Membrane protein extraction and purification using styrene-maleic acid (SMA) co-polymer:effect of variations in polymer structure

The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene to maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA) which vary in size and shape were used. Our results show that several polymers can be used to extract membrane proteins comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular weight (7.5-10 kDa) is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification SMA 2000 was found to be the best choice for yield, purity and function. However the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

Lipid analytic of environmental and cultural samples

Membrane lipids of marine planktonic archaea have provided unique insights into archaeal ecology and paleoceanography. However, past studies of archaeal lipids in suspended particulate matter (SPM) and sediments mainly focused on a small class of fully saturated glycerol dibiphytanyl glycerol tetraether (GDGT) homologues identified decades ago. The apparent low structural diversity of GDGTs is in strong contrast to the high diversity of metabolism and taxonomy among planktonic archaea. Furthermore, adaptation of archaeal lipids in the deep ocean remains poorly constrained. We report the archaeal lipidome in SPM from diverse oceanic regimes. We extend the known inventory of planktonic archaeal lipids to include numerous unsaturated archaeal ether lipids (uns-AELs). We further reveal i) different thermal regulations and polar headgroup compositions of membrane lipids between the epipelagic (<= 100 m) and deep (> 100 m) populations of archaea; ii) stratification of unsaturated GDGTs with varying redox conditions; and iii) enrichment of tetra-unsaturated archaeol and fully saturated GDGTs in epipelagic and deep oxygenated waters, respectively. Such stratified lipid patterns are consistent with the typical distribution of archaeal phylotypes in marine environments. We thus provide an ecological context for GDGT-based paleoclimatology and bring about the potential use of uns-AELs as biomarkers for planktonic Euryarchaeota. This article is protected by copyright. All rights reserved.

How curvature-generating proteins build scaffolds on membrane nanotubes

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex sub-cellular structures, and many other cellular phenomena. They form three-dimensional assemblies, which act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we employ various experimental, theoretical and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein-membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. 

An investigation of the molecular pharmacology of G protein-coupled receptor 35

G protein-coupled receptors (GPCRs) are seven-pass integral membrane proteins that act as transducers of extracellular signals across the lipid bilayer. Their location and involvement in basic and pathological physiological processes has secured their role as key targets for pharmaceutical intervention. GPCRs are targeted by many of the best-selling drugs on the market and there are a substantial number of GPCRs that are yet to be characterised; these could offer interest for therapeutic targeting. GPR35 is one such receptor that, as a result of gene knockout and genome wide association studies, has attracted interest through its association with cardiovascular and gastrointestinal disease. Elucidation of the basic physiological function of GPR35 has, however, been difficult due a paucity of potent and selective ligands in addition to a lack of consensus on the endogenous ligand. Herein, a focussed drug discovery effort was carried out to identify agonists of GPR35. Various in vitro cellular assays were employed in conjunction with N- or C-terminally manipulated forms of the receptor to investigate GPR35’s signalling profile and to provide an assay format suitable for the characterisation of newly identified ligands. Although GPR35 associates with both Gαi/o and Gα13 families of small heterotrimeric G proteins, the G protein-independent β-arrestin-2 recruitment format was found to be the most suited to drug screening efforts. Small molecule compound screening, carried out in conjunction with the Medical Research Council Technology, identified compound 1 as the most potent ligand of human GPR35 reported at that time. However, the lower efficacy and potency of compound 1 at the rodent species orthologues of GPR35 prevented its use in in vivo studies. A subsequent effort, carried out with Novartis, focused on mast cell stabilisers as putative agonists of GPR35, revealed lodoxamide and bufrolin as highly potent agonists that activated human and rat GPR35 with equal potency. This finding offered–for the first time–the opportunity to employ the same GPR35 ligand between species at a similar concentration, an important factor to consider when translating rodent in vivo functional studies to those in man. Additionally, using molecular modelling and site directed mutagenesis studies, these newly identified compounds were used to aid characterisation of the ligand binding pockets of human and rat GPR35 to reveal the molecular basis of species selectivity at this receptor. In summary, this research effort presents GPR35 tool compounds that can now be used to dissect the basic biology of GPR35 and investigate its contribution to disease.

Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.

