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International audience; Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system. The lipase had a maximal activity at pH 6 and the specific activity was estimated to be about 45 micromol min(-1) mg(-1) protein using triolein as a substrate. Knock-out mutant analysis showed no phenotype during germination indicating that this enzyme is fully dispensable for TAG storage breakdown during germination. Northern blot analyses indicated that the transcript is present in all tissues tested.

The amoeba-infecting Mimivirus is the largest known double-stranded DNA virus, with a 400 nm particle size, comparable to that of mycoplasma. The complete sequence of its 1.2 Mbp genome has recently been determined [Raoult et al. (2004 ▶), Science, 306, 1344–1350] and revealed numerous genes that were not expected to be found in a virus, such as genes encoding translation components, including 4-amino-acyl tRNA synthetases and homologues to various translation initiation, elongation and termination factors. A comprehensive structural and functional study of these Mimivirus gene products was initiated, as they may hold important clues about the origin of DNA viruses. Here, the first preliminary crystallographic and functional results obtained on one of these targets, Mimivirus TyrRS, are reported. Preliminary phasing was obtained using an original combination of homology modelling and normal mode analysis. Experimental evidence that Mimivirus tyrosyl tRNA synthetase recombinant gene product does indeed activate tyrosine is also presented.

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [ 3 H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [ 3 H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA · liter −1 · h −1 and from 0.76 to 0.23 nmol of 3 H-FFA · liter −1 · h −1 , respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26μ g of C · liter −1 · h −1 ). However, the ratio of MUF-oleate activities to [ 3 H]triolein activities, which was constant at the offshore stations (3.8 ± 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.

Shigella infections lead to severe inflammation associated with destruction of colonic mucosa. We assessed the effect of in vivo blockade of CD14 on the outcome of experimental Shigella infection in rabbits. A total of 17 rabbits were divided into two groups: 8 received a single i.v. dose of anti-rabbit CD14 monoclonal antibody prior to infection with an invasive Shigella flexneri strain; the remainder served as controls. The anti-CD14-treated rabbits exhibited more severe tissue destruction and a 50-fold increase in bacterial invasion of the intestinal mucosa when compared to controls. Similar numbers of polymorphonuclear leukocytes were recruited to the intestinal mucosa in both groups despite the massive bacterial invasion seen in the CD14-blocked group. No statistically significant differences were seen in levels of IL-1β nor in the ratio of IL-1RA/IL-1β for either group. In contrast, higher quantities of TNF-α were observed in the CD14-blocked group. To conclude, anti-CD14 treatment had a detrimental effect on the capacity of Shigella-infected animals to clear the infection.

Liver disease is frequent in patients taking home parenteral nutrition (HPN), but its cause remains unclear. Ongoing inflammation was implicated in HPN-associated cholestasis, so we examined the relation between liver-enzyme concentrations and circulating inflammatory and immune markers in these patients. In 17 HPN patients and 10 age- and sex-matched control subjects, we examined erythrocyte sedimentation rate, blood neopterin, soluble interleukin (IL)--2 receptors, circulating tumor necrosis factor-alpha, IL-6, aspartate and alanine aminotransferases, alkaline phosphatases, and gamma-glutamyltranspeptidase (GGT) concentrations. Fourteen of 17 patients had abnormal liver function tests with an increase in alkaline phosphatases (P0.001), gamma-glutamyltranspeptidase (P0.01), and aspartate aminotransferase (P0.01). Alkaline phosphatases were positively correlated to erythrocyte sedimentation rate, neopterin, tumor necrosis factor-alpha, and IL-6. gamma-Glutamyltranspeptidase was positively linked to tumor necrosis factor-alpha and soluble IL-2 receptors. There was no link between aminotransferases and inflammatory parameters. Liver-enzyme concentrations were correlated to the amount of total intravenous calories and calories originating from carbohydrates but not to infused lipids (median infused lipids x kg(-1) body weight x d(-1) = 0.62 g) in contrast to recently published data. Our results confirmed that the number of infused calories contributes to liver toxicity in HPN patients and strongly suggested that sustained inflammation is probably a key factor in worsening HPN-associated cholestasis.

