Academic literature on the topic 'Erythrocyte membrances'

The phospholipid and fatty acid compositions of the host infected erythrocyte plasma membrane (IEPM) have been determined for erythrocytes infected with the human malaria parasite Plasmodium falciparum. IEPM were prepared by selective lysis of the host erythrocyte (but not of the parasite membranes) with 0.1% saponin, followed by differential centrifugation. The purity of the IEPM was determined by measuring the membrane-specific enzyme markers acetylcholinesterase, glutamate dehydrogenase and lactate dehydrogenase, and by immunoelectron microscopy using monoclonal antibodies specific for human erythrocyte glycophorin A (4E7) and for a 195 kDa parasite membrane glycoprotein (Pf6 3B10.1). Both approaches demonstrated that the host erythrocyte plasma membrane preparation was free from contamination by parasite membranes. During intra-erythrocytic development of the parasite, the phospholipid composition of the erythrocyte membrane was strikingly altered. IEPM contained more phosphatidylcholine (38.7% versus 31.7%) and phosphatidylinositol (2.1% versus 0.8%) and less sphingomyelin (14.6% versus 28.0%) than normal uninfected erythrocytes. Similar alterations in phospholipid composition were determined for erythrocyte membranes of parasitized cells isolated by an alternative method utilizing polycationic polyacrylamide microbeads (Affigel 731). The total fatty acid compositions of the major phospholipids in IEPM were determined by g.l.c. The percentage of polyunsaturated fatty acids in normal erythrocyte phospholipids (39.4%) was much higher than in phospholipids from purified parasites (23.3%) or IEPM (24.0%). The unsaturation index of phospholipids in IEPM was considerably lower than in uninfected erythrocytes (107.5 versus 161.0) and was very similar to that in purified parasites (107.5 versus 98.5). Large increases in palmitic acid (C16:0) (from 21.88% to 31.21%) and in oleic acid (C18:1) (from 14.64% to 24.60%), and major decreases in arachidonic acid (C20:4) (from 17.36% to 7.85%) and in docosahexaenoic acid (C22:6) (from 4.34% to 1.8%) occurred as a result of infection. The fatty acid profiles of individual phospholipid classes from IEPM resembled in many instances the fatty acid profiles of parasite phospholipids rather than those of uninfected erythrocytes. Analysis of IEPM from P. falciparum-infected erythrocytes (trophozoite stage) revealed that, during intra-erythrocytic maturation of the parasite, the host erythrocyte phospholipid composition was markedly refashioned. These alterations were not dependent on the method used to isolate the IEPM, with similar results obtained using either a saponin-lysis method or binding to Affigel beads. Since mature erythrocytes have negligible lipid synthesis and metabolism, these alterations must occur as a result of parasite-directed metabolism of erythrocyte lipids and/or trafficking of lipids between the parasite and erythrocyte membranes.

Monoclonal antibodies against the rodent malaria parasite, Plasmodium yoelii, have been prepared and characterized by indirect immunofluorescence on acetone-fixed infected mouse erythrocytes. The antibody of clone K2 reacted strongly with late trophozoites and schizonts, whereas it did so weakly and diffusely with ring forms and early trophozoites. Strong fluorescence was confined to granular structures in schizonts and merozoites. Parasites that invaded erythrocytes in vitro lost the strong fluorescence. Instead, immunofluorescence appeared in the membranes of erythrocytes infected in vitro with merozoites. Erythrocytes infected with more than one merozoite had intensified immunofluorescence in their membranes. Staining of the invaded erythrocytes with 4′,6-diamidino-2-phenylindole (DAPI) hydrochloride demonstrated that membranes of all the invaded erythrocytes acquired the P. yoelii antigen. These results suggest that the P. yoelii antigen in merozoites is translocated into erythrocyte membranes upon entry into the host cell. Immunofluorescence continued to appear in membranes of infected erythrocytes throughout the intra-erythrocytic parasite growth. Staining of unfixed infected erythrocytes with the K2 antibody failed to detect the parasite antigen. In contrast, immunofluorescence was present in unfixed membranes of erythrocyte ghosts, which had been spontaneously formed after rupture of schizont-infected erythrocytes by merozoite release. No immunofluorescence appeared in either acetone-fixed or unfixed ghosts of normal erythrocytes. These results suggest the antigenic determinant of the P. yoelii antigen is exposed at the cytoplasmic surface of the infected erythrocyte membrane. Immunoprecipitation has revealed that the K2 antibody recognizes a 160 X 10(3) Mr P. yoelii antigen.