Adipocyte size fluctuation, mechano-active lipid droplets and caveolae

International audience

A biochemical analysis into the co-translational folding of a G protein-coupled receptor; GPR35

The folding and targeting of membrane proteins poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane (TM) domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer. The biogenesis of a mature membrane protein takes place through the insertion and integration into the lipid bilayer. A number of TM proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. Although studies into the folding and targeting of a number of membrane proteins have been carried out to date, there is little information on one of the largest class of eukaryotic membrane proteins; the G-protein-coupled receptors (GPCRs). This project studies the early folding events of the human ortholog of GPR35. To analyse the structure of the 1st TM domain, intermediates were generated and assessed by the biochemical method of pegylation (PEG-MAL). A structurally-similar microbial opsin (Bacterioopsin) was also used to investigate the differences in the early protein folding within eukaryotic and prokaryotic translation systems. Results showed that neither the 1st TM domain of GPR35 nor Bacterioopsin were capable of compacting in the ribosome tunnel before their N-terminus reached the ribosome exit point. The results for this assay remained consistent whether the proteins were translated in a eukaryotic or prokaryotic translation system. To examine the communication mechanism between the ribosome, the nascent chain and the protein targeting pathway, crosslinking experiments were carried out using the homobifunctional lysine cross-linker BS3. Specifically, the data generated here show that the nascent chain of GPR35 reaches the ribosomal protein uL23 in an extended conformation and interacts with the SRP protein as it exits the ribosome tunnel. This confirms the role of SRP in the co-translational targeting of GPR35. Using these methods insights into the early folding of GPCRs has been obtained. Further experiments using site-directed mutagenesis to reduce hydrophobicity in the 1st TM domain of GPR35, highlighted the mechanisms by which GPCRs are targeted to the endoplasmic reticulum. Confirming that hydrophobicity within the signal anchor sequence is essential of SRP-dependent targeting. Following the successful interaction of the nascent GPR35 and SRP, GPR35 is successfully targeted to ER membranes, shown here as dog pancreas microsomes (DPMs). Glycosylation of the GPR35 N-terminus was used to determine nascent chain structure as it is inserted into the ER membrane. These glycosylation experiments confirm that TM1 has obtained its compacted state whilst residing in the translocon. Finally, a site-specific cross-linking approach using the homobifunctional cysteine cross-linker, BMH, was used to study the lateral integration of GPR35 into the ER. Cross-linking of GPR35 TM1 and TM2 could be detected adjacent to a protein of ~45kDa, believed to be Sec61α. The loss of this adduct, as the nascent chain extends, showed the lateral movement of GPR35 TM1 from the translocon was dependent on the subsequent synthesis of TM2.

Release currents of IP₃ receptor channel clusters and concentration profiles

We simulate currents and concentration profiles generated by Ca2+ release from the endoplasmic reticulum (ER) to the cytosol through IP3 receptor channel clusters. Clusters are described as conducting pores in the lumenal membrane with a diameter from 6 nm to 36 nm. The endoplasmic reticulum is modeled as a disc with a radius of 1–12 mm and an inner height of 28 nm. We adapt the dependence of the currents on the trans Ca2+ concentration (intralumenal) measured in lipid bilayer experiments to the cellular geometry. Simulated currents are compared with signal mass measurements in Xenopus oocytes. We find that release currents depend linearly on the concentration of free Ca2+ in the lumen. The release current is approximately proportional to the square root of the number of open channels in a cluster. Cytosolic concentrations at the location of the cluster range from 25 μM to 170 μM. Concentration increase due to puffs in a distance of a few micrometers from the puff site is found to be in the nanomolar range. Release currents decay biexponentially with timescales of < 1 s and a few seconds. Concentration profiles decay with timescales of 0.125–0.250 s upon termination of release.

Distribution of neutral prilocaine in a phospholipid bilayer: Insights from molecular dynamics simulations

In this work, we report a 20-ns constant pressure molecular dynamics simulation of prilocaine (PLC), in amine-amide local anesthetic, in a hydrated liquid crystal bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine. The partition of PLC induces the lateral expansion of the bilayer and a concomitant contraction in its thickness. PLC molecules are preferentially found in the hydrophobic acyl chains region, with a maximum probability at similar to 12 angstrom from the center of the bilayer (between the C(4) and C(5) methylene groups). A decrease in the acyl chain segmental order parameter, vertical bar S-CD vertical bar, compared to neat bilayers, is found, in good agreement with experimental H-2-NMR studies. The decrease in vertical bar S-CD vertical bar induced by PLC is attributed to a larger accessible volume per lipid in the acyl chain region. (C) 2008 Wiley Periodicals, Inc.

