Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter ∼0.1 and 0.2 μm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 °C, this temperature corresponding closely to the heat capacity maxima (T_em) of DNPC MLVs and LUVs (T_em ≈21 °C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T_em. This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans→gauche isomerization.     

Oxidized phospholipids as potential molecular targets for antimicrobial peptides

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.     

Binding of LL-37 to model biomembranes: insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Amyloid-type fiber formation in control of enzyme action: interfacial activation of phospholipase A2

The lag-burst behavior in the action of phospholipase A₂ (PLA₂) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T_m of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA₂, evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA₂, involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form “mature” fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.     

Membranes: a meeting point for lipids, proteins and therapies

Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.     

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.     

Differences in Muscle and Adipose Tissue Gene Expression and Cardio-Metabolic Risk Factors in the Members of Physical Activity Discordant Twin Pairs

High physical activity/aerobic fitness predicts low morbidity and mortality. Our aim was to identify the most up-regulated gene sets related to long-term physical activity vs. inactivity in skeletal muscle and adipose tissues and to obtain further information about their link with cardio-metabolic risk factors. We studied ten same-sex twin pairs (age range 50–74 years) who had been discordant for leisure-time physical activity for 30 years. The examinations included biopsies from m. vastus lateralis and abdominal subcutaneous adipose tissue. RNA was analyzed with the genome-wide Illumina Human WG-6 v3.0 Expression BeadChip. For pathway analysis we used Gene Set Enrichment Analysis utilizing active vs. inactive co-twin gene expression ratios. Our findings showed that among the physically active members of twin pairs, as compared to their inactive co-twins, gene expression in the muscle tissue samples was chronically up-regulated for the central pathways related to energy metabolism, including oxidative phosphorylation, lipid metabolism and supportive metabolic pathways. Up-regulation of these pathways was associated in particular with aerobic fitness and high HDL cholesterol levels. In fat tissue we found physical activity-associated increases in the expression of polyunsaturated fatty acid metabolism and branched-chain amino acid degradation gene sets both of which associated with decreased ‘high-risk’ ectopic body fat and plasma glucose levels. Consistent with other findings, plasma lipidomics analysis showed up-regulation of the triacylglycerols containing the polyunsaturated fatty acids. Our findings identified skeletal muscle and fat tissue pathways which are associated with the long-term physical activity and reduced cardio-metabolic disease risk, including increased aerobic fitness. In particular, improved skeletal muscle oxidative energy and lipid metabolism as well as changes in adipocyte function and redistribution of body fat are associated with reduced cardio-metabolic risk.     

Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems

The Yersinia enterocolitica O:3 lipopotysaccharide O-antigen is a homopotymer of 6-deoxy-L-altrose. The cloned rfb region was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementatton experiments showed that eight of the genes, organized into two operons, rfbABC and rfbDEFGH, are essential for 0-antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar to Salmonella proteins involved in the dTDP-l -rhamnose biosynthesis. Rhamnose and 6-deoxy-l -altrose are C3-epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT of Escherichia coli. This and transposon mutagenesis showed that RfbD and RfbE function as O-antigen exporters.     

Responses by the capercaillie Tetrao urogallus, and the willow grouse Lagopus lagopus, to the green matter available in early spring

Food matter eaten in the first snowless spots early in the spring by capercaillie Tetrao urogallus and willow grouse Lagopus lagopus was studied in Finnish Forest Lapland. When the snow disappears, both species change to feeding mainly on the plants available in the snowless spots, although male capercaillie does not exploit this nutritious diet to the same extent as female capercaillie and willow grouse. Selection in favour of nitrogen and phosphorus-rich food items, i.e. Betula pubescens , male catkins Eriophorum vaginatum , flower buds, and Equisetum spp., stems and tips, seems to be characteristic especially of the female capercaillie, whose winter diet is poor in these elements. Female capercaillie also feeds on more plant species or different parts of plants at one time than willow grouse, while no differences were recorded between male and female willow grouse in the composition of their spring food. The crowberry Empetrum hermaphroditum , almost regularly produces a bountiful berry crop in northern Finland, and since it overwinters well, it represents an easy source of energy and water available almost every spring. The quality and/or the quantity of the green food matter available early in the spring may fluctuate considerably and be of importance for short-term fluctuations in the populations of these tetraonid species.