Importance of Lipid Composition in the Membrane Dynamics of Urothelial Umbrella Cells

The studies on membrane dynamics in urinary bladder umbrella cells were focused on proteins as important factors for maintaining the permeability barrier and their role as pathways modulators for the discoidal/fusiform endocytic vesicles which is one of the main features of the umbrella cells. However, our workgroup has dedicated to the study of lipid membrane composition and its biological impact in the urothelium. We first studied the lipid membrane composition of rat urothelium modified my dietary treatments differentiated in their fatty acid composition. Changes of lipid composition were related to the asymmetric unit membrane organization and permeability. Finally, we observed that the lipid composition was critically related to the intracellular pathways of discoidal/fusiform endocytic vesicles and their content. The purpose of this communication is to summarize the importance of lipids in the membrane organization and permeability of the apical plasma membrane and endocytic vesicles of umbrella cells.Fil: Grasso, Ernesto Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

?2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ?2-microglobulin (?2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ?2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ?2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ?2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ?2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ?2m amyloid-associated osteoarticular tissue destruction in DRA

Effect of plant-based feed ingredients on osmoregulation in the Atlantic salmon lens

Lenses of adult Atlantic salmon fed with a plant oil and plant protein-based diet (plant diet) were compared to lenses of fish fed a diet based on traditional marine ingredients (marine diet) with respect to biochemical composition and functionality ex vivo. After 12 months of feeding, plant diet-fed fish had smaller lenses with higher water contents and lower concentrations of histidine (His) and N-acetylhistidine (NAH) than fish fed with the marine diet. Cataract development in both dietary groups was minimal and no differences between the groups were observed. Lens fatty acid and lipid class composition differed minimally, although a significant increase in linoleic acid was observed. The lenses were examined for their ability to withstand osmotic disturbances ex vivo. Culture in hypoosmotic and hyperosmotic media led to increase and decrease of lens volume, respectively. Lenses from plant diet-fed fish were less resistant to swelling and shrinking, released less NAH into the culture medium, and accumulated His and NAH at higher rates than lenses from marine diet-fed fish. Culture in hypoosmotic medium resulted in higher cataract scores than in control and hyperosmotic medium. mRNA expression of selected genes, including glutathione peroxidase 4 and SPARC (secreted protein acidic and rich in cysteine), was affected by diet and osmotic treatment. It can be concluded that lenses of farmed Atlantic salmon are affected by the diet composition, both in biochemical composition and physiological functionality in relation to osmoregulation

Association between cholesterol-phospholipid vesicles and cholesterol crystals in human gallbladder bile

Rapid aggregation of cholesterol-phospholipid vesicles in gallbladder bile seems to be the first event in the production of cholesterol crystals, a prerequisite for cholesterol gallstone formation. We examined the amount of these vesicles in 33 human gallbladder biles in relation to biliary lipid composition and to the presence of cholesterol crystals. Biliary microscopy detected cholesterol crystals in all 19 biles from patients with cholesterol gallstones but in none of 14 biles from patients with pigment stones. Gel chromatography was used to separate vesicles and micelles in the native bile with an eluting buffer containing 10 mM sodium cholate to prevent disruption of micellar lipids. Cholesterol, phospholipid and bile salt concentrations were measured in every fraction collected. Bile acid, phospholipid, cholesterol and total lipid concentrations were not significantly different in samples with and without cholesterol crystals. The cholesterol saturation index (1.4 ± 0.11 vs. 1.0 ± 0.08) was significantly (p < 0.01) higher in biles with crystals than without crystals. Gel filtration revealed a vesicular peak in addition to micellar fraction in 18 (23.1 ± 3.2% of total cholesterol) of the 19 biles with crystals but only in three (15.7 ± 2.4% of total cholesterol) of 14 biles without crystals. There was no relation between biliary lipid concentration or the cholesterol saturation index and the percentage of vesicular cholesterol in biles with or without crystals. The close association of vesicles and crystals in human gallbladder bile supports the contention that vesicles are important in the initial nucleation of cholesterol monohydrate crystals

Identifying the Onset of Phase Separation in Quaternary Lipid Bilayer Systems from Coarse-Grained Simulations

Understanding the (de)mixing behavior of multicomponent lipid bilayers is an important step towards unraveling the nature of spatial composition heterogeneities in cellular membranes and their role in biological function. We use coarse-grained molecular dynamics simulations to study the composition phase diagram of a quaternary mixture of phospholipids and cholesterol. This mixture is known to exhibit both uniform and coexisting phases. We compare and combine different statistical measures of membrane structure to identify the onset of phase coexistence in composition space. An important element in our approach is the dependence of composition heterogeneities on the size of the system. While homogeneous phases can be structured and display long correlation lengths, the hallmark behavior of phase coexistence is the scaling of the apparent correlation length with system size. Because the latter cannot be easily varied in simulations, our method instead uses information obtained from observation windows of different sizes to accurately distinguish phase coexistence from structured homogeneous phases. This approach is built on very general physical principles, and will be beneficial to future studies of the phase behavior of multicomponent lipid bilayers

Carbohydrate and lipid composition of vegetables, and bioavalability assessed in a rat model: Impact different cultivation systems

