Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

Stabilization of Phospholipid Coatings in Capillary Electrophoresis

The development of a simple method of coating a semi-permanent phospholipid layer onto a capillary for electrochromatography use was the focus of this study. The work involved finding good coating conditions, stabilizing the phospholipid coating, and examining the effect of adding divalent cations, cetyltrimethylammonium bromide, and polyethylene glycol (PEG)-lipids on the stability of the coating. Since a further purpose was to move toward more biological membrane coatings, the capillaries were also coated with cholesterol-containing liposomes and liposomes of red blood cell ghost lipids. Liposomes were prepared by extrusion, and large unilamellar vesicles with a diameter of about 100 nm were obtained. Zwitterionic phosphatidylcholine (PC) was used as a basic component, mainly 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) but also eggPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Different amounts of sphingomyelin, bovine brain phosphatidylserine, and cholesterol were added to the PC. The stability of the coating in 40 mM N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) solution at pH 7.4 was studied by measuring the electroosmotic flow and by separating neutral steroids, basic proteins, and low-molar-mass drugs. The presence of PC in the coating solution was found to be essential to achieving a coating. The stability of the coating was improved by the addition of negative phosphatidylserine, cholesterol, divalent cations, or PEGylated lipids, and by working in the gel-state region of the phospholipid. Study of the effect on the PC coating of divalent metal ions calcium, magnesium, and zinc showed a molar ratio of 1:3 PC/Ca2+ or PC/Mg2+ to give increased rigidity to the membrane and the best coating stability. The PEGylated lipids used in the study were sterically stabilized commercial lipids with covalently attached PEG chains. The vesicle size generally decreased when PEGylated lipids of higher molar mass were present in the vesicle. The predominance of discoidal micelles over liposomes increased PEG chain length and the average size of the vesicles thus decreased. In the capillary electrophoresis (CE) measurements a highly stable electroosmotic flow was achieved with 20% PEGylated lipid in the POPC coating dispersion, the best results being obtained for disteroyl PEG (3000) conjugates. The results suggest that smaller particles (discoidal micelles) result in tighter packing and better shielding of silanol groups on the silica wall. The effect of temperature on the coating stability was investigated by using DPPC liposomes at temperatures above (45 C) and below (25 C) the main phase transition temperature. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was heavily suppressed and the PC coating was stabilized. Also dispersions of DPPC with 0−30 mol% of cholesterol and sphingomyelin in different ratios, which more closely resemble natural membranes, resulted in stable coatings. Finally, the CE measurements revealed that a stable coating is formed when capillaries are coated with liposomes of red blood cell ghost lipids.

The high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.

Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.

The role of phospholipid transfer protein and cholesteryl ester transfer protein in reverse cholesterol transport

In atherosclerosis, cholesterol accumulates in the vessel wall, mainly in the form of modified low-density lipoprotein (LDL). Macrophages of the vessel wall scavenge cholesterol, which leads to formation of lipid-laden foam cells. High plasma levels of high-density lipoprotein (HDL) protect against atherosclerosis, as HDL particles can remove peripheral cholesterol and transport it to the liver for excretion in a process called reverse cholesterol transport (RCT). Phospholipid transfer protein (PLTP) remodels HDL particles in the circulation, generating prebeta-HDL and large fused HDL particles. In addition, PLTP maintains plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. Most of the cholesteryl ester transfer protein (CETP) in plasma is bound to HDL particles and CETP is also involved in the remodeling of HDL particles. CETP enhances the heteroexchange of cholesteryl esters in HDL particles for triglycerides in LDL and very low-density lipoprotein (VLDL). The aim of this thesis project was to study the importance of endogenous PLTP in the removal of cholesterol from macrophage foam cells by using macrophages derived from PLTP-deficient mice, determine the effect of macrophage-derived PLTP on the development of atherosclerosis by using bone marrow transplantation, and clarify the role of the two forms of PLTP, active and inactive, in the removal of cholesterol from the foam cells. In addition, the ability of CETP to protect HDL against the action of chymase was studied. Finally, cholesterol efflux potential of sera obtained from the study subjects was compared. The absence of PLTP in macrophages derived from PLTP-deficient mice decreased cholesterol efflux mediated by ATP-binding cassette transporter A1. The bone marrow transplantation studies showed that selective deficiency of PLTP in macrophages decreased the size of atherosclerotic lesions and caused major changes in serum lipoprotein levels. It was further demonstrated that the active form of PLTP can enhance cholesterol efflux from macrophage foam cells through generation of prebeta-HDL and large fused HDL particles enriched with apoE and phospholipids. Also CETP may enhance the RCT process, as association of CETP with reconstituted HDL particles prevented chymase-dependent proteolysis of these particles and preserved their cholesterol efflux potential. Finally, serum from high-HDL subjects promoted more efficient cholesterol efflux than did serum derived from low-HDL subjects which was most probably due to differences in the distribution of HDL subpopulations in low-HDL and high-HDL subjects. These studies described in this thesis contribute to the understanding of the PLTP/CETP-associated mechanisms underlying RCT.