Lipid remodelling in the reef-building honeycomb worm, Sabellaria alveolata, reflects acclimation and local adaptation to temperature

Acclimation and adaptation, which are key to species survival in a changing climate, can be observed in terms of membrane lipid composition. Remodelling membrane lipids, via homeoviscous adaptation (HVA), counteracts membrane dysfunction due to temperature in poikilotherms. In order to assess the potential for acclimation and adaptation in the honeycomb worm, Sabellaria alveolata, a reef-building polychaete that supports high biodiversity, we carried out common-garden experiments using individuals from along its latitudinal range. Individuals were exposed to a stepwise temperature increase from 15 °C to 25 °C and membrane lipid composition assessed. Our results suggest that S. alveolata was able to acclimate to higher temperatures, as observed by a decrease in unsaturation index and 20:5n-3. However, over the long-term at 25 °C, lipid composition patterns are not consistent with HVA expectations and suggest a stress response. Furthermore, unsaturation index of individuals from the two coldest sites were higher than those from the two warmest sites, with individuals from the thermally intermediate site being in-between, likely reflecting local adaptation to temperature. Therefore, lipid remodelling appears limited at the highest temperatures in S. alveolata, suggesting that individuals inhabiting warm environments may be close to their upper thermal tolerance limits and at risk in a changing climate.

Étude de propriétés physico-chimiques des membranes lipidiques chargées d’acide palmitique/stérol et de stéarylamine/cholestérol