Because the use of live attenuated mutants of Shigella spp. represents a promising approach to protection against bacillary dysentery (M. E. Etherridge, A. T. M. Shamsul Hoque, and D. A. Sack, Lab. Anim. Sci. 46:61–66, 1996), it becomes essential to rationalize this approach in animal models in order to optimize attenuation of virulence in the vaccine candidates, as well as their route and mode of administration, and to define the correlates of protection. In this study, we have compared three strains of Shigella flexneri 5—the wild-type M90T, an aroC mutant, and a double purE aroC mutant—for their pathogenicity, immunogenicity, and protective capacity. Protection against keratoconjunctivitis, induced by wild-type M90T, was used as the protection read out in guinea pigs that were inoculated either intranasally or intragastrically. Following intranasal immunization, the aroC mutant elicited weak nasal tissue destruction compared to M90T and achieved protection correlated with high levels of local anti-lipopolysaccharide immunoglobulin A (IgA), whereas the purE aroC double mutant, which also elicited weak tissue destruction, was not protective and elicited a low IgA response. Conversely, following intragastric immunization, only the M90T purE aroC double mutant elicited protection compared to both the aroC mutant and the wild-type strain. This mutant caused mild inflammatory destruction, particularly at the level of Peyer's patches, but it persisted much longer within the tissues. This could represent an essential parameter of the protective response that, in this case, did not clearly correlate with high anti-lipopolysaccharide IgA titers.

The relationship between microalbuminuria and tissue-type plasminogen activator antigen (tPA-ag) and fibrinogen was evaluated in non-diabetic subjects. Subjects were participants of the D.E.S.I.R. (Data from an Epidemiological Study on the Insulin Resistance syndrome) Study. Analyses were carried out on 2248 women and 2402 men for fibrinogen and on 272 women and 284 men for tPA-ag. Microalbuminuria was defined as urinary albumin concentration greater than 20 mg/l. Men with microalbuminuria had a 6% higher fibrinogen concentration than those without (3.07 g/l (95% confidence interval: 2.99,3.15) vs. 2.89 g/l (2.87,2.91), adjusted for age and smoking). This relationship existed in hypertensive as well as non-hypertensive subjects. The association between microalbuminuria and tPA-ag existed only in hypertensive men, those with microalbuminuria having a 21% higher tPA-ag than those without (4.39 ng/ml (3.70,5.08) vs. 3.63 ng/ml (3.32,3.94), adjusted for age and smoking). Adjustment for other risk markers for cardiovascular disease did not change the results. There was no relationship between microalbuminuria and these haemostatic factors in women. The results of this study suggest that in non-diabetic men, microalbuminuria is associated with fibrinogen, but with tPA-ag only when concomitant with hypertension.

Lipid transfer proteins (LTPs) are small, basic and abundant proteins in higher plants. They are capable of binding fatty acids and of transferring phospholipids between membranes in vitro. LTPs from this family contain a signal peptide and are secreted in the cell wall. Their biological function is presently unknown. LTPs have been suggested to participate to cutin assembly and to the defense of the plants against pathogens. A genetic approach should prove useful to provide clues on their in vivo functions. Here, the characterization of the LTP gene family in Arabidopsis thaliana is described. At least 15 genes were identified, their map position determined and the expression pattern characterized for six of them. All the sequences exhibit the typical features of plant LTPs. The molecular weight is close to 9 kDa, the isoelectric point is near 9 (except for three acidic LTPs), and typical amino acid residues such as cysteines are conserved. Genomic DNA blotting hybridization experiments performed using ltp1 to ltp6 as probes indicate that ltps form distinct 1-3 gene subfamilies which do not cross hybridize. Expression studies indicate that all the genes tested are expressed in flowers and siliques, but not in roots. Ltp1, ltp5 and ltp2 are expressed significantly in leaves, while ltp6 is detected only in 2-4-week-old leaves. In addition, ltp4 and ltp3 are strongly upregulated by abscisic acid (ABA). Tandem repeats can be noted concerning ltp1 and ltp2 on chromosome 2, ltp3 and ltp4 on chromosome 5 and ltp5 and ltp12 on chromosome 3. While ltp7, ltp8 and ltp9 map at the same position on chromosome 2, the other genes are dispersed throughout the genome. The characterization of the Arabidopsis ltp gene family will permit to initiate a genetic approach for determining the in vivo function(s) of these proteins.

International audience; Caspases are intracellular proteases that mediate mammalian cell apoptosis. Caspase-1 (Casp-1) is a unique caspase because it activates the proinflammatory cytokines interleukin (IL)-1beta and IL-18. Shigella flexneri, the etiological agent of bacillary dysentery, induces macrophage apoptosis, which requires Casp-1 and results in the release of mature IL-1beta and IL-18. Here we show that casp-1(-/-) mice infected with S. flexneri do not develop the acute inflammation characteristic of shigellosis and are unable to resolve the bacterial infection. Using casp-1(-/-) mice supplemented with recombinant cytokines and experiments with IL-1beta(-/-) and IL-18(-/-) mice, we show that IL-1beta and IL-18 are both required to mediate inflammation in S. flexneri infections. Together, these data demonstrate the importance of Casp-1 in acute inflammation and show the different roles of its substrates, IL-1beta and IL-18, in this response.