Phospholipid cytochrome c interactions

The ability of the peripherally associated membrane protein cytochrome c (cyt c) to bind phospholipids in vitro was studied using fluorescence spectroscopy and large unilamellar liposomes. Previous work has shown that cyt c can bind phospholipids using two distinct mecha- nisms and sites, the A-site and the C-site. This binding is mediated by electrostatic or hydrophobic interactions, respectively. Here, we focus on the mechanism underlying these interactions. A chemically modified cyt c mutant Nle91 was used to study the ATP-binding site, which is located near the evolutionarily invariant Arg 91 on the protein surface. This site was also demonstrated to mediate phospholipid binding, possibly by functioning as a phospholipid binding site. Circular dichroism spectroscopy, time resolved fluorescence spectroscopy of zinc- porphyrin modified [Zn2+-heme] cyt c and liposome binding studies of the Nle91 mutant were used to demonstrate that ATP induces a conformational change in membrane- bound cyt c. The ATP-induced conformational changes were mediated by Arg 91 and were most pronounced in cyt c bound to phospholipids via the C-site. It has been previously reported that the hydrophobic interaction between phospho- lipids and cyt c (C-site) includes the binding of a phospholipid acyl chain inside the protein. In this mechanism, which is known as extended phospholipid anchorage, the sn-2 acyl chain of a membrane phospholipid protrudes out of the membrane surface and is able to bind in a hydrophobic cavity in cyt c. Direct evidence for this type of bind- ing mechanism was obtained by studying cyt c/lipid interaction using fluorescent [Zn2+- heme] cyt c and fluorescence quenching of brominated fatty acids and phospholipids. Under certain conditions, cyt c can form fibrillar protein-lipid aggregates with neg- atively charged phospholipids. These aggregates resemble amyloid fibrils, which are involved in the pathogenesis of many diseases. Congo red staining of these fibers con- firmed the presence of amyloid structures. A set of phospholipid-binding proteins was also found to form similar aggregates, suggesting that phospholipid-induced amyloid formation could be a general mechanism of amyloidogenesis.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

External signal-activated liposomal drug delivery systems

Modern drug discovery gives rise to a great number of potential new therapeutic agents, but in some cases the efficient treatment of patient may not be achieved because the delivery of active compounds to the target site is insufficient. Thus, drug delivery is one of the major challenges in current pharmaceutical research. Numerous nanoparticle-based drug carriers, e.g. liposomes, have been developed for enhanced drug delivery and targeting. Drug targeting may enhance the efficiency of the treatment and, importantly, reduce unwanted side effects by decreasing drug distribution to non-target tissues. Liposomes are biocompatible lipid-based carriers that have been studied for drug delivery during the last 40 years. They can be functionalized with targeting ligands and sensing materials for triggered activation. In this study, various external signal-assisted liposomal delivery systems were developed. Signals can be used to modulate drug permeation or release from the liposome formulation, and they provide accurate control of time, place and rate of activation. The study involved three types of signals that were used to trigger drug permeation and release: electricity, heat and light. Electrical stimulus was utilized to enhance the permeation of liposomal DNA across the skin. Liposome/DNA complex-mediated transfections were performed in tight rat epidermal cell model. Various transfection media and current intensities were tested, and transfection efficiency was evaluated non-invasively by monitoring the concentration of secreted reporter protein in cell culture medium. Liposome/DNA complexes produced gene expression, but electrical stimulus did not enhance the transfection efficiency significantly. Heat-sensitive liposomal drug delivery system was developed by coating liposomes with biodegradable and thermosensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate polymer. Temperature-triggered liposome aggregation and contents release from liposomes were evaluated. The cloud point temperature (CP) of the polymer was set to 42 °C. Polymer-coated liposome aggregation and contents release were observed above CP of the polymer, while non-coated liposomes remained intact. Polymer precipitates above its CP and interacts with liposomal bilayers. It is likely that this induces permeabilization of the liposomal membrane and contents release. Light-sensitivity was introduced to liposomes by incorporation of small (< 5 nm) gold nanoparticles. Hydrophobic and hydrophilic gold nanoparticles were embedded in thermosensitive liposomes, and contents release was investigated upon UV light exposure. UV light-induced lipid phase transitions were examined with small angle X-ray scattering, and light-triggered contents release was shown also in human retinal pigment epithelial cell line. Gold nanoparticles absorb light energy and transfer it into heat, which induces phase transitions in liposomes and triggers the contents release. In conclusion, external signal-activated liposomes offer an advanced platform for numerous applications in drug delivery, particularly in the localized drug delivery. Drug release may be localized to the target site with triggering stimulus that results in better therapeutic response and less adverse effects. Triggering signal and mechanism of activation can be selected according to a specific application.