We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.

Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking α-spectrin, ankyrin, protein 4.2, protein 4.1, β-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.

PURPOSE: To study the influence of human placenta extract (HPE) and its individual fractions on the thermal stability of human erythrocyte membrane. METHODS: HPE fractions were isolated by gel chromatography. Thermal hemolysis of erythrocytes, exposed to 55°C was measured spectrophotometrically. Cytosol microvscosity and barrier function of erythrocyte membranes at hyperthermia were investigated by EPR spin probe TEMPON. Thermal denaturation of erythrocyte membrane proteins were studied by differential scanning calorimetry. RESULTS: Pre-treatment of erythrocytes with HPE or its fractions inhibited thermal hemolysis. Low-molecular fractions (below 4 kDa and 12-20 kDa) were the most effective in thermal hemolysis inhibition ((31.7±3.3) % and (31.5±3.2) %, respectively). The latter fractions markedly reduced the hyperthermia (55°C)-induced permeability of erythrocytes for ferricyanide ions and inhibited the thermo-induced structural transitions in erythrocyte membrane between 40 and 50°C, which are associated with cytoskeletal proteins. HPE fractions reversibly increased the denaturation temperatures of erythrocyte membrane proteins, except that of spectrin, and enlarged the enthalpy of denaturation of all membrane proteins. CONCLUSIONS: HPE and its individual fractions increased the thermal stability of erythrocyte membranes and erythrocytes. This effect was attributed to the reversible binding of some low molecular ingredient of HPE to the integral proteins and consequent stabilization of their interaction with under-membrane cytoskeleton.

The aim of this study was to characterize the interaction of chosen catechins ((+)-catechin, (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCG)) with human erythrocytes and their protective effects against oxidative damage of erythrocytes. Uptake of the catechins by erythrocytes was studied by fluorimetry, their interaction with erythrocyte membrane was probed by changes in erythrocyte osmotic fragility and in membrane fluidity evaluated with spin labels, while protection against oxidative damage was assessed by protection against hemolysis induced by permanganate and protection of erythrocyte membranes against lipid peroxidation and protein thiol group oxidation. Catechin uptake was similar for all the compounds studied. Accumulation of catechins in the erythrocyte membrane was demonstrated by the catechin-induced increase in osmotic resistance and rigidification of the erythrocyte membrane detected by spin labels 5-doxyl stearic acid and 16-doxyl stearic acid. (−)-Epigallocatechin and EGCG inhibited erythrocyte acetylcholinesterase (mixed-type inhibition). Catechins protected erythrocytes against permanganate-induced hemolysis, oxidation of erythrocyte protein thiol groups, as well as membrane lipid peroxidation. These results contribute to the knowledge of the beneficial effects of catechins present in plant-derived food and beverages.

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 μM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

Erythrocyte membrane interactions with insulin fibrils (amyloid) have been investigated using centrifugation, fluorescence spectroscopy, light scattering, and flow cytometric techniques. The results indicate that insulin fibrils are having moderate affinity to erythrocyte membrane. However, analysis of the apparent dissociation constants of human erythrocyte membranes (leaky and resealed vesicles) with amyloid insulin reveal that the insulin binding is drastically reduced on attaining the fibrillar state compared with native insulin. To understand the role of insulin receptors on erythrocytes binding to amyloid, we have studied the interaction of biotinylated forms of denatured and amyloidic insulin with erythrocytes. FITC-streptavidin was used as a counter staining in flow cytometry measurements. We found that insulin fibrils bind 10 times more with erythrocyte membranes than with amylin and denatured insulin.Key words: insulin amyloid, erythrocyte membrane, amyloid binding, flow cytometry, dissociation constant.

The practical importance for the pharmaceutical and cosmetics industries of the interactions between biological membranes and surfactant molecules has led to intensive research within this area. The interactions of non-ionic surfactant n-octyl-β-D-glucopyranoside (OG) with the human and rat erythrocyte membranes were studied. The in vitro hemolytic and antihemolytic activities were determined by employing a method in which both erythrocytes were added to the hypotonic medium containing OG at different concentrations, and the amount of haemoglobin released was determined. noctyl- β-D-glucopyranoside was found to have a biphasic effect on both types of erythrocyte membrane. We also investigated the interactions of OG with the erythrocyte membrane in isotonic medium; the dose-dependent curves show similar behaviour in both human and rat erythrocytes. Our results showed that OG has greater antihemolytic potency on rat than on human erythrocytes; furthermore, rat erythrocytes were more sensitive than human erythrocytes to hypotonic shock. How the different lipoprotein structure of these erythrocytes determines a difference in antihemolytic activity is discussed.