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was ex- tracted from Parinari glaberrimum seed kernels and the pu- rified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluores- cent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13,15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of de- tergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween ® 20, Brij ® 35, Dobanol ® , n-dodecylglucoside) above their critical micellar concentration dramatically in- creases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quanti- ties as low as 0.1 ng of pure pancreatic lipase could be de- tected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples. —Beis- son, F., N. Ferte, J. Nari, G. Noat, V. Arondel, and R. Verger. Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings. J. Lipid Res. 1999. 40: 2313-2321.

Infection by the enteric bacterial pathogen Shigella results in intense mucosal inflammation and destruction of the colonic and rectal epithelium in infected humans. Initial bacterial translocation occurs through the follicle-associated epithelium. Previous experiments suggest that interleukin-1 (IL-1) is crucial to trigger inflammation, particularly in the follicular zones. During the first 4 hours of infection in a rabbit ligated-loop model of intestinal invasion, there are two salient characteristics: (i) a high concentration of IL-1α and IL-1β, both in infected Peyer's patch tissue and in the corresponding efferent mesenteric blood, and (ii) a very low level of expression of IL-1 receptor antagonist (IL-1ra). These may reflect a combination of regulation of expression and secretion of IL-1α, IL-1β, and IL-1ra by both resident and recruited phagocytes and the induction of mononuclear phagocyte apoptosis by Shigella . This low IL-1ra/IL-1 ratio likely accounts for the rapid, uncontrolled inflammation characteristic of shigellosis.

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.

In shigellosis, the network of cellular interactions mediated by a balance of pro- and anti-inflammatory cytokines or chemokines is clearly tipped toward acute destructive inflammation of intestinal tissues by the bacterial invader. This work has addressed the role played by interleukin-8 (IL-8) in a rabbit model of intestinal invasion by Shigella flexneri . IL-8, which is largely produced by the epithelial cells themselves, appears to be a major mediator of the recruitment of polymorphonuclear leukocytes (PMNs) to the subepithelial area and transmigration of these cells through the epithelial lining. Neutralization of IL-8 function by monoclonal antibody WS-4 caused a decrease in the amount of PMNs streaming through the lamina propria and the epithelium, thus significantly attenuating the severity of epithelial lesions in areas of bacterial invasion. These findings are in agreement with our previous work (31). In contrast to the PMNs, the bacteria displayed increased transepithelial translocation, as well as overgrowth in the lamina propria and increased passage into the mesenteric blood. By mediating eradication of bacteria at their epithelial entry site, although at the cost of severe epithelial destruction, IL-8 therefore appears to be a key chemokine in the control of bacterial translocation.

We report here that the Shigella invasion plasmid antigen (Ipa)B, which is sufficient to induce apoptosis in macrophages, binds to caspase (Casp)-1, but not to Casp-2 or Casp-3. Casp-1 is activated and its specific substrate interleukin-1beta is cleaved shortly after Shigella infection. Macrophages isolated from Casp-1 knock-out mice are not susceptible to Shigella-induced apoptosis, although they respond normally to other apoptotic stimuli. Shigella kills macrophages from casp-3, casp-11, and p53 knock-out mice as well as macrophages overexpressing Bcl-2. We propose that Shigella induces apoptosis by directly activating Casp-1 through IpaB, bypassing signal transduction events and caspases upstream of Casp-1. Taken together these data indicate that Shigella-induced apoptosis is distinct from other forms of apoptosis and seems uniquely dependent on Casp-1.

We have investigated the capacity of live attenuated Shigella flexneri strains to act as vectors for the induction of local and systemic antibody responses against heterologous epitopes. The S. flexneri IpaC antigen was selected as a carrier protein into which the C3 neutralizing epitope of the poliovirus VP1 protein was inserted in eight sites distributed along IpaC. The resulting IpaC-C3 hybrid proteins were expressed from recombinant plasmids in the S. flexneri 2a vaccine candidate, SC602. Their production was similar to that of wild-type IpaC. All of the hybrid proteins but one were secreted as efficiently as wild-type IpaC. Immunization of mice with each of the recombinant SC602 derivatives reveals that one construct is able to induce serum and local anti-C3 antibodies, showing that at least one permissive site of insertion within IpaC can be defined. Furthermore, mouse-to-mouse variability in the anti-C3 response indicates that the amount of hybrid proteins produced in the host by SC602 should be improved for optimal use of S. flexneri live attenuated strains as mucosal vectors for foreign epitopes.

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nona-peptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPL-GALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.

While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, lipid droplets are delineated by a half-unit membrane whose protein constituents are poorly understood, except in the specialized case of oleosins, which are associated with seed lipid droplets. Recently, we identified a new class of lipid-droplet associated proteins called LDAPs that localize specifically to the lipid droplet surface within plant cells and share extensive sequence similarity with the small rubber particle proteins (SRPPs) found in rubber-accumulating plants. Here, we provide additional evidence for a role of LDAPs in lipid accumulation in oil-rich fruit tissues, and further explore the functional relationships between LDAPs and SRPPs. In addition, we propose that the larger LDAP/SRPP protein family plays important roles in the compartmentalization of lipophilic compounds, including triacylglycerols and polyisoprenoids, into lipid droplets within plant cells. Potential roles in lipid droplet biogenesis and function of these proteins also are discussed.

