Enhanced lipid extraction from unbroken microalgal cells using enzymes

The marine microalga Nannochloropsis sp. was chosen as a model organism to investigate the feasibility of using cell wall-degrading enzymes to enhance the recovery of intracellular lipids. An enzyme cocktail containing galactomannanase, 1,4-β-cellobiosidase and β-glucosidase as main components was prepared from commercial enzyme preparations. The effects of pretreatment time (P), enzyme dosage (D), pH and temperature (T) on the amount of extracted lipids were investigated using response surface methodology. Under the best conditions (P = 90 min, D = 1.3 mg g–1, pH = 5, T = 36°C) over 70% of the lipids present in the microalga were recovered. SEM and TEM characterization of enzyme-treated microalgae showed extensive cell damage with significant disruption of the cell wall and release of algal material. Overall, the results of this study strongly support the use of commercial enzyme preparations to improve lipid recovery from microalgae and provide useful information on the influence of process conditions on the treatment efficiency

Lateral phase separation in mixtures of lipids and cholesterol

In an effort to understand "rafts" in biological membranes, we propose phenomenological models for saturated and unsaturated lipid mixtures, and lipid-cholesterol mixtures. We consider simple couplings between the local composition and internal membrane structure, and their influence on transitions between liquid and gel membrane phases. Assuming that the gel transition temperature of the saturated lipid is shifted by the presence of the unsaturated lipid, and that cholesterol acts as an external field on the chain melting transition, a variety of phase diagrams are obtained. The phase diagrams for binary mixtures of saturated/unsaturated lipids and lipid/cholesterol are in semi-quantitative agreement with the experiments. Our results also apply to regions in the ternary phase diagram of lipid/lipid/cholesterol systems

Manipulation of lipid rafts in neuronal cells

Lipid rafts are specialized plasma membrane micro-domains highly enriched in cholesterol, sphingolipids and glycosylphosphatidylinositol (GPI) anchored proteins. Lipid rafts are thought to be located in the exofacial leaflet of plasma membranes. Functionally, lipid rafts are involved in intracellular trafficking of proteins and lipids, secretory and endocytotic pathways, signal transduction, inflammation and in cell-surface proteolysis. There has been substantial interest in lipid rafts in brain, both with respect to normal functioning and with certain neurodegenerative diseases. Based on the impact of lipid rafts on multitude biochemical pathways, modulation of lipid rafts is used to study related disease pathways and probably offers a target for pharmacological intervention. Lipid rafts can be targeted by modulation of its main components, namely cholesterol and sphingolipids. Other approaches include the modulation of membrane dynamics and it has been reported that protein-lipid interactions can vary the occurrence and composition of these membrane micro-domains. The present review summarizes the possibilities to modulate lipid rafts with focus on neuronal cells. Keywords: Lipid raft, cholesterol, membrane fluidity, statin, cyclodextrine, docosahexaenoic acid

Lipid Peroxidation After Intracortical Injection of Ferric Chloride Increases the Incidence of Seizures in Young Rats

Clinical studies have shown that the incidence of early posttraumatic seizures ishigher in children than in adults and it has been proposed that iron-induced lipidperoxidation has an important role in the development of epileptogenic foci. In this study,we examined some of the hypothesized reasons for the difference in the incidence ofearly posttraumatic seizures between young and adult rats. Twelve young and twelveadult rats were randomized into 4 groups. Group 1 and 2 were control groups, eachcomprising of 6 young rats and 6 adult rats respectively and were given intracorticalinjections of normal saline. Group 3 and 4 were injury groups, again comprising 6 youngrats and 6 adult rats respectively and were given intracortical injections of FeCl3. All ratswere observed for 6 hours post injection for the occurrence of seizures and were thenkilled. The injected hemispheres were extirpated and tested for malondialdehyde (MDA)level and superoxide dismutase (SOD) activity as indices of oxidative damage. Resultsshowed that seizures were observed only in Group 3. Increased MDA level and decreasedSOD activity were observed in Group 3 (ANOVA, p<0.001). Increased MDA levels anddecreased SOD activity were significantly higher in rats with seizures (Group 3) than inthose without seizures (independent t-test, p<0.001). We conclude was that differentlevels of lipid peroxidation induced by intracortical ferric chloride injection may accountfor the different seizure incidence between young and adult rat

Dynamics of Vesicle Formation from Lipid Droplet: Mechanism and Controllability

A coarse-grained model developed by Marrink et al. [J. Phys. Chem. B 111, 7812 (2007)] is applied to investigate vesiculation of lipid [dipalmitoylphosphatidylcholine (DPPC)] droplets in water. Three kinds of morphologies of micelles are found with increasing lipid droplet size. When the initial lipid droplet is smaller, the equilibrium structure of the droplet is a spherical micelle. When the initial lipid droplet is larger, the lipid ball starts to transform into a disk micelle or vesicle. The mechanism of vesicle formation from a lipid ball is analyzed from the self-assembly of DPPC on the molecular level, and the morphological transition from disk to vesicle with increasing droplet size is demonstrated. Importantly, we discover that the transition point is not very sharp, and for a fixed-size lipid ball, the disk and vesicle appear with certain probabilities. The splitting phenomenon, i.e., the formation of a disk/vesicle structure from a lipid droplet, is explained by applying a hybrid model of the Helfrich membrane theory. The elastic module of the DPPC bilayer and the smallest size of a lipid droplet for certain formation of a vesicle are successfully predicted.Comment: 22 pages, 11 figures Submitted to J. Chem. Phy