The intra-membrane particles (IMPs) observed on the fracture face of frozen erythrocyte membranes are thought to correspond primarily to “band 3” tetramers or dimers. Some recent studies correlating the inhibition of water diffusion in erythrocytes by p-chloromercuribenzene sulfonate (PCMBS) with the binding of 203Hg to erythrocyte membrane proteins has enabled band 3 and the polypeptides in band 4.5 to be identified as the proteins associated with the channels for water permeation in human erythrocytes. A further characterization of the effects of the incubation of human erythrocyte membranes with PCMBS and N-ethylmaleimide (NEM) has been performed as previously described. Experimental conditions have been previously described.A comparison was made of the appearance of freeze-etched membranes of control erythrocytes and erythrocytes with the sulphydryl reagents. It was found that on many of the control and NEM-treated cells, small (50-100 nm) elevated patches could be seen (Fig. 1, 2 and 3). These are present on both fracture and etch faces and are devoid of any intramembrane particles. The patch elevations were never observed in the membranes of PCMBS-treated cells (Fig. 4).

Human erythrocytes are highly specialised cells boasting numerous features to maximise gas carriage, exchange and delivery around the body. The role of the highly proteinaceous red cell membrane in these processes is vital. Some membrane proteins such as the Rh-associated glycoprotein (RhAG) and aquaporin-1 (AQP1) are postulated to form gas channels, and disorders affecting membrane proteins can have extensive effects on normal red cell function. In this work, the role and interactions of Rh proteins are probed using rare variant Rh-deficient erythrocytes. The RhCE polypeptide is required for normal expression of other members of the Rh complex. Lack of RhCE is associated with depressed expression of CD44, an adhesion molecule, and may alter expression of proteins involved in complement such as decay acceleration factor and Iymphocyte function-associated antigen 3. Absence of RhAG prevents Rh complex expression and is found to affect band 3 macrocomplex proteins GPA and protein 4.2, highlighting the important role for RhAG in the macrocomplex. AQP1 is increased in the absence of RhAG, which supports the hypothesis that they share similar functions. The hereditary stomatocytoses are disorders that affect the ion permeability of red cell membranes. This work comprises a study of the pleiotropic disorder stomatin-deficient cryohydrocytosis (sdCHC), which is caused by mutations in the red cell glucose transporter, glut1. The mutant proteins show minimal glucose transport and increased permeability to cations when expressed heterologously in Xenopus laevis oocytes - consistent with the disease phenotype. sdCHC erythrocytes have very reduced amounts of stomatin, a monotopic membrane protein, a feature shared by other very leaky red cells. This is accompanied by a concomitant increase in stomatin-like protein 2, whose function in red cells is currently unknown. The fates of stomatin proteins in normal and leaky cells are investigated throughout erythropoiesis and found to differ between cation-leaky phenotypes.