The oil palm fruit mesocarp contains high lipase activity that increases free fatty acids and necessitates post-harvest inactivation by heat treatment of fruit bunches. Even before heat treatment the mesocarp lipase activity causes consequential oil losses and requires costly measures to limit free fatty acids quantities. Here we demonstrate that elite low-lipase lines yield oil with substantially less free fatty acids than standard genotypes, allowing more flexibility for post-harvest fruit processing and extended ripening for increased yields. We identify the lipase and its gene cosegregates with the low-/high-lipase trait, providing breeders a marker to rapidly identify potent elite genitors and introgress the trait into major cultivars. Overall, economic gains brought by wide adoption of this material could represent up to one billion dollars per year. Expected benefits concern all planters but are likely to be highest for African smallholders who would be more able to produce oil that meets international quality standards., Lipase in the mesocarp of oil palm fruits reduces the quality of oil and leads to decreased yield. In this study, the authors identify the gene encoding the mesocarp lipase, raising the possibility of breeding palm genotypes that produce better quality oil.

The effect of human recombinant interleukin-1 receptor antagonist on intestinal inflammation, tissue destruction, and bacterial invasion during experimental shigellosis caused by Shigella flexneri was studied in the rabbit-ligated loop infection model. Intravenous infusion of the inhibitor at a dose of 2 mg/kg per h, was initiated 30 min before intestinal loops were ligated and infected, and continued during the 8-h period of infection. The animals treated with IL-1 receptor antagonist showed a striking decrease in inflammation, destruction, and bacterial invasion of their tissues, both at the level of the villous intestine and Peyer's patches. This is conclusive evidence that interleukin-1 plays a critical role in the pathogenesis of shigellosis. This proinflammatory cytokine is here proposed as a major trigger of the inflammatory reaction which is characteristic of this invasive disease of the intestine, due to the particular interaction existing between S. flexneri and macrophages.

National audience

Previous genetic evidence suggested that the fad8 and fad7 genes of Arabidopsis thaliana encode chloroplast membrane-associated omega-3 desaturases. A putative fad8 cDNA was isolated by heterologous hybridization using a gene encoding an endoplasmic reticulum-localized omega-3 desaturase (fad3) as a probe. The cDNA encodes a protein of 435 amino acid residues with a molecular mass of 50,134 D. Constitutive expression of the cDNA in transgenic plants of a fad7 mutant resulted in genetic complementation of the mutation, indicating that the fad7 and fad8 gene products are functionally equivalent. Expression of the fad8 cDNA in transgenic plants often resulted in the co-suppression of both the endogenous fad7 and fad8 genes in spite of the fact that these two genes share only about 75% nucleotide identity. In contrast to all other known plant desaturases, including fad7, the steady-state level of fad8 mRNA is strongly increased in plants grown at low temperature. This suggests that the role of fad8 is to provide increased omega-3 desaturase activity in plants that are exposed to low growth temperature. The fad8-1 mutation created a premature stop codon 149 amino acids from the amino-terminal end of the fad8 open reading frame, suggesting that this mutation results in a complete loss of fad8 activity.

Oil palm can accumulate up to 90% oil in its mesocarp, the highest level observed in the plant kingdom. In contrast, the closely related date palm accumulates almost exclusively sugars. To gain insight into the mechanisms that lead to such an extreme difference in carbon partitioning, the transcriptome and metabolite content of oil palm and date palm were compared during mesocarp development. Compared with date palm, the high oil content in oil palm was associated with much higher transcript levels for all fatty acid synthesis enzymes, specific plastid transporters, and key enzymes of plastidial carbon metabolism, including phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. Transcripts representing an ortholog of the WRI1 transcription factor were 57-fold higher in oil palm relative to date palm and displayed a temporal pattern similar to its target genes. Unexpectedly, despite more than a 100-fold difference in flux to lipids, most enzymes of triacylglycerol assembly were expressed at similar levels in oil palm and date palm. Similarly, transcript levels for all but one cytosolic enzyme of glycolysis were comparable in both species. Together, these data point to synthesis of fatty acids and supply of pyruvate in the plastid, rather than acyl assembly into triacylglycerol, as a major control over the storage of oil in the mesocarp of oil palm. In addition to greatly increasing molecular resources devoted to oil palm and date palm, the combination of temporal and comparative studies illustrates how deep sequencing can provide insights into gene expression patterns of two species that lack genome sequence information.

