Comparison of ethanol tolerance between potential cyanobacterial production hosts.

Cyanobacteria are photosynthetic prokaryotes that have been extensively studied as potential autotrophic biotechnological hosts for the production of different carbon-based end-products directly from atmospheric CO2. While commercially competitive applications do not yet exist, the production of ethanol in cyanobacteria is the most mature technology, endorsed by relatively high production yields and established status of ethanol in the global biofuel market. Within this concept, the aim here was to systematically compare ethanol tolerance of different commonly used cyanobacterial strains and substrains, in order to assess their relative potential for biotechnological production platforms. The comparison revealed clear strain-specific differences in ethanol toxicity, with growth inhibition GI50 values ranging between 3 g L-1 (0.4% V/V) and 28 g L-1 (3.5% V/V). The most tolerant wild-type strains were Synechocystis sp. PCC 6803 (substrain A) and Synechococcus sp. PCC 7002, which did not show any apparent effect in growth below ethanol concentrations 9.2 g L-1 (1.2% V/V). In comparison to typical biotechnological yeast strains used for ethanol fermentation, these values are clearly lower but still around the same order of magnitude. The results also underlined the challenges in direct number-based comparison between cyanobacterial strains and culture conditions due to inconsistencies in respect to chlorophyll content, cell morphology and optical properties.

Triacylglycerol regioisomers in human milk resolved with an algorithmic novel electrospray ionization tandem mass spectrometry method.

A highly sensitive mass spectrometric (MS) method was developed and validated to analyze ratios of regioisomeric triacylglycerols (TAGs) in fats and oils. UPLC resolution of lithiated TAGs followed by daughter scan MS/MS of positive ions revealed several indicative ions for quantitative analysis. Reference TAGs containing C14-C20 fatty acids (FAs) showed good linear response. Analysis of Finnish and Chinese pooled human milk samples revealed hundreds of regioisomeric TAGs. At least 64mol% of the TAGs were quantified with relative standard deviation <17%. When present in the same TAG molecule together with C18 FAs, palmitic acid was typically in the sn-2 position. When together with FAs 10:0, 12:0, 14:0, 20:1 and 20:2, the sn-2 preference of 16:0 was less clear. Oleic acid occupied typically the sn-1/sn-3 positions but when together with FAs 20:1, 20:2, 18:2, 14:1, 12:0 or 10:0 the positioning of 18:1 did not follow these rules.

Faecalibacterium prausnitzii treatment improves hepatic health and reduces adipose tissue inflammation in high-fat fed mice.

Faecalibacterium prausnitzii is considered as one of the most important bacterial indicators of a healthy gut. We studied the effects of oral F. prausnitzii treatment on high-fat fed mice. Compared to the high-fat control mice, F. prausnitzii-treated mice had lower hepatic fat content, aspartate aminotransferase and alanine aminotransferase, and increased fatty acid oxidation and adiponectin signaling in liver. Hepatic lipidomic analyses revealed decreases in several species of triacylglycerols, phospholipids and cholesteryl esters. Adiponectin expression was increased in the visceral adipose tissue, and the subcutaneous and visceral adipose tissues were more insulin sensitive and less inflamed in F. prausnitzii-treated mice. Further, F. prausnitzii treatment increased muscle mass that may be linked to enhanced mitochondrial respiration, modified gut microbiota composition and improved intestinal integrity. Our findings show that F. prausnitzii treatment improves hepatic health, and decreases adipose tissue inflammation in mice and warrant the need for further studies to discover its therapeutic potential.

Enantioselective chromatography in analysis of triacylglycerols common in edible fats and oils.

Enantiomers of racemic triacylglycerol (TAG) mixtures were separated using two chiral HPLC columns with a sample recycling system and a UV detector. A closed system without sample derivatisation enabled separation and identification by using enantiopure reference compounds of eleven racemic TAGs with C12-C22 fatty acids with 0-2 double bonds. The prolonged separation time was compensated for by fewer pretreatment steps. Presence of one saturated and one unsaturated fatty acid in the asymmetric TAG favoured the separation. Enantiomeric resolution, at the same time with stronger retention of TAGs, increased with increasing fatty acid chain length in the sn-1(3) position. Triunsaturated TAGs containing oleic, linoleic or palmitoleic acids did not separate. The elution order of enantiomers was determined by chemoenzymatically synthesised enantiopure TAGs with a co-injection method. The method is applicable to many natural fats and oils of low unsaturation level assisting advanced investigation of lipid synthesis and metabolism.

Membrane curvature effects on glycolipid transfer protein activity.

The glycolipid transfer protein (GLTP) is monomeric in aqueous solutions, and it binds weakly to membrane interfaces with or without glycolipids. GLTP is a surface-active protein and adsorbs to exert a maximal surface pressure value of 19 mN/m. The change in surface pressure following GLTP adsorption decreased linearly with initial surface pressure. The exclusion pressure for different phospholipids and sphingolipids was between 23 and 31 mN/m, being clearly highest for the negatively charged dipalmitoyl-phosphatidylserine. This can be explained by electrostatic forces when GLTP is positively charged at neutral pH (isoelectric point = 9.0) and by phosphatidylserine being negatively charged. If GLTP is injected under a palmitoyl-galactosylceramide monolayer above 30 mN/m, the presence of GLTP leads to a decrease in the surface pressure as a function of time. This suggests that GLTP is able to remove glycolipids from the monolayer without penetrating the monolayer. On the other hand, if phospholipid vesicles with or without glycolipids are also present in the subphase, no change in the surface pressure takes place. This suggests that GLTP in the presence of curved membranes is not able to transfer from or to planar membranes. We also show that transfer of fluorescently labeled galactosylceramide is faster from small highly curved palmitoyl-oleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylcholine bilayer vesicles but not from palmitoyl-sphingomyelin vesicles regardless of the size.

Membrane interaction and activity of the glycolipid transfer protein.

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles.

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC

Structural evidence for adaptive ligand binding of glycolipid transfer protein.

Glycolipids participate in many important cellular processes and they are bound and transferred with high specificity by glycolipid transfer protein (GLTP). We have solved three different X-ray structures of bovine GLTP at 1.4 angstroms, 1.6 angstroms and 1.8 angstroms resolution, all with a bound fatty acid or glycolipid. The 1.4 angstroms structure resembles the recently characterized apo-form of the human GLTP but the other two structures represent an intermediate conformation of the apo-GLTPs and the human lactosylceramide-bound GLTP structure. These novel structures give insight into the mechanism of lipid binding and how GLTP may conformationally adapt to different lipids. Furthermore, based on the structural comparison of the GLTP structures and the three-dimensional models of the related Podospora anserina HET-C2 and Arabidopsis thaliana accelerated cell death protein, ACD11, we give structural explanations for their specific lipid binding properties.

Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity.

Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.

Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes.

The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C16. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to 'lipid raft' like membranes, suggesting that the protein could be involved in raft assembly.

Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2.

This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded by SCP-X. The presented structural models of apo-AtSCP-2 and the ligand-bound conformation of AtSCP-2 reveal remarkable similarity with two of the structurally known SCP-2s, the SCP-2-like domain of human DBP and the rabbit SCP-2, correspondingly. The AtSCP-2 models in both forms have a similar hydrophobic ligand-binding tunnel, which is extremely suitable for lipid binding. AtSCP-2 showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC) from donor membranes to acceptor membranes. The transfer of BODIPY-PC was almost completely inhibited after addition of 1-palmitoyl 2-oleoyl phosphatidylcholine or ergosterol. Dimyristoyl phosphatidic acid, stigmasterol, steryl glucoside, and cholesterol showed a moderate to marginal ability to lower the BODIPY-PC transfer rate, and the single chain palmitic acid and stearoyl-coenzyme A did not affect transfer at all. Expression analysis showed that AtSCP-2 mRNA is accumulating in most plant tissues. Plasmids carrying fusion genes between green fluorescent protein and AtSCP-2 were transformed with particle bombardment to onion epidermal cells. The results from analyzing the transformants indicate that AtSCP-2 is localized to peroxisomes.

A calorimetric study of binary mixtures of dihydrosphingomyelin and sterols, sphingomyelin, or phosphatidylcholine.

The thermotropic properties of binary mixtures of D-erythro-n-palmitoyl-dihydrosphingomyelin (16:0-DHSM), D-erythro-n-palmitoyl-sphingomyelin (16:0-SM), cholesterol, lathosterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were studied by differential scanning calorimetry. Addition of sterol to 16:0-DHSM and 16:0-SM bilayers resulted in a progressive decrease in both the T(m) and the enthalpy of the main transition. The sterol-induced broad components in 16:0-DHSM endotherms had markedly lower enthalpies than those induced in 16:0-SM. Pretransitions recorded in 16:0-DHSM and 16:0-SM membranes responded differently to low concentrations of cholesterol. The presence of 5 mol % cholesterol increased the pretransition temperature in 16:0-SM bilayers, whereas it decreased the temperature in 16:0-DHSM membranes. Lathosterol behaved in general as cholesterol with regard to its effects on the thermotropic behavior of both sphingolipids, but it appeared to form more stable sterol-rich domains, as seen from the higher T(m) of the broad component, in comparison to cholesterol. Thermograms recorded on binary mixtures of 16:0-SM:16:0-DHSM and DPPC:16:0-DHSM showed that 16:0-SM mixed nearly ideally with 16:0-DHSM, whereas DPPC mixing was less ideal in a 16:0-DHSM membrane. In conclusion, we observed that 16:0-DHSM interactions with sterols differed from that seen with 16:0-SM, and that 16:0-DHSM mixed better with 16:0-SM than DPPC, which indicates that DHSM could function as a membrane organizer within laterally condensed domains.

Properties of palmitoyl phosphatidylcholine, sphingomyelin, and dihydrosphingomyelin bilayer membranes as reported by different fluorescent reporter molecules.

The properties of vesicle membranes prepared from 16:0-SM, 16:0-DHSM, or DPPC were characterized using steady-state and time-resolved fluorescence spectroscopy and different fluorescent reporter molecules. The acyl-chain region was probed using free and phospholipid-bound 1,6-diphenyl-1,3,5-hexatriene. 16:0-DHSM was found to be the more ordered than both DPPC and 16:0-SM 5 degrees C below and above melting temperature. Interfacial properties of the phospholipid bilayers were examined using 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan), 6-propionyl-2-dimethyl-amino-naphthalene (Prodan), and dansyl-PE. Laurdan and Prodan reported that the two sphingomyelin (SM) membrane interfaces were clearly different from the DPPC membrane interface, whereas the two SM membrane interfaces had more similar properties (both in gel and liquid-crystalline phase). Prodan partition studies showed that membrane resistance to Prodan partitioning increased in the order: 16:0-SM < DPPC < 16:0-DHSM. The degree to which dansyl-PE is exposed to water reflects the structural properties of the membrane-water interface. By comparing the lifetime of dansyl-PE in water and deuterium oxide solution, we could show that the degree to which the dansyl moiety was exposed to water in the membranes increased in the order: 16:0-SM < DPPC < 16:0-DHSM. In conclusion, this study has shown that DHSM forms more ordered bilayers than acyl-chain matched SM or phosphatidylcholine, even in the liquid-crystalline state.