Undercovering the molecular mechanisms of lipid signalling at ER-PM contact sites in tomato (Solanum lycopersicum) under abiotic stress conditions

Abiotic stresses cause large reductions in crop production. Therefore, is important to understand how plants respond in order to develop varieties with increased resistance. Lipid-transport proteins (LTP) are emerging as key players of lipid signaling in response to numerous stresses. Specifically, SYT1, a protein first identified by its role in abiotic stress tolerance, is now recognized as an endoplasmic reticulum-plasma membrane contact site tether capable. Our recent data support that SYT1 in involved on non-vesicular lipid-transport of diacyl glycerol (DAG) through its SMP domain. This data together with the interaction of SYT1 with a diacyl glycerol kinase (DGK) suggest a lipid signaling pathway where the product of phospholipase C, diacylglycerol, might be simultaneously translocated from the plasma membrane to the endoplasmic-reticulum by SYT1 and phosphorylated to phosphatidic acid by DGK at the plasma membrane. Using in vitro biochemical approaches we are investigating the affinity of specific lipid species transported by SYT1 using lipid-competition assays, where a fluorescent lipid competes for SYT1 binding-pocket with different lipid species. Using bioinformatic we are obtaining insight into the lipid signal pathway involving PHOSPHOLIPASE C (PLC), DIACYLGLYCEROL KINASE (DGK) and SYNAPTOTAGMIN1 (SYT1) in tomatoUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Identification of plasma lipid biomarkers for prostate cancer by lipidomics and bioinformatics

Background: Lipids have critical functions in cellular energy storage, structure and signaling. Many individual lipid molecules have been associated with the evolution of prostate cancer; however, none of them has been approved to be used as a biomarker. The aim of this study is to identify lipid molecules from hundreds plasma apparent lipid species as biomarkers for diagnosis of prostate cancer. Methodology/Principal Findings: Using lipidomics, lipid profiling of 390 individual apparent lipid species was performed on 141 plasma samples from 105 patients with prostate cancer and 36 male controls. High throughput data generated from lipidomics were analyzed using bioinformatic and statistical methods. From 390 apparent lipid species, 35 species were demonstrated to have potential in differentiation of prostate cancer. Within the 35 species, 12 were identified as individual plasma lipid biomarkers for diagnosis of prostate cancer with a sensitivity above 80%, specificity above 50% and accuracy above 80%. Using top 15 of 35 potential biomarkers together increased predictive power dramatically in diagnosis of prostate cancer with a sensitivity of 93.6%, specificity of 90.1% and accuracy of 97.3%. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) demonstrated that patient and control populations were visually separated by identified lipid biomarkers. RandomForest and 10-fold cross validation analyses demonstrated that the identified lipid biomarkers were able to predict unknown populations accurately, and this was not influenced by patient's age and race. Three out of 13 lipid classes, phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (ePE) and ether-linked phosphatidylcholine (ePC) could be considered as biomarkers in diagnosis of prostate cancer. Conclusions/Significance: Using lipidomics and bioinformatic and statistical methods, we have identified a few out of hundreds plasma apparent lipid molecular species as biomarkers for diagnosis of prostate cancer with a high sensitivity, specificity and accuracy

Lipid Metabolism

Perilipin 2, also known as Adipose differentiation-relation protein or PLIN2, is a lipid droplet-binding protein present in almost every tissue. The absence of PLIN2 upregulates hepatic very low-density lipoprotein secretion, relieves hepatosteatosis, and improves whole body insulin resistance in mice. Despite of the importance in mediating lipid metabolism, the regulation of PLIN2 itself remains largely unknown. Previous reports have shown that X-box binding protein 1 (XBP1) is an important regulator of lipogenesis. XBP1 is a transcription factor that recognizes and binds to a consensus sequence, 5’-TGACGTGG-3’. Interestingly, when we looked through the promoter region of mouse Plin2 gene, we found that the consensus sequence is present in the Plin2 promoter. Therefore, we hypothesize that XBP1 might directly bind to Plin2 promoter and regulate the Plin2 expression. To test our hypothesis, we will perform the luciferase assay to examine whether the Plin2 promoter activity is regulated by XBP1. We first designed forward and reverse PCR primers, which include BglII and BamHI restriction enzyme sites respectively, to amplify the Plin2 promoter region (from -1100 to +40). We performed PCR and cloned the Plin2 promoter to a TA vector. The TA vector was then sequenced to exclude any point mutations. After sequencing, we sub cloned the Plin2 promoter into a vector containing a luciferase reporter. In the future, we will transfect 293T, a human embryonic kidney cell line, with the Plin2 promoter-luciferase vector we generated. We will compare the Plin2 promoter activity by measuring the luminescence in the presence or absence of XBP1