Lipid Gymnastics: Evidence of Complete Acyl Chain Reversal in Oxidized Phospholipids from Molecular Simulations

In oxidative environments, biomembranes contain oxidized lipids with short, polar acyl chains. Two stable lipid oxidation products are PoxnoPC and PazePC. PoxnoPC has a carbonyl group, and PazePC has an anionic carboxyl group pendant at the end of the short, oxidized acyl chain. We have used MD simulations to explore the possibility of complete chain reversal in OXPLs in POPC-OXPL mixtures. The polar AZ chain of PazePC undergoes chain reversal without compromising the lipid bilayer integrity at concentrations up to 25% OXPL, and the carboxyl group points into the aqueous phase. Counterintuitively, the perturbation of overall membrane structural and dynamic properties is stronger for PoxnoPC than for PazePC. This is because of the overall condensing and ordering effect of sodium ions bound strongly to the lipids in the PazePC simulations. The reorientation of AZ chain is similar for two different lipid force fields. This work provides the first molecular evidence of the “extended lipid conformation” in phospholipid membranes. The chain reversal of PazePC lipids decorates the membrane interface with reactive, negatively charged functional groups. Such chain reversal is likely to exert a profound influence on the structure and dynamics of biological membranes, and on membrane-associated biological processes.     

The magnetic retina: light-dependent and trigeminal magnetoreception in migratory birds

Recent advances have brought much new insight into the physiological mechanisms and required characteristics of the sensory molecules that enable birds to use magnetic fields for orientation. European robins almost certainly have two magnetodetection senses, one associated with the ophthalmic branch of the trigeminal nerve, and one based on light-dependent radical-pair processes in both eyes. The first brain areas processing magnetic information from each of these two senses have been identified. It has been experimentally verified that Earth-strength magnetic fields can affect photo-induced chemical reactions and that these reactions can respond to magnetic field direction. Diagnostic behavioural experiments have provided clues to identify putative magnetoreceptive molecules in the retina. We discuss the implications of these and other recent findings and outline crucial open questions with an emphasis on the light-dependent mechanism.     

Energetic consequences of a major change in habitat use: endangered Brent Geese Branta bernicla hrota losing their main food resource

Coastal seagrasses are declining at increasing rates worldwide, forcing herbivores previously reliant on these habitats to abandon them in search of alternative ways to fulfil their daily energy budgets. After two decades of declining seagrass abundance in Mariager Fjord, Denmark, the Svalbard breeding population of Light‐bellied Brent Geese Branta bernicla hrota has experienced substantial changes in habitat use at this traditional autumn staging area. Declines in seagrasses have caused birds to depend increasingly on Sea Lettuce Ulva lactuca in recent years, and forced birds into terrestrial habitats such as saltmarsh and winter wheat. In contrast to those birds exploiting aquatic habitats, birds relying on these new habitats showed higher energy expenditure and failed to balance their energy budget. Eelgrass (Zostera) was energetically superior to other food resources, with marine Ulva being second best. Predicted body mass development under two different scenarios indicate that present habitat use resulted in a midwinter body mass around 122 g lower than just 20 years ago, equivalent to c. 9.4% of Brent Goose body weight. Even after controlling for inter‐annual differences in thermoregulatory costs, the effect of changes in habitat use translated into a body mass reduction of c. 56 g, which could adversely affect survival and future reproduction. Flyway‐wide declines in Zostera abundance and further reductions in traditional habitats due to climate change give cause to reassess projected population trends and consequent management implications for the East Atlantic flyway population of Light‐bellied Brent Geese.     

Temperature-controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers

Temperature-controlled atomic force microscopy (AFM) has been used to visualize and study the structure and kinetics of ripple phases in one-component dipalmitoylphosphatidylcholine (DPPC) and two-component dimyristoylphosphatidylcholine-distearoylphosphatidylcholine (DMPC-DSPC) lipid bilayers. The lipid bilayers are mica-supported double bilayers in which ripple-phase formation occurs in the top bilayer. In one-component DPPC lipid bilayers, the stable and metastable ripple phases were observed. In addition, a third ripple structure with approximately twice the wavelength of the metastable ripples was seen. From height profiles of the AFM images, estimates of the amplitudes of the different ripple phases are reported. To elucidate the processes of ripple formation and disappearance, a ripple-phase DPPC lipid bilayer was taken through the pretransition in the cooling and the heating direction and the disappearance and formation of ripples was visualized. It was found that both the disappearance and formation of ripples take place virtually one ripple at a time, thereby demonstrating the highly anisotropic nature of the ripple phase. Furthermore, when a two-component DMPC-DSPC mixture was heated from the ripple phase and into the ripple-phase/fluid-phase coexistence temperature region, the AFM images revealed that several dynamic properties of the ripple phase are important for the melting behavior of the lipid mixture. Onset of melting is observed at grain boundaries between different ripple types and different ripple orientations, and the longer-wavelength metastable ripple phase melts before the shorter-wavelength stable ripple phase. Moreover, it was observed that the ripple phase favors domain growth along the ripple direction and is responsible for creating straight-edged domains with 60 degrees and 120 degrees angles, as reported previously.     

