) in children's urine by high-performance liquid chromatography with electrochemical detection has been developed. Conjugated NADA was measured as the free compound after enzymatic hydrolysis and purification on alumina. The total analysis time is 25 min. The results show a linearity of the whole assay from 0.005 to 20 mumol/l NADA; the sensitivity is 0.2 pmol per 20 microliters injected sample. Mean recoveries of 96.7 and 86.6% were obtained for free and total NADA, respectively. Modifications of the retention times (between 2 and 50 min) induced by changes in the eluent were determined. Conjugated NADA accounted for about 90% of the total excretion of NADA. These results suggest that this compound could play an important part in neuroblastoma; its concentration is thirteen times higher in children with neuroblastoma than in normal subjects.

The following two sentences, appearing on p 2642, need to be modified: “Using a new dedicated method, Daboussi et al. [232] targeted the diatom model organism Phaeodactylum tricornutum and used zinc finger nucleases, meganucleases, and transcription activator-like effector nucleases to edit Phaeodactylum tricornutum by inducing targeted mutagenesis of certain genes responsible for HVM production. This led to formation of a mutant diatom Tn 19745_1 that produces 45 times more lipid than the control and further increased the HVM production when grown in nutrient-stressed media.” These sentences should read (changes are indicated in bold) “Using a new dedicated method, Daboussi et al. [232] targeted the diatom model organism Phaeodactylum tricornutum and used zinc finger nucleases, meganucleases, and transcription activator-like effector nucleases TALEN to edit Phaeodactylum tricornutum by inducing targeted mutagenesis of certain genes responsible for HVM production. This led to formation of a mutant diatom Tn 19745_1 that produces 45 times more TAG than the control and further increased the HVM production when grown in nutrient-stressed media.” The authors would like to apologize for any inconvenience caused to the readers by these changes.

Whole blood and urinary levels of serotonin (5-hydroxytryptamine; 5-HT) and the derivative urinary 5-hydroxyindoleacetic acid (5-HIAA) were measured in normal and autistic subjects. An association was tested between autism and a marker coding for the 5-HT2A serotonergic receptor gene. Significant group (high urinary 5-HT and low whole blood 5-HT in autism) and age effects (urinary 5-HT decrease with age) were found. Moreover, whole blood 5-HT levels were correlated with clinical state. No differences in allele and genotype frequencies for the 5-HT2A receptor marker were found in this autistic population compared with age-matched healthy students.

Family studies and epidemiologic data in autism show the involvement of genetic factors in the etiology of this syndrome. The frequent association of X chromosome with mental retardation and behavior disturbances raises the question of its implication in the etiology of autism. Several markers of X chromosome were tested in autistic and control populations by association study. The autistic population was submitted to an extensive clinical examination. For the DXS287 marker, chi 2 analysis showed a different allele distribution between control and patient groups. This difference was enhanced when children with the most severe autistic behaviors and the least serious cognitive disorders were selected for statistical comparison. To our knowledge, this is the first association study described using markers of X chromosome in infantile autism. These preliminary results encourage our research on this chromosome, which could be considered as a significant genetic component of the multifactorial etiology of autism.

Twin studies and familial aggregation studies indicate that genetic factors could play a role in infantile autism. In an earlier study, we identified a possible positive association between autism and a c-Harvey-ras (HRAS) oncogene marker at the 3' end of the coding region. In an attempt to confirm this finding, we studied a larger population, well-characterized clinically and genetically. We report a positive association between autism and two HRAS markers, the 3' marker used in the initial study and an additional marker in exon 1.

The aim of this study was to explore two markers of the Insuline (INS) gene and one marker of the Insuline-Like Growth Factor 2 (IGF2) gene by Polymerase Chain Reaction (PCR) in larger samples of autistic and control children

An accurate and precise isocratic high-performance liquid chromatographic technique for the analysis of urinary vanillactic acid (VLA) and plasma dihydroxyphenylalanine (DOPA), especially at low concentrations (pmol/l) for VLA and nmol/l for DOPA), is described. The compounds were purified in a single step, (on an anion exchanger for VLA and on aluminium oxide for DOPA), separated by ion-pair reversed-phase liquid chromatography, and detected electrochemically. A single analysis was complete within 18 min. Mean recoveries of 103 and 81% were obtained for VLA and DOPA, respectively, and the limits of detection were 42 and 76 pmol/l, respectively. The mean values of the intra-assay coefficient of variation were 14 and 7.1% for VLA and DOPA, respectively, and the mean values of the inter-assay coefficient of variation were 15.7 and 11.6%, respectively. Modifications of the retention times (between 2 and 42 min) induced by changes in the eluent were determined. Reference values for normal children and children with neuroblastoma or various tumours are given.