Environmental as well as cultivation factors may greatly influence the chemical composition of plants. The main factors affecting chemical composition of foodstuff is level and type of fertilizer (conventional and organic cultivation systems), location or soiltype and year of harvest. Organic foods are defined as products which are produced under controlled cultivation conditions characterized by the absence of synthetic fertilizers and very restricted use of pesticides. Dietary carbohydrates constitute a major fraction of most feedstuffs and can be divided according to glycosidic linkage into sugars (mono- and disaccharides), oligosaccharides, starch and non-starch polysaccharides (NSP). The bulk of disaccharides and starch will be broken down by the action of pancreatic and mucosal enzymes in the small intestine, while there are no enzymes capable of cleaving some types of oligosaccharides and NSP. A fraction of starch (resistant starch) may also pass the small intestine undegraded either because the starch is physically inaccessible, the starch has a structure that resist amylolysis or the starch is retrograded after heat treatment. Lignin is not a carbohydrate but is tightly associated to cell wall polysaccharides. The term dietary fibre (DF) is used for cell wall and storage NSP and lignin. Adequate intake of dietary fibre are generally accepted as linked to health benefit into a protective role in large bowel cancer, diabetes, coronary heart disease and the issue of faecal bulking. Linoleic (C18:2 n-6) and α-linolenic (C18:3 n-3) are essential fatty acids, which cannot be synthesized in the mammalian organism, and therefore must be supplied in the diet of animals and man. These fatty acids are precursors for the important longer chain higher polyunsaturated fatty acids of the n-6 and n-3 families. Although fats are essential part of the diet, but if consumed in excess, they may exert negative effects on human weight change. Potatoes, carrots, peas, green kale, apple, and rapeseed were grown by three different cultivation strategies, i.e. organic (ORG), conventional (CON), or semi-organic (ORG+) farming system. Each ingredient was treated as for application for human consumption: potatoes, mature, soaked peas and kale were boiled and raw carrots and apples were shredded, and the food was then freeze-dried and packed into airtight bags. Rapeseed oil was produced from the air-dried rapeseeds of the three cultivation treatments, and the residual was discarded. The carbohydrate fraction of the ingredients except rapeseed oil was analysed into: starch, sugars, oligosaccharides and all its constituents and lignin. Likewise the dietary lipids of all ingredients were extracted and the long-chain fatty acids determined by GLC. The ingredients were mixed with a standard synthetic mixture and were formulated to meet the NRC requirements for rats and used in a balance experiments for measuring the bioavalability of the ingredients. Carbohydrate and lignin were predominant dietary constituents with value from 584 g/kg DM in kale to 910 g/kg DM in potatoes. Triacylglycerol was the major lipid class in pea with 82 % of total fatty acids in contrast to apple with only 35 % of fatty acids of the ether extract

Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.Fil: Coria, Mariela Janet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; ArgentinaFil: Carmona Viglianco, Yamila Virginia. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Marra, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Bioquímicas de La Plata; Argentina; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Médicas; ArgentinaFil: Gomez-Mejiba, Sandra Esther. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Oklahoma Medical Research Foundation; Estados UnidosFil: Ramirez, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Oklahoma Medical Research Foundation; Estados UnidosFil: Anzulovich Miranda, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; ArgentinaFil: Gimenez, Maria Sofia. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentin

Anomalously Slow Domain Growth in Fluid Membranes with Asymmetric Transbilayer Lipid Distribution

The effect of asymmetry in the transbilayer lipid distribution on the dynamics of phase separation in fluid vesicles is investigated numerically for the first time. This asymmetry is shown to set a spontaneous curvature for the domains that alter the morphology and dynamics considerably. For moderate tension, the domains are capped and the spontaneous curvature leads to anomalously slow dynamics, as compared to the case of symmetric bilayers. In contrast, in the limiting cases of high and low tensions, the dynamics proceeds towards full phase separation.Comment: 4 pages, 5 figure

Neuronal human BACE1 knock-in induces systemic diabetes in mice

Acknowledgements The authors thank S. Tammireddy (Diabetes and Cardiovascular Science, University of the Highlands and Islands, Inverness, UK) for technical support with the lipidomics component. Funding We would like to thank R. Simcox, Romex Oilfield Chemicals, for financial support for KP, and acknowledge additional contributions from the Scottish Alzheimer’s Research UK network for the lipidomics work. The College of Life Science and Medicine, University of Aberdeen, sponsored the imaging study. MD was funded by British Heart Foundation and Diabetes UK; NM was funded by a British Heart Foundation Intermediate Fellowship; KS was funded by a European Foundation for the Study of Diabetes/Lilly programme grant; and RD was funded by an Institute of Medical Sciences PhD studentship.Peer reviewedPublisher PDFPublisher PD

Microcavity supported lipid membranes: versatile platforms for building asymmetric lipid bilayers and for protein recognition

Microcavity supported lipid bilayers (MSLB) are contact-free membranes suspended across aqueousfilled pores that maintain the lipid bilayer in a highly fluidic state and free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer significant opportunity for modelling membrane asymmetry, protein-membrane interactions and aggregation at the membrane interface. Herein, we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity and cholera toxin- ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phase, and ternary phase separated membrane compositions. Fluorescence lifetime correlation spectroscopy (FLCS) was used to determine the lateral mobility of lipid and protein, and electrochemical impedance spectroscopy (EIS) enabled detection of protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have profound impact on membrane electrochemical resistance where the resistance of a ternary symmetric phase separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition at the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coefficient for MSLBs was observed to be 2.5 fold faster for asymmetric MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that interplay of lipid packing across both membrane leaflets and concentration of GM1 both affect the extent of cholera toxin aggregation and consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asymmetric, MSLBs offer the opportunity for building greater biomimicry into biophysical models and the approach described demonstrates the value of MSLBs in studying aggregation and membrane associated multivalent interactions prevalent in many carbohydrates mediated processes