Functional screening of a retroviral peptide library for MHC class I presentation

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.     

Linked foreign T-cell help activates self-reactive CTL and inhibits tumor growth

Transgenic mice expressing membrane-bound OVA under the rat insulin promoter, RIP-mOVA, has previously been suggested to display deletional tolerance toward the dominant CTL epitope, SIINFEKL, and provide an elegant model system to test the hypothesis that the lack of T cell help contributes to the tolerance. To understand how the CD8 tolerance is maintained in these mice, a set of neo-self-Ags, OVA, modified to contain a foreign Th peptide, were constructed and tested for their ability to induce CTL responses in RIP-mOVA mice. Immunization with these Th peptide-modified OVA molecules and not with the wild-type OVA induced self-reactive CTLs recognizing dominant CTL peptide, SIINFEKL. Importantly, immunization with the modified OVA constructs also prevented the growth of OVA-expressing tumors in transgenic mice. Since endogenous OVA Th peptides did not contribute toward breaking self CTL tolerance, these results also highlighted a very robust CD4 T cell tolerance toward OVA in RIP-mOVA mice that has not been previously described. These results therefore provide direct evidence that it is the tolerance in the CD4 Th cell compartment that helps maintain the CTL tolerance against self-Ag in these mice. Since the CTL tolerance can be broken or bypassed by foreign Th peptides inserted into a self Ag, potential of using this approach in generating effective therapeutic cancer vaccines is discussed.     

Magnetoreception and its use in bird navigation

Recent advances have brought new insight into the physiological mechanisms that enable birds and other animals to use magnetic fields for orientation. Many birds seem to have two magnetodetection senses, one based on magnetite near the beak and one based on light-dependent radical-pair processes in the bird's eye(s). Among the most exciting recent results are: first, behavioural responses of birds experiencing oscillating magnetic fields. Second, the occurrence of putative magnetosensory molecules, the cryptochromes, in the eyes of migratory birds. Third, detection of a brain area that integrates specialised visual input at night in night-migratory songbirds. Fourth, a putative magnetosensory cluster of magnetite in the upper beak. These and other recent findings have important implications for magnetoreception; however, many crucial open questions remain.     

A thermodynamic study of the effects of cholesterol on the interaction between liposomes and ethanol

The association of ethanol with unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes of varying cholesterol content has been investigated by isothermal titration calorimetry over a wide temperature range (8-45 degrees C). The calorimetric data show that the interaction of ethanol with the lipid membranes is endothermic and strongly dependent on the phase behavior of the mixed lipid bilayer, specifically whether the lipid bilayer is in the solid ordered (so), liquid disordered (ld), or liquid ordered (lo) phase. In the low concentration regime (<10 mol%), cholesterol enhances the affinity of ethanol for the lipid bilayer compared to pure DMPC bilayers, whereas higher levels of cholesterol (>10 mol%) reduce affinity of ethanol for the lipid bilayer. Moreover, the experimental data reveal that the affinity of ethanol for the DMPC bilayers containing small amounts of cholesterol is enhanced in the region around the main phase transition. The results suggest the existence of a close relationship between the physical structure of the lipid bilayer and the association of ethanol with the bilayer. In particular, the existence of dynamically coexisting domains of gel and fluid lipids in the transition temperature region may play an important role for association of ethanol with the lipid bilayers. Finally, the relation between cholesterol content and the affinity of ethanol for the lipid bilayer provides some support for the in vivo observation that cholesterol acts as a natural antagonist against alcohol intoxication.     

Phase I study of TNFα AutoVaccIne in Patients with metastatic cancer

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNF in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNF proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNF antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 g) of TNF vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNF antibody titres were measured by both a RIA, using soluble native TNF as the antigen, and by an ELISA using immobilized partly denatured TNF. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNF in the ELISA, whereas, antibody titres against native TNF in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNF. In conclusion, TNF vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNF, evaluation of safety and tumour responses to the TNF vaccine was compromised.     

Biophysical mechanisms of phospholipase A2 activation and their use in liposome-based drug delivery

Secretory phospholipase A2 (PLA2) is a ubiquitous water-soluble enzyme found in venom, pancreatic, and cancerous fluid. It is also known to play a role in membrane remodeling processes as well as in cellular signaling cascades. PLA2 is interfacially active and functions mainly on organized types of substrate, e.g. micelles and lipid bilayers. Hence the activity of the enzyme is modulated by the lateral organization and the physical properties of the substrate, in particular the structure in the nanometer range. The evidence for nano-scale structure and lipid domains in bilayers is briefly reviewed. Results obtained from a variety of experimental and theoretical studies of PLA2 activity on lipid-bilayer substrates are then presented which provide insight into the biophysical mechanisms of PLA2 activation on lipid bilayers and liposomes of different composition. The insight into these mechanisms has been used to propose a novel principle for liposomal drug targeting, release, and absorption triggered by secretory PLA2.