Electron transport systems exist in the plasma membranes of all cells. Although not well characterised they play roles in cell growth and proliferation, hormone responses and other cell signalling events, but perhaps their most important role, especially in erythrocytes, is enabling the cell to respond to changes in both intra- and extracellular redox environments. Human erythrocytes possess a transmembrane electron transport capability that mediates the transfer of reducing equivalents from reduced intracellular species to oxidised extracellular species and is concomitant with proton extrusion. In the work for this thesis I showed that erythrocyte membranes contain a transmembrane WST-1 (water soluble tetrazolium-1) reductase activity that uses reducing equivalents from intracellular NADH to reduce extracellular WST-1. The rate of WST-1 reduction was increased by the presence of phenazine methosulfate and, although of low activity, it showed similar properties to a previously reported transmembrane NADH-oxidase activity. 1H NMR experiments showed that WST-1 was reversibly bound to the membrane and/or proteins in the membrane within the timeframe of the NMR experiment, confirming the location of the WST-1 reduction. Preliminary attempts to purify NADH:WST-1 reductase and NADH:ferricyanide reductase activities from the erythrocyte plasma membrane were inconclusive. The protein(s) responsible for the reduction of these oxidants appear to be of low abundance in the plasma membrane and may be part of a larger protein complex. Further work on the isolation of these redox activities is required before the protein(s) involved can be identified with any confidence. The ability of cells to export electrons suggests that an electron import mechanism might also exist to re-establish the cell�s redox-buffering equilibrium under conditions of oxidative stress. This hypothesis was tested in glucose-deprived erythrocytes using reduced glutathione and NADH as extracellular electron donors. It was shown that neither reduced glutathione nor NADH donated reducing equivalents through a transmembrane redox system. Extracellular NADH was, however, able to produce profound changes in starvation metabolism and methaemoglobin reduction rates. The addition of extracellular NADH caused a six-fold increase in the rate of lactate production above that observed in glucose-starved controls, together with a concomitant decrease in pyruvate production. In erythrocytes containing high levels of methaemoglobin, extracellular NADH increased the rate of methaemoglobin reduction in both the presence and absence of glucose. These results were explained by the leakage of lactate dehydrogenase from erythrocytes due to an admittedly low level of haemolysis. This caused the displacement of the intracellular pseudo-equilibrium of the lactate dehydrogenase reaction via transmembrane exchange of lactate, allowing the conversion of extracellular pyruvate to lactate and resulted in an increase in intracellular NADH concentrations. The latter increased the rate of methaemoglobin reduction. In conclusion, the work described in this thesis showed that erythrocyte membranes do not contain mechanisms for importing electrons or reducing equivalents from extracellular reduced glutathione or NADH. Erythrocytes do, however, contain an electron export system which can reduce extracellular oxidants such as WST-1 and the activity of this system depends on an intricate balance between intracellular antioxidants and enzyme activities. There is much still to be learnt about plasma membrane redox systems, little is known, for example, about the protein composition, mechanism of action, and the in vivo conditions under which these systems are most active.