Les stérosomes, des vésicules artificielles composées d’amphiphiles monoalkylés et d’un grand pourcentage de stérols, sont prometteurs dans plusieurs domaines comme les industries pharmaceutiques et alimentaires. Il existe des stérosomes chargés négativement, positivement et neutres. Dans ce mémoire, nous avons approfondi nos connaissances sur les propriétés physico-chimiques des stérosomes chargés : acide palmitique (PA)/stérol et stéarylamine (SA)/cholestérol (Chol). Premièrement, afin de mesurer la diffusion latérale de PA dans les membranes PA/stérol (30/70 mol/mol) par RMN à gradients pulsés, nous avons tenté de former des bicouches liquide-ordonnées (lo) orientées magnétiquement avec ce mélange. En s'inspirant de l’idée que l’ajout de 1,2-dihexanoyl-sn-glycéro-3-phosphocholine (DHPC), un lipide à courtes chaînes, dans le système 1,2-dimyristoyl-sn-glycéro-3-phosphocholine (DMPC) mène à la formation de bicouches orientées, nous avons étudié la formulation PA perdeutéré/acide hexanoïque (HA)/Chol avec une proportion molaire de 25/18/57 à plusieurs températures; aucune formation de bicouches orientées n’a été observée. Ce résultat pourrait être expliqué par la solubilisation partielle de HA en milieu aqueux. Alors, une quantité insuffisante serait insérée dans la bicouche pour induire son orientation. La formulation PA perdeutéré/DHPC/Chol n’a pas conduit, elle non plus, à des bicouches orientées magnétiquement à des températures et concentrations lipidiques variées. En étudiant le mélange DMPC/DHPC/Chol (67/17/14), nous avons remarqué que la présence de Chol inhibait l'orientation magnétique des bicouches. Tandis que le mélange DMPC/DHPC/stigmastérol (SS) avec les proportions molaires 67/19/14 et 72/21/7 conduisait à des bicouches orientées avec leur normale (n) perpendiculaire au champ magnétique à 40 °C et 50 °C. Ces résultats suggèrent que le mélange PA/SS avec une proportion de lipide à courtes chaînes, HA et DHPC, pourrait mener à des bicouches orientées magnétiquement. Le mélange PA/Chol avec un lipide à courtes chaînes pourrait aussi être étudié en présence des lanthanides. Deuxièmement, nous avons examiné la possibilité de moduler la libération de matériel encapsulé dans des liposomes essentiellement composés de PA et d’un stérol. Il est connu que le mélange PA/Chol (30/70) à pH ≥ 7,5 forme des liposomes très peu perméables. Il est avantageux de pouvoir moduler la perméabilité pour avoir un contrôle sur le temps de libération de leur contenu, qui est un paramètre de grande importance pour les formulations liposomales de médicaments. D’abord, il a été montré que l’acide oléique (OA)/Chol (30/70) est capable de former des vésicules, ce qui n’avait jamais été prouvé auparavant. Par contre, les bicouches OA/Chol (30/70) ne sont pas plus perméables que les bicouches PA/Chol (30/70). L’ajout de 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphatidylcholine (POPC) dans le mélange PA/Chol n’augmente pas plus la perméabilité. En effet, les cinétiques de relargage de calcéine des vésicules PA/POPC/Chol (15/27.5/57.5), POPC/Chol (40/60) et POPC étaient très semblables à celle de PA/Chol (30/70). Il a été remarqué que les études littéraires se contredisent à propos de la perméabilité à la calcéine des bicouches de phosphatidylcholine (PC). L’explication de ces divergences est inconnue pour le moment. En remplaçant la moitié de la proportion molaire de Chol par le cholate de sodium (SC) dans le mélange PA/Chol (30/70), la membrane n’était pas plus apte à libérer son contenu. Il se pourrait que le SC se retrouvant dans la bicouche n’induit pas une diminution d’empilement. Il est aussi possible que le SC ne s'insère pas dans la membrane à cause de son hydrophilie considérable et il pourrait alors former seul des micelles. En remplaçant complètement le Chol par le sulfate de cholestérol (SChol), un stérol chargé négativement, et en préparant les vésicules à un bas pH, la formulation PA/SChol (30/70) mène à une très grande perméabilité à pH 7.5; le relargage est provoqué par un saut de pH. Nos travaux suggèrent qu'il serait possible de moduler la perméabilité des liposomes en les préparant avec le mélange PA/SChol/Chol en variant les proportions entre 30/63/7 à 30/70/0. Le diagramme pH-composition du mélange PA/SChol/Chol indique que ces proportions conduisent, à pH 7.4, à la coexistence de phases solide et lo en différentes proportions, ce qui pourrait moduler la perméabilité membranaire. Troisièmement, les résultats de perméabilité obtenus avec la calcéine et les difficultés survenues lors de l’extrusion des vésicules encapsulant cette sonde nous ont amené à nous demander si la calcéine interagit avec les bicouches chargées. L’impact de certains anions, dont la calcéine, a été examiné sur les bicouches chargées positivement SA/Chol (50/50). La calorimétrie différentielle à balayage (DSC, de l’anglais differential scanning calorimetry), indique qu’il n’y a aucune transition entre 25 et 90 °C pour les liposomes SA/Chol (50/50) à pH = 7.4. L’ajout de chlorure de sodim (375 mM) n’a pas mené à la formation d’agrégats et aucune transition n’a été observée sur le thermogramme. La formation d’agrégats macroscopiques instantanément après l’ajout d’hydrogénophosphate de sodium (125 mM), de sulfate de sodium (125 mM) et de calcéine (3 mM) a été observée. Une transition a été observée sur les thermogrammes en présence de ces sels. Les agrégats observés pourraient être associés à la transition de phase. L’effet des anions sur la température et l’enthalpie de transition suivent le même ordre que la série d’Hofmeister : sulfate > hydrogénophosphate > chlorure (pas de pic). La calcéine avait l’impact le plus prononcé sur l’agrégation; ceci illustre que la calcéine n’est pas une sonde fluorescente inerte avec le mélange SA/Chol. Elle pourrait être un chaotrope volumineux. De plus, les interactions SA-calcéine plus fortes, menant à l’agrégation des vésicules, que les interactions PC-calcéine pourraient s’expliquer par le fait que la SA est chargée positivement.

Étude de propriétés physico-chimiques des membranes lipidiques chargées d’acide palmitique/stérol et de stéarylamine/cholestérol