Complement system and alcoholic liver disease

Alcoholic liver disease (ALD) is a well recognized and growing health problem worldwide. ALD advances from fatty liver to inflammation, necrosis, fibrosis and cirrhosis. There is accumulating evidence that the innate immune system is involved in alcoholic liver injury. Within the innate and acquired immune systems, the complement system participates in inflammatory reactions and in the elimination of invading foreign, as well as endogenous apoptotic or injured cells. The present study aimed at evaluating the role of the complement system in the development of alcoholic liver injury. First, in order to study the effects of chronic ethanol intake on the complement system, the deposition of complement components in liver and the expression of liver genes associated with complement in animals with alcohol-induced liver injury were examined. It was demonstrated that chronic alcohol exposure leads to hepatic deposition of the complement components C1, C3, C8 and C9 in the livers of rats. Liver gene expression analysis showed that ethanol up-regulated the expression of transcripts for complement factors B, C1qA, C2, C3 and clusterin. In contrast, ethanol down-regulated the expression of the complement regulators factor H, C4bp and factor D and the terminal complement components C6, C8α and C9. Secondly, the role of the terminal complement pathway in the development of ALD was evaluated by using rats genetically deficient in the complement component C6 (C6-/-). It was found that chronic ethanol feeding induced more liver pathology (steatosis and inflammatory changes) in C6-/- rats than in wild type rats. The hepatic triacylglyceride content and plasma alanine aminotransferase activity increased in C6-/- rats, supporting the histopathological findings and elevation of the plasma pro-/anti-inflammatory TNF-/IL-10 ratio was also more marked in C6-/- rats. Third, the role of the alternative pathway in the development of alcoholic liver steatosis was characterized by using C3-/- mice. In C3-/- mice ethanol feeding tended to reduce steatosis and had no further effect on liver triacylglyceride, liver/body weight ratio nor on liver malondialdehyde level and serum alanine aminotransferase activity. In C3-/- mice alcohol-induced liver steatosis was reduced also after an acute alcohol challenge. In both wild type and C3-/- mice ethanol markedly reduced serum cholesterol and ApoA-I levels, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid binding proteins and fatty acid -oxidation enzymes. In contrast, exclusively in C3-/- mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated the expression of transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. In conclusion, this study has demonstrated that the complement system is involved in the development of alcohol-induced liver injury. Chronic alcohol exposure causes local complement activation and induction of mRNA expression of classical and alternative pathway components in the liver. In contrast expression of the terminal pathway components and soluble regulators were decreased. A deficient terminal complement pathway predisposes to alcoholic liver damage and promotes a pro-inflammatory cytokine response. Complement component C3 contributes to the development of alcohol-induced fatty liver and its consequences by affecting regulatory and specific transcription factors of lipid homeostasis.

Effects of recreational use and fragmentation on the understorey vegetation and soil microbial communities of urban forests in southern Finland

The impacts of fragmentation and recreational use on the hemiboreal urban forest understorey vegetation and the microbial community of the humus layer (the phospholipid fatty acid (PLFA) pattern, microbial biomass and microbial activity, measured as basal respiration) were examined in the greater Helsinki area, southern Finland. Trampling tolerance of 1) herb-rich OMT, 2) mesic MT, and 3) sub-xeric VT forests (in decreasing order of fertility) was studied by comparing relative understorey vegetation cover (urban/untrampled reference ratio) of the three forest types. The trampling tolerance of forest vegetation increased with the productivity of the site (sub-xeric < mesic < herb-rich). Wear of understorey vegetation correlated positively with the number of residents (i.e., recreational pressure) around the forest patch. An increase of 15000 residents within a radius of 1 km around a forest patch was associated with ca. 30% decrease in the relative understorey vegetation cover. The cover of dwarf shrub Vaccinium myrtillus in particular decreased with increasing levels of wear. The cover of mosses in urban forests was less than half of that in untrampled reference areas. Cover of tree saplings, mainly Sorbus aucuparia, and some resilient herbs was higher than in the reference areas. In small urban forest fragments, broad-leaved trees, grasses and herbs were more abundant and mosses were scarcer than in larger urban forest areas. Thus, due to trampling and edge effects, resilient herb and grass species are replacing sensitive dwarf shrubs, mosses and lichens in urban forests. Differences in the soil microbial community structure were found between paths and untrampled areas and the effects of paths extended more than one meter from the paths. Paths supported approximately 25-30% higher microbial biomass with a transition zone of at least 1 m from the path edge. However, microbial activity per unit of biomass was lower on paths than in untrampled areas. Furthermore, microbial biomass and activity were 30-45% lower at the first 20 m into the forest fragments, due to low moisture content of humus near the edge. The decreased microbial activity detected at forest edges and paths implies decreased litter decomposition rates, and thus, a change in nutrient cycling. Changes in the decomposition and nutrient supply may in turn affect the diversity and function of plant communities in urban forests. Keywords: boreal forest vegetation, edge effects, phospholipid fatty acids, trampling, urban woodlands, wear

Phospholipids of lipid-containing bacteriophages and their transbilayer distribution

In this study we used electro-spray ionization mass-spectrometry to determine phospholipid class and molecular species compositions in bacteriophages PM2, PRD1, Bam35 and phi6 as well as their hosts. To obtain compositional data of the individual leaflets, phospholipid transbilayer distribution in the viral membranes was studied. We found that 1) the membranes of all studied bacteriophage are enriched in PG as compared to the host membranes, 2) molecular species compositions in the phage and host membranes are similar, and 3) phospholipids in the viral membranes are distributed asymmetrically with phosphatidylglycerol enriched in the outer leaflet and phosphatidylethanolamine in the inner one (except Bam35). Alternative models for selective incorporation of phospholipids to phages and for the origins of the asymmetric phospholipid transbilayer distribution are discussed. Notably, the present data are also useful when constructing high resolution structural models of bacteriophages, since diffraction methods cannot provide a detailed structure of the membrane due to high motility of the lipids and lack of symmetric organization of membrane proteins.

Venous thromboembolism during pregnancy and the impact of thrombophilia in pregnancy complications

Venous thromboembolism (VTE) is the greatest single cause of maternal mortality in pregnant women in developed countries. Pregnancy is a hypercoagulable state and brings about an enhanced risk of deep venous thrombosis (DVT) in otherwise healthy women. Traditionally, unfractionated heparin (UFH) has been used for treatment of DVT during pregnancy. We showed in our observational study that low molecular weight heparin (LMWH) is as effective and safe as UFH in the treatment of DVT during pregnancy. Although DVT during pregnancy is often massive, increasing the risk of developing long-term consequences, namely post-thrombotic syndrome (PTS), only 11% of all patients had confirmed PTS 3 4 years after DVT. In our studies the prevalence of PTS was not dependent on treatment (UFH vs LMWH). Low molecular weight heparin is more easily administered, few laboratory controls are required and the hospital stay is shorter, factors that lower the costs of treatment. Cervical insufficiency is defined as repeated very preterm delivery during the second or early third trimester. Infection is a well-known risk factor of preterm delivery. We found overpresentation of thrombophilic mutations (FV Leiden, prothrombin G20210A)among 42 patients with cervical insufficiency compared with controls (OR 6.7, CI 2.7 18.4). Thus, thrombophilia might be a risk factor of cervical insufficiency possibly explained by interaction of coagulation and inflammation processes. The presence of antiphospholipid (aPL) antibodies increases the risk for recurrent miscarriage (RM). Annexins are proteins which all bind to anionic phospholipids (PLs) preventing clotting on vascular phospholipid surfaces. Plasma concentrations of circulating annexin IV and V were investigated in 77 pregnancies at the beginning of pregnancy among women with a history of RM, and in connection to their aPL antibody status. Control group consisted unselected pregnant patients (n=25) without history of adverse pregnancy outcome. Plasma levels of annexin V were significantly higher at the beginning (≤5th week) of pregnancy in women with aPL antibodies compared with those without aPL antibodies (P=0.03). Levels of circulating annexin V were also higher at the 6th (P= 0.01) and 8th week of pregnancy in subjects with aPL antibodies (P=0.01). Results support the hypothesis that aPL could displace annexin from anionic phospholipid surfaces of syncytiotrophoblasts (STBs) and may exert procoagulant activities on the surfaces of STBs Recurrent miscarriage (RM) has been suggested to be caused by mutations in genes coding for various coagulation factors resulting in thrombophilia. In the last study of my thesis were investigated the prevalence of thrombomodulin (TM) and endothelial protein C receptor polymorphism EPCR among 40 couples and six women suffering RM. This study showed that mutations in the TM or EPCR genes are not a major cause of RM in Finnish patients.

Functional role of the Mso1p-Sec1p complex in membrane fusion regulation

Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins. They perform an essential, albeit poorly understood function in SNARE complex formation in membrane fusion. In addition to the SNARE complex components, only a few SM protein binding proteins are known. Typically, their binding modes to SM proteins and their contribution to the membrane fusion regulation is poorly characterised. We identified Mso1p as a novel Sec1p interacting partner. It was shown that Mso1p and Sec1p interact at sites of polarised secretion and that this localisation is dependent on the Rab GTPase Sec4p and its GEF Sec2p. Using targeted mutagenesis and N- and C-terminal deletants, it was discovered that the interaction between an N-terminal peptide of Mso1p and the putative Syntaxin N-peptide binding area in Sec1p domain 1 is important for membrane fusion regulation. The yeast Syntaxin homologues Sso1p and Sso2p lack the N-terminal peptide. Our results show that in addition to binding to the putative N-peptide binding area in Sec1p, Mso1p can interact with Sso1p and Sso2p. This result suggests that Mso1p can mimic the N-peptide binding to facilitate membrane fusion. In addition to Mso1p, a novel role in membrane fusion regulation was revealed for the Sec1p C-terminal tail, which is missing in its mammalian homologues. Deletion of the Sec1p-tail results in temperature sensitive growth and reduced sporulation. Using in vivo and in vitro experiments, it was shown that the Sec1p-tail mediates SNARE complex binding and assembly. These results propose a regulatory role for the Sec1p-tail in SNARE complex formation. Furthermore, two novel interaction partners for Mso1p, the Rab GTPase Sec4p and plasma membrane phospholipids, were identified. The Sec4p link was identified using Bimolecular Fluorescence Complementation assays with Mso1p and the non-SNARE binding Sec1p(1-657). The assay revealed that Mso1p can target Sec1p(1-657) to sites of secretion. This effect is mediated via the Mso1p C-terminus, which previously has been genetically linked to Sec4p. These results and in vitro binding experiments suggest that Mso1p acts in cooperation with the GTP-bound form of Sec4p on vesicle-like structures prior to membrane fusion. Mso1p shares homology with the PIP2 binding domain of the mammalian Munc18 binding Mint proteins. It was shown both in vivo and in vitro that Mso1p is a phospholipid inserting protein and that this insertion is mediated by the conserved Mso1p amino terminus. In vivo, the Mso1p phospholipid binding is needed for sporulation and Mso1p-Sec1p localisation at the sites of secretion at the plasma membrane. The results reveal a novel layer of membrane fusion regulation in exocytosis and propose a coordinating role for Mso1p in connection with membrane lipids, Sec1p, Sec4p and SNARE complexes in this process.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.