Mestrado em Análises Clínicas
MSc in Clinical Analysis
PORTUGUÊS: A prevalência da obesidade está crescendo em todo o mundo, sendo este aumento particularmente notório na infância e adolescência. Os alimentos ricos em calorias e o estilo de vida sedentário são duas das características do estilo de vida moderno, que pode estar por de trás desta pandemia. A obesidade pediátrica é particularmente importante em nosso país: uma alta prevalência de sobrepeso / obesidade (30%) tem sido descrita em crianças e adolescentes portugueses, em comparação com seus pares de outros países europeus. A obesidade tem sido associada a várias outras doenças, como hipertensão, diabetes mellitus tipo 2, síndrome metabólica e doenças cardiovasculares. Além disso, a inflamação é conhecida por desempenhar um papel vital nessas doenças, ou seja, na iniciação e progressão da doença arteriosclerótica vascular, na resistência à insulina, e dislipidemia. Mais recentemente, a obesidade tem sido proposta como um estado de baixo grau inflamatório crónico. De fato, O tecido adiposo é uma importante fonte de várias substâncias que estão ligadas à resposta inflamatória e à imunidade - as adipoquinas. As dietas de indivíduos obesos são, geralmente, ricos em gorduras saturadas e ácidos gordos (AG) trans, e pobres em AG polinsaturados (AGPI), especialmente, em AGPI do tipo ω 3. Este tipo de hábitos alimentares causa alterações endógenas no metabolismo lipídico, e pode levar a mudanças na constituição em AG em diferentes tecidos do corpo. A composição lipídica das membranas celulares é importante para as propriedades reológicas e físico-químicas das células, influenciando a actividade dos canais protéicos e das bombas da membrana, assim como de transportadores e receptores membranares. Deste modo, o perfil lipídico da membrana é importante na modulação da sinalização celular e de várias funções biológicas da célula. Os lípidos da membrana dos glóbulos vermelhos estão em equilíbrio constante com lípidos e lipoproteínas plasmáticos. Apesar dos níveis dos lípidos plasmáticos serem altamente influenciados pelo estado de jejum, os níveis e tipo de lípidos da membrana eritrocitária reflectem o equilíbrio lipídico por períodos mais longos. Os glóbulos vermelhos (GV), por tratarem-se de células não-nucleadas, apresentam uma capacidade de biossíntese e mecanismos de defesa muito limitados. Portanto, quando expostos a stress físico e / ou químico, o eritrócito sofre e acumula o dano imposta por aquelas fontes durante sua vida em circulação. Em condições de stress oxidativo a membrana do eritrócito, pode sofrer danos em seus lípidos e proteínas. Deste modo, o eritrócito é um bom modelo para estudar os danos oxidativos dos lípidos e proteínas que ocorrem em estados pró-inflamatórios e oxidativas, e também pode fornecer um importante modelo para estudar o impacto dos hábitos alimentares na composição dos lípidos e proteínas das membranas celulares. O objectivo deste estudo foi analisar o impacto da obesidade no perfil lipídico, no metabolismo da glicose e na inflamação, bem como o impacto da obesidade, e das alterações a ela associadas, na composição da membrana do GV. Foram estudadas 34 crianças e adolescentes obesos [15 (44,1%), com idade média de 14,1 anos (8-17)] do Hospital S. João e do Hospital Infantil Maria Pia. O grupo total foi dividido de acordo com o percentil do índice de massa corporal (IMC) em 17 obesos, 8 sobre-pesos e 9 controles. Obesidade foi definida como um IMC superior ao percentil 95, ajustados para idade e sexo, segundo " gráficos de crescimento do Centro de Controle de Doenças de 2000". Sobrepeso foi considerado para os percentis de IMC igual ou superior a 85 e inferior a 95; e controles quando o IMC era inferior ao percentil 85, ajustado para sexo e idade. Os três grupos estavam equilibrados para a idade, sexo e estágio de maturação sexual de Tanner. Foram determinados os níveis circulantes de triglicerídeos, colesterol, colesterol de lipoproteína de alta densidade, colesterol de lipoproteína de baixa densidade, lipoproteína (a), apolipoproteína A, apolipoproteína B, proteína C-reativa, glicose e insulina. Um estudo hematológico básico foi realizado. A membrana eritrocitária foi estudada com a determinação de marcadores de lesão eritrocitária: hemoglobina ligada a membrana, carbonilação proteica, peroxidação lipídica e perfil de banda 3 – agregados de alto peso molecular, monómeros e fragmentos proteolíticos. O perfil de ácidos gordos da membrana foi também determinado. Os indivíduos obesos apresentaram, quando comparados com os controlos, alterações para um perfil lipídico mais aterogénico, um aumento da resistência à insulina e da inflamação. Assim, houve um aumento geral dos marcadores de risco de doença cardiovascular (DCV). Nenhuma diferença significativa foi encontrada no eritrograma ou nos marcadores de lesão eritrocitária. Quanto ao perfil de AG da membrana eritrocitária, os AG insaturados apresentaram uma tendência para o aumento, enquanto os AG saturados mostraram uma tendência para diminuir com a obesidade. Apesar disso, o AG beénico ácido (22:0) apresentou um aumento significativo nos obesos, em comparação com os controles. Uma proporção crescente de 20:0, 18:3n3, 20:3n6e 22:4n6 foram encontrados para indivíduos com sobrepeso e obesos, em relação aos controlos. Estes AG, que aumentaram com a obesidade, apresentaram as associações mais significativas com os marcadores de DVC estudados e que estão alterados com a obesidade na nossa população. Mais estudos são necessários para esclarecer as associações entre as alterações do perfil de AG da membrana eritrocitária e os marcadores de risco de DCV. Um estudo envolvendo mais participantes poderia ajudar a esclarecer algumas tendências observadas. Quanto à análise da membrana lipídica, algumas abordagens interessantes poderiam ser feitas, como analisar separadamente os AG ligados a fosfolípidos e ésteres de colesterol, ou analisar individualmente cada folheto da membrana plasmática (interno e externo). Além disso, a optimização da técnica de separação e identificação dos AG seria fundamental, pois no presente estudo não pudemos avaliar AG importante, como por exemplo o EPA (ácido eicosapentaenoico - 20:5n3) e GLA (ácido gama linolénico - 18:3 n6), devido a limitações técnicas.
ENGLISH: The prevalence of obesity is growing worldwide and in childhood the increase is particularly striking. The caloric rich foods and the reduced physical exercise practice are two of characteristics of the modern lifestyle that may underlie this pandemia. Childhood obesity is particularly important in our country, as a high prevalence of overweight/obesity (over 30%) has been reported for Portuguese children, as compared to other European countries. Obesity has been associated with several other diseases, such as hypertension, type 2 diabetes mellitus, metabolic syndrome and cardiovascular diseases. Moreover, inflammation is known to play a vital role in those diseases, namely, in the initiation and progression of the atherosclerotic vascular disease, in insulin resistance, and in dyslipidemia. More recently, obesity has been proposed as a chronic low grade inflammatory condition. The white adipose tissue is an important source of several substances that are linked to inflammatory response and to immunity - the adipokines. The diets of obese individuals are, usually, rich in saturated and trans fatty acids (FA), and poor in polyunsaturated FA (PUFA), especially, in ω 3 PