Metabolite profiling and quantitative genetics of natural variation for flavonoids in Arabidopsis

Little is known about the range and the genetic bases of naturally occurring variation for flavonoids. Using Arabidopsis thaliana seed as a model, the flavonoid content of 41 accessions and two recombinant inbred line (RIL) sets derived from divergent accessions (Cvi-0×Col-0 and Bay-0×Shahdara) were analysed. These accessions and RILs showed mainly quantitative rather than qualitative changes. To dissect the genetic architecture underlying these differences, a quantitative trait locus (QTL) analysis was performed on the two segregating populations. Twenty-two flavonoid QTLs were detected that accounted for 11-64% of the observed trait variations, only one QTL being common to both RIL sets. Sixteen of these QTLs were confirmed and coarsely mapped using heterogeneous inbred families (HIFs). Three genes, namely TRANSPARENT TESTA (TT)7, TT15, and MYB12, were proposed to underlie their variations since the corresponding mutants and QTLs displayed similar specific flavonoid changes. Interestingly, most loci did not co-localize with any gene known to be involved in flavonoid metabolism. This latter result shows that novel functions have yet to be characterized and paves the way for their isolation.     

Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCD_max (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPR_min (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.     

Lack of Liver X Receptors Leads to Cell Proliferation in a Model of Mouse Dorsal Prostate Epithelial Cell

Recent studies underline the implication of Liver X Receptors (LXRs) in several prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In order to understand the molecular mechanisms involved, we derived epithelial cells from dorsal prostate (MPECs) of wild type (WT) or Lxrαβ−/− mice. In the WT MPECs, our results show that LXR activation reduces proliferation and correlates with the modification of the AKT-survival pathway. Moreover, LXRs regulate lipid homeostasis with the regulation of Abca1, Abcg1 and Idol, and, in a lesser extent, Srebp1, Fas and Acc. Conversely cells derived from Lxrαβ−/− mice show a higher basal phosphorylation and consequently activation of the survival/proliferation transduction pathways AKT and MAPK. Altogether, our data point out that the cell model we developed allows deciphering the molecular mechanisms inducing the cell cycle arrest. Besides, we show that activated LXRs regulate AKT and MAPK transduction pathways and demonstrate that LXRs could be good pharmacological targets in prostate disease such as cancer.     

Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3'-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.     

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l⁻¹), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l⁻¹ h⁻¹) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.     

Phospholipidosis and down-regulation of the PI3-K/PDK-1/Akt signalling pathway are vitamin E inhibitable events associated with 7-ketocholesterol-induced apoptosis

Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (alpha-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively.     

Temporal and spatial genetic structure in Vitellaria paradoxa (shea tree) in an agroforestry system in southern Mali

Ten microsatellite loci were used to investigate the impact of human activity on the spatial and temporal genetic structure of Vitellaria paradoxa (Sapotaceae), a parkland tree species in agroforestry systems in southern Mali. Two stands (forest and fallow) and three cohorts (adults, juveniles and natural regeneration) in each stand were studied to: (i) compare their levels of genetic diversity (gene diversity, HE; allelic richness, Rs; and inbreeding, FIS); (ii) assess their genetic differentiation (FST); and (iii) compare their levels of spatial genetic structuring. Gene diversity parameters did not vary substantially among stands or cohorts, and tests for bottleneck events were nonsignificant. The inbreeding coefficients were not significantly different from zero in most cases (FIS = -0.025 in forest and 0.045 in fallow), suggesting that the species is probably outbreeding. There was a weak decrease in F(IS) with age, suggesting inbreeding depression. Differentiation of stands within each cohort was weak (FST = 0.026, 0.0005, 0.010 for adults, juveniles and regeneration, respectively), suggesting extensive gene flow. Cohorts within each stand were little differentiated (FST = -0.001 and 0.001 in forest and fallow, respectively). The spatial genetic structure was more pronounced in fallow than in forest where adults showed no spatial structuring. In conclusion, despite the huge influence of human activity on the life cycle of Vitellaria paradoxa growing in parkland systems, the impact on the pattern of genetic variation at microsatellite loci appears rather limited, possibly due to the buffering effect of extensive gene flow between unmanaged and managed populations.