Les stérosomes, des vésicules artificielles composées d’amphiphiles monoalkylés et d’un grand pourcentage de stérols, sont prometteurs dans plusieurs domaines comme les industries pharmaceutiques et alimentaires. Il existe des stérosomes chargés négativement, positivement et neutres. Dans ce mémoire, nous avons approfondi nos connaissances sur les propriétés physico-chimiques des stérosomes chargés : acide palmitique (PA)/stérol et stéarylamine (SA)/cholestérol (Chol). Premièrement, afin de mesurer la diffusion latérale de PA dans les membranes PA/stérol (30/70 mol/mol) par RMN à gradients pulsés, nous avons tenté de former des bicouches liquide-ordonnées (lo) orientées magnétiquement avec ce mélange. En s'inspirant de l’idée que l’ajout de 1,2-dihexanoyl-sn-glycéro-3-phosphocholine (DHPC), un lipide à courtes chaînes, dans le système 1,2-dimyristoyl-sn-glycéro-3-phosphocholine (DMPC) mène à la formation de bicouches orientées, nous avons étudié la formulation PA perdeutéré/acide hexanoïque (HA)/Chol avec une proportion molaire de 25/18/57 à plusieurs températures; aucune formation de bicouches orientées n’a été observée. Ce résultat pourrait être expliqué par la solubilisation partielle de HA en milieu aqueux. Alors, une quantité insuffisante serait insérée dans la bicouche pour induire son orientation. La formulation PA perdeutéré/DHPC/Chol n’a pas conduit, elle non plus, à des bicouches orientées magnétiquement à des températures et concentrations lipidiques variées. En étudiant le mélange DMPC/DHPC/Chol (67/17/14), nous avons remarqué que la présence de Chol inhibait l'orientation magnétique des bicouches. Tandis que le mélange DMPC/DHPC/stigmastérol (SS) avec les proportions molaires 67/19/14 et 72/21/7 conduisait à des bicouches orientées avec leur normale (n) perpendiculaire au champ magnétique à 40 °C et 50 °C. Ces résultats suggèrent que le mélange PA/SS avec une proportion de lipide à courtes chaînes, HA et DHPC, pourrait mener à des bicouches orientées magnétiquement. Le mélange PA/Chol avec un lipide à courtes chaînes pourrait aussi être étudié en présence des lanthanides. Deuxièmement, nous avons examiné la possibilité de moduler la libération de matériel encapsulé dans des liposomes essentiellement composés de PA et d’un stérol. Il est connu que le mélange PA/Chol (30/70) à pH ≥ 7,5 forme des liposomes très peu perméables. Il est avantageux de pouvoir moduler la perméabilité pour avoir un contrôle sur le temps de libération de leur contenu, qui est un paramètre de grande importance pour les formulations liposomales de médicaments. D’abord, il a été montré que l’acide oléique (OA)/Chol (30/70) est capable de former des vésicules, ce qui n’avait jamais été prouvé auparavant. Par contre, les bicouches OA/Chol (30/70) ne sont pas plus perméables que les bicouches PA/Chol (30/70). L’ajout de 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphatidylcholine (POPC) dans le mélange PA/Chol n’augmente pas plus la perméabilité. En effet, les cinétiques de relargage de calcéine des vésicules PA/POPC/Chol (15/27.5/57.5), POPC/Chol (40/60) et POPC étaient très semblables à celle de PA/Chol (30/70). Il a été remarqué que les études littéraires se contredisent à propos de la perméabilité à la calcéine des bicouches de phosphatidylcholine (PC). L’explication de ces divergences est inconnue pour le moment. En remplaçant la moitié de la proportion molaire de Chol par le cholate de sodium (SC) dans le mélange PA/Chol (30/70), la membrane n’était pas plus apte à libérer son contenu. Il se pourrait que le SC se retrouvant dans la bicouche n’induit pas une diminution d’empilement. Il est aussi possible que le SC ne s'insère pas dans la membrane à cause de son hydrophilie considérable et il pourrait alors former seul des micelles. En remplaçant complètement le Chol par le sulfate de cholestérol (SChol), un stérol chargé négativement, et en préparant les vésicules à un bas pH, la formulation PA/SChol (30/70) mène à une très grande perméabilité à pH 7.5; le relargage est provoqué par un saut de pH. Nos travaux suggèrent qu'il serait possible de moduler la perméabilité des liposomes en les préparant avec le mélange PA/SChol/Chol en variant les proportions entre 30/63/7 à 30/70/0. Le diagramme pH-composition du mélange PA/SChol/Chol indique que ces proportions conduisent, à pH 7.4, à la coexistence de phases solide et lo en différentes proportions, ce qui pourrait moduler la perméabilité membranaire. Troisièmement, les résultats de perméabilité obtenus avec la calcéine et les difficultés survenues lors de l’extrusion des vésicules encapsulant cette sonde nous ont amené à nous demander si la calcéine interagit avec les bicouches chargées. L’impact de certains anions, dont la calcéine, a été examiné sur les bicouches chargées positivement SA/Chol (50/50). La calorimétrie différentielle à balayage (DSC, de l’anglais differential scanning calorimetry), indique qu’il n’y a aucune transition entre 25 et 90 °C pour les liposomes SA/Chol (50/50) à pH = 7.4. L’ajout de chlorure de sodim (375 mM) n’a pas mené à la formation d’agrégats et aucune transition n’a été observée sur le thermogramme. La formation d’agrégats macroscopiques instantanément après l’ajout d’hydrogénophosphate de sodium (125 mM), de sulfate de sodium (125 mM) et de calcéine (3 mM) a été observée. Une transition a été observée sur les thermogrammes en présence de ces sels. Les agrégats observés pourraient être associés à la transition de phase. L’effet des anions sur la température et l’enthalpie de transition suivent le même ordre que la série d’Hofmeister : sulfate > hydrogénophosphate > chlorure (pas de pic). La calcéine avait l’impact le plus prononcé sur l’agrégation; ceci illustre que la calcéine n’est pas une sonde fluorescente inerte avec le mélange SA/Chol. Elle pourrait être un chaotrope volumineux. De plus, les interactions SA-calcéine plus fortes, menant à l’agrégation des vésicules, que les interactions PC-calcéine pourraient s’expliquer par le fait que la SA est chargée positivement.

Fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids in model membranes is connected not to lipid mobility but to probe location

Nitrobenzoxadiazole (NBD)-labeled lipids are popular fluorescent membrane probes. However, the understanding of important aspects of the photophysics of NBD remains incomplete, including the observed shift in the emission spectrum of NBD-lipids to longer wavelengths following excitation at the red edge of the absorption spectrum (red-edge excitation shift or REES). REES of NBD-lipids in membrane environments has been previously interpreted as reflecting restricted mobility of solvent surrounding the fluorophore. However, this requires a large change in the dipole moment (Dm) of NBD upon excitation. Previous calculations of the value of Dm of NBD in the literature have been carried out using outdated semi-empirical methods, leading to conflicting values. Using up-to-date density functional theory methods, we recalculated the value of Dm and verified that it is rather small (B2 D). Fluorescence measurements confirmed that the value of REES is B16 nm for 1,2-dioleoyl-sn-glycero-3- phospho-L-serine-N-(NBD) (NBD-PS) in dioleoylphosphatidylcholine vesicles. However, the observed shift is independent of both the temperature and the presence of cholesterol and is therefore insensitive to the mobility and hydration of the membrane. Moreover, red-edge excitation leads to an increased contribution of the decay component with a shorter lifetime, whereas time-resolved emission spectra of NBD-PS displayed an atypical blue shift following excitation. This excludes restrictions to solvent relaxation as the cause of the measured REES and TRES of NBD, pointing instead to the heterogeneous transverse location of probes as the origin of these effects. The latter hypothesis was confirmed by molecular dynamics simulations, from which the calculated heterogeneity of the hydration and location of NBD correlated with the measured fluorescence lifetimes/REES. Globally, our combination of theoretical and experiment-based techniques has led to a considerably improved understanding of the photophysics of NBD and a reinterpretation of its REES in particular.

The Use of Pacific Menhaden Oil and Palm Oil on Lipid Metabolism Profile in Blood Serum of Native Cockerels

Cholesterol is an important component of cell membrane, plasma lipoprotein and precursor of steroid synthesis in biologically crucial bile salt and steroid hormones.  Cholesterol level in the blood exceeding the 300 mg/100 ml can result in the incident of cardiac disorder. Cholesterol, HDL (High Density of Lipoprotein), LDL (Low Density of Lipo protein) biosynthesis in the blood plasma of broiler chicken can be manipulated by the use of Pacific Menhaden Oil and Palm Oil.  Therefore, The aim of the current experiment was to investigate the effect of  long chain fatty acid from Pacific Menhaden Oil (PMO) and Palm Oil (PO) use in feed on the blood level of cholesterol, HDL and LDL of native cockerels. Four kinds of ration which contained different levels of PMO and PO were formulated.  They were R0 (control, without PMO and PO); R1 (10% PO); R2  (10% PMO) and R3 (5% PMO and 5% PO).  A total of 72 native cockerels, aged two weeks were kept in 24 pens, three cockerels in each pen.  Each experimental unit were assingned at random to receive one of the four rations in completely randomized designed with six replicates. The use of PMO and PO in feed did not significantly affect (P>0.05) the concentration of cholesterol, HDL, LDL and triglyceride in the blood.  Although statistically was not significantly significant, Cockerels fed R2 had the highest concentration of HDL (39.36 mg/dl) and the lowest concentration of LDL (9.10) in the blood. (Animal Production 7(2): 67-73 (2005) Key Words: Pacific Menhaden Oil, Palm Oil, Cholesterol, HDL, LDL, Triglyceride

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.