Date palm is one of the most economically important woody crops cultivated in the Middle East and North Africa and is a good candidate for improving agricultural yields in arid environments. Nonetheless, long generation times (5-8 years) and dioecy (separate male and female trees) have complicated its cultivation and genetic analysis. To address these issues, we assembled a draft genome for a Khalas variety female date palm, the first publicly available resource of its type for a member of the order Arecales. The ∼380 Mb sequence, spanning mainly gene-rich regions, includes >25,000 gene models and is predicted to cover ∼90% of genes and ∼60% of the genome. Sequencing of eight other cultivars, including females of the Deglet Noor and Medjool varieties and their backcrossed males, identified >3.5 million polymorphic sites, including >10,000 genic copy number variations. A small subset of these polymorphisms can distinguish multiple varieties. We identified a region of the genome linked to gender and found evidence that date palm employs an XY system of gender inheritance.

Mutations at the fad7 locus of Arabidopsis thaliana (previously called fadD) cause decreased desaturation of dienoic fatty acids in chloroplast lipids in plants grown at elevated temperatures. This suggested that the fad7 locus encodes a chloroplast omega-3 desaturase that catalyzes the desaturation of lipid-linked 18:2 and 16:2 fatty acids. In order to clone the fad7 gene, it was first genetically mapped relative to the flanking restriction fragment length polymorphism markers 4547 and 2488A on chromosome 3, and yeast artificial chromosomes covering the locus were identified. A putative desaturase cDNA clone that was isolated by low stringency heterologous probing with a cDNA for an endoplasmic reticulum-localized omega-3 desaturase (fad3) hybridized to the yeast artificial chromosomes and could not be resolved from the locus by restriction fragment length polymorphism mapping. Expression of the cDNA in transgenic fad7 mutant plants resulted in restoration of wild type fatty acid composition and suppression of a previously observed effect of the fad7 mutation on chloroplast number, indicating genetic complementation. The structural gene contained seven introns within a coding sequence of 1338 base pairs, which encodes a 446-amino acid polypeptide of 51,172 daltons. The amino-terminal region of the fad7 gene product contained a consensus chloroplast transit peptide. Except for the amino-terminal domain, the deduced amino acid sequence of the fad7 gene product had high homology to the fad3 gene product, indicating that fad7 encodes an omega-3 desaturase and that the two genes arose from a common ancestral gene. There was no apparent effect of growth temperature on the steady-state levels of fad7 mRNA in wild type plants.

Un colloque sur les lipides de plantes et les oxylipines s’est tenu a Strasbourg les 10 et 11 juillet 2001, dans le cadre des Journees de la Societe Francaise de Physiologie Vegetale. Plus de 85 chercheurs ont participe a ce colloque et 45 communications ont ete faites. La plupart ont porte sur la synthese des sterols et des isoprenoides, le metabolisme des lipides, les relations entre le stress thermique et les lipides membranaires ainsi que sur les oxylipines. Les conferences plenieres ont ete donnees par le Pr Heinz sur les cerebrosides et les sterols glucosides, le Dr R. Verger sur les lipases et le Dr I. Feussner sur les oxylipines. Une table ronde s’est tenue pour discuter de la constitution d’un reseau reunissant les specialistes des lipides des plantes en France. L’idee a ete accueillie favorablement par tous les participants et il a ete envisage de creer un site « web » contenant un annuaire des chercheurs, des cahiers de protocoles et une base de donnees bibliographiques. Ceci se fera en collaboration etroite avec les chercheurs europeens et americains de la specialite.

Context. The High Frequency Instrument (HFI) is one of the two focal instruments of the Planck mission. It will observe the whole sky in six bands in the 100 GHz–1 THz range.\ud \ud Aims. The HFI instrument is designed to measure the cosmic microwave background (CMB) with a sensitivity limited only by fundamental sources: the photon noise of the CMB itself and the residuals left after the removal of foregrounds. The two high frequency bands will provide full maps of the submillimetre sky, featuring mainly extended and point source foregrounds. Systematic effects must be kept at negligible levels or accurately monitored so that the signal can be corrected. This paper describes the HFI design and its characteristics deduced from ground tests and calibration.\ud \ud Methods. The HFI instrumental concept and architecture are feasible only by pushing new techniques to their extreme capabilities, mainly: (i) bolometers working at 100 mK and absorbing the radiation in grids; (ii) a dilution cooler providing 100 mK in microgravity conditions; (iii) a new type of AC biased readout electronics and (iv) optical channels using devices inspired from radio and infrared techniques.\ud \ud Results. The Planck-HFI instrument performance exceeds requirements for sensitivity and control of systematic effects. During ground-based calibration and tests, it was measured at instrument and system levels to be close to or better than the goal specification.

Lipides acyle dans Arabidopsis et toutes les autres plantes ont une myriade de fonctions diverses. Il s'agit notamment de fournir la barriere de diffusion de base des membranes qui separe les cellules et les organites subcellulaires. Cette fonction implique seuls plus de 10 classes de lipides membranaires, y compris les phospholipides, galactolipides, et sphingolipides, et au sein de chaque classe, les variations dans la composition de la chaine acyle augmenter le nombre de structures a plusieurs centaines de possibles especes moleculaires. Lipides acyle dans le sous forme de compte triacylglycerol de 35% du poids de graines d'Arabidopsis et representent leur forme majeure de carbone et de stockage d'energie. Une couche de cutine et les cires cuticulaires qui limite la perte d'eau et offre une protection contre les invasions par des agents pathogenes et autres stress recouvre toute la surface aerienne d'Arabidopsis. Des fonctions similaires sont fournis par la suberine et des cires associes qui sont localisees dans les racines, les teguments et les zones d'abscission et sont produites en reponse a une blessure. Ce chapitre se concentre sur les voies metaboliques qui sont associes a la biosynthese et la degradation des lipides acyle mentionnes ci-dessus. Ces voies, les enzymes et les genes sont egalement presentes en detail dans un site Web associe (ARALIP: http://aralip.plantbiology.msu.edu/ ). Protocoles et methodes utilises pour l'analyse des lipides Arabidopsis sont fournis. Enfin, un resume detaille de la composition des lipides Arabidopsis est fourni en trois chiffres et 15 tables.

National audience; Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.; Lipides acyle dans Arabidopsis et toutes les autres plantes ont une myriade de fonctions diverses. Il s'agit notamment de fournir la barrière de diffusion de base des membranes qui sépare les cellules et les organites subcellulaires. Cette fonction implique seuls plus de 10 classes de lipides membranaires, y compris les phospholipides, galactolipides, et sphingolipides, et au sein de chaque classe, les variations dans la composition de la chaîne acyle augmenter le nombre de structures à plusieurs centaines de possibles espèces moléculaires. Lipides acyle dans le sous forme de compte triacylglycérol de 35% du poids de graines d'Arabidopsis et représentent leur forme majeure de carbone et de stockage d'énergie. Une couche de cutine et les cires cuticulaires qui limite la perte d'eau et offre une protection contre les invasions par des agents pathogènes et autres stress recouvre toute la surface aérienne d'Arabidopsis. Des fonctions similaires sont fournis par la subérine et des cires associés qui sont localisées dans les racines, les téguments et les zones d'abscission et sont produites en réponse à une blessure. Ce chapitre se concentre sur les voies métaboliques qui sont associés à la biosynthèse et la dégradation des lipides acyle mentionnés ci-dessus. Ces voies, les enzymes et les gènes sont également présentés en détail dans un site Web associé (ARALIP: http://aralip.plantbiology.msu.edu/ ). Protocoles et méthodes utilisés pour l'analyse des lipides Arabidopsis sont fournis. Enfin, un résumé détaillé de la composition des lipides Arabidopsis est fourni en trois chiffres et 15 tables.

Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen. To map the B-cell epitopes of this protein, the gene encoding IpaC was cloned and expressed at a high level in Escherichia coli. The partially purified recombinant protein was used to raise rabbit polyclonal antisera and murine monoclonal antibodies. A lambda gt11 ipaC gene library was screened with the antisera and antibodies. Recombinant DNA clones producing specific antigenic determinants were isolated, and the sequence of their DNA inserts was determined. The amino acid sequence of each determinant was deduced from the minimal overlap of DNA inserts of multiple antibody-positive DNA clones. Two distinct epitopes, located between amino acid residues 25 and 33 and 90 and 97, were identified. Two additional B-cell epitopes which were located between residues 297 and 349, near the carboxy-terminal end of the protein, were characterized. Each of these epitopes was also recognized by sera from convalescent humans and monkeys. Therefore, it seems likely that these epitopes are relevant to the humoral response against IpaC during natural infection.

A low lipase activity from a crude extract of Arabidopsis seedlings was assayed using three sensitive methods (radiolabelled triacylglycerols, commercial resorufin ester and triacylglycerols containing the naturally fluorescent parinaric acid as substrates). The specific activity of the extract was found to be similar using the three methods. However, the plant lipase activity measured using the radioactivity and the fluorescence assays could be abolished by heating the extract, contrary to the apparent activity measured using the commercial colorimetric assay. Unlike the radioactivity assay, the fluorescence assay can be monitored continuously. The parinaric acid-based method is therefore the only one to provide a sensitive, specific and continuous assay.

Genetic and molecular data now available on the pathogenic properties of Shigella flexneri allow rational design of live attenuated vaccine strains. The genes required at given steps of the infection process can be selectively mutated to impair the bacterium's capacity to interact with intestinal epithelial cells and/or survive within intestinal tissues in general. We have tested two mutations in S. flexneri serotype 5a (M90T) which, alone or in combination, have yielded promising results when evaluated as vaccine prototypes in orally infected macaque monkeys. The first mutation, icsA, blocks intracellular and cell-to-cell spread of the micro-organism. This mutant (SC560) appeared reasonably well tolerated and elicited protection against homologous challenge. The second mutation, ompB, disconnects the bacterium from one of its major environmental regulatory factors, osmolarity. This mutant (SC433) still caused slight dysenteric symptoms in vaccinees. It was also perfectly protective. When these two mutations were combined, the double mutant (SC445), was perfectly tolerated but failed to protect one out of five animals. These studies bring interesting prospects of the possibility of immunizing against shigellosis. In addition to providing new possibilities for vaccine design, construction and evaluation of these mutants allowed substantial progress in understanding the pathogenesis of shigellosis.

In the oil palm (Elaeis guineensis Jacq.), an active endogenous lipase is responsible for the massive release of free fatty acids (FFA) in the mesocarp of fruits at maturity. This can lead to the production of oils with unacceptable acid values. We have investigated the lipase activities in 39 genotypes of oil palm presently used in breeding programs. While most E. guineensis genotypes exhibited high lipase activity values, four of them with negligible activities were identified.We analyzed in situ lipolysis in the mesocarp upon severe wounding of fruits. The FFA content of high-lipase fruits ranged from 17 to 55%, while all low-lipase fruits contained less than 7.5% FFA. The analysis of oil from fallen overripe fruits indicated that all lowlipase genotypes contained less than 1.2% FFA (oil acidity ,5% FFA, FAO-WHO international norms), while all but one high-lipase genotypes had FFA contents much higher than the acceptable standards. Because the identified low-lipase lines are of high agronomical value and at least one of them is an elite genotype, it will soon be possible to provide farmers with new low-lipase oil palm lines allowing significant savings on labor costs, without any need for further breeding. (Resume d'auteur)

The unripe fruit of babaco (Vasconcellea 9 heilbornii; syn. Carica pentagona) contains a latex, similar to that in Carica papaya, which exhibits lipolytic activity. Herein, the regioselectivity, stereoselectivity and typoselectivity in both hydrolysis and acyltransfer reactions of babaco latex lipases were studied and compared to those of Carica papaya latex. In hydrolysis, both biocatalysts are 1,3-regioselective with ratios for 1,2-2,3-diacylglycerols/1,3- diacylglycerol of 6.5 and 21 for babaco and papaya, respectively. In contrast, papaya latex had a slight sn- 3 stereopreference. Babaco latex displayed a higher activity on triacylglycerols with short chain and unsaturated fatty acids.

The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase.

BACKGROUND: The effect of lifestyle changes in cohorts of free-living populations has been surprisingly little evaluated. DESIGN: A longitudinal study. METHODS: In the French Data from an Epidemiological Study on the Insulin Resistance (D.E.S.I.R) study of 1958 men and 2028 women, aged 30-65 years, the impact of 3-year changes in lifestyle habits (sporting activity, physical activity at home and at work, alcohol drinking, smoking) on metabolic syndrome parameters [insulin, glucose, high-density lipoprotein (HDL) cholesterol, triglycerides, systolic blood pressure, waist circumference] and on body mass index (BMI) were investigated. RESULTS: In men, 3-year increases in sporting activity were associated with a lowering of insulin, glucose, systolic blood pressure and waist circumference (all P < 0.05). For women, the only effect was on lowering waist circumference (P < 0.03). Increases in physical activity at home were beneficially associated with HDL-cholesterol, triglycerides, waist circumference and BMI changes (all P < 0.05) in men, but had no apparent effect in women. Decreases in alcohol intake only had an effect in men, with decreases in HDL-cholesterol and systolic blood pressure (P < 0.05), whereas decreasing cigarette smoking in men was associated with significant increases in insulin, glucose, triglycerides, waist and BMI (P < 0.001), and in women HDL-cholesterol, waist circumference and BMI increased (P < 0.02). These results were mainly caused by those who had stopped smoking. CONCLUSIONS: Increases in physical activity over the 3-year period were associated with beneficial effects on syndrome parameters, particularly in men. Smoking cessation and alcohol moderation produced mixed effects on these parameters.

To compare the sensitivities of BMI, waist circumference and waist hip ratio (WHR) in identifying subjects who should be screened for diabetes and/or for obesity-associated dyslipidaemia. Cross-sectional study. Central-western France. More than 3000 men and women, aged 40–64 years, from the French study: data from an epidemiological study on the insulin resistance syndrome (D.E.S.I.R.). Sensitivity and specificity for screened diabetes (fasting plasma glucose⩾7.0 mmol/l) and screened dyslipidaemia (triglycerides⩾2.3 mmol/l and/or HDL-cholesterol

International audience; OBJECTIVE: To compare the sensitivities of BMI, waist circumference and waist hip ratio (WHR) in identifying subjects who should be screened for diabetes and/or for obesity-associated dyslipidaemia. DESIGN: Cross-sectional study. SETTING: Central-western France. PARTICIPANTS: More than 3000 men and women, aged 40-64 years, from the French study: data from an epidemiological study on the insulin resistance syndrome (D.E.S.I.R.). MAIN OUTCOME MEASURES: Sensitivity and specificity for screened diabetes (fasting plasma glucose>or=7.0 mmol/l) and screened dyslipidaemia (triglycerides>or=2.3 mmol/l and/or HDL-cholesterol or=27/26 kg/m(2) (men/women) or waist >or=96/83 cm or WHR>or=0.96/0.83. These values were chosen as thresholds. In men, BMI had a nonsignificantly higher sensitivity than waist or WHR for both diabetes and dyslipidaemia (77 vs 74 and 66% P

International audience; The core of the High Frequency Instrument (HFI) on-board the Planck satellite consists of 52 bolometric detectors cooled at 0.1 Kelvin. In order to achieve such a low temperature, the HFI cryogenic architecture consists in several stages cooled using different active coolers. These generate weak thermal fluctuations on the HFI thermal stages. Without a dedicated thermal control system these fluctuations could produce unwanted systematic effects, altering the scientific data. The HFI thermal architecture allows to minimise these systematic effects, thanks to passive and active control systems described in this paper. The passive and active systems are used to damp the high and low frequency fluctuations respectively. The last results regarding the tests of the HFI passive and active thermal control systems are presented here. The thermal transfer functions measurement between active coolers and HFI cryogenic stages will be presented first. Then the stability of the temperatures obtained on the various cryogenic stages with PID regulations systems will be checked through analysis of their power spectrum density.

In 2001 the "National Cholesterol and Education Program Expert Panel" gave a clinical definition of the metabolic syndrome. The frequency of this syndrome at baseline and its incidence and persistence at three years is studied in a French population.2109 men and 2184 women from the D.E.S.I.R. longitudinal cohort study (Data from an Epidemiological Study on the Insulin Resistance syndrome) in central-western France, aged 30 to 64 years, were examined at inclusion and three years later.Evaluation of the frequencies, incidences and persistence of the metabolic syndrome and its abnormalities. This syndrome is defined by the presence of three or more of five abnormalities: waist circumference102/88 cm (men/women); triglycerideso r=1.69 mmol/l, HDL-cholesterol1.04/1.29 mmol/l (men/women); systolic/diastolic blood pressureor =130 and/or 85 mmHg; fasting plasma glucoseor =6.1 mmol/l.At baseline, 10% of men and 7% of women had the metabolic syndrome. If the syndrome was defined to include a treatment in the abnormalities (for diabetes, hypertension, dyslipidemia), the syndrome frequencies increased to 16% and 11%. However only 12% and 8% respectively, had this syndrome both at inclusion and at three years. High blood pressure was the most frequent abnormality: 70% and 47% in men and women respectively, at inclusion. The most stable abnormality was high waist circumference (80% persisted), hyperglycaemia the least stable (60% persisted). Hyperinsulinaemia did not cluster closely with this syndrome.The age-specific frequency of the syndrome is more than 2.5 times higher in the US than in this French cohort and this ratio increased with age. The higher frequencies of abdominal obesity and low HDL-cholesterol in women than in men suggest that these gender-specific thresholds may need to be refined.

Central venous catheter (CVC) infection is the most frequent complication during home parenteral nutrition (HPN). We prospectively assessed incidence and catheter-related sepsis (CRS)-associated factors in the 42 adult patients enrolled in our HPN centre since its opening.Age, frequency of infusions, CVC type, autonomy or nurse/family aid, underlying disease, involved infectious organism(s), hospital stay, efficacy of antibiotic-lock and other infectious complications, were studied.CRS occurred 39 times (3/1000 days of HPN). In 37/39 cases, it was proven by both peripheral and central blood cultures. In 56% of patients, clinical signs were discrete, delaying diagnosis. Individual factors like learning potency, underlying disease (especially chronic intestinal obstruction with bacterial overgrowth), and length of remaining colon and small intestine, were slightly associated with higher CRS incidence. Usually, one organism (S. epidermidis; 51%) was detected. A total of 14 CVC were immediately removed. In the others, antibiotic-lock was more effective in patients having tunnelled catheters (TC, 50%) than implanted devices (25%; P0.05). Mean hospital stay was 22+/-15 days, which was influenced by 3 patients presenting associated osteomyelitis.CRS incidence was 3/1000 days of HPN. Clinical symptoms were often discrete, suggesting importance of rigorous survey. Individual apprenticeship and risk for higher bacterial translocation seem associated to higher CRS incidence. CVC sterilization was more frequent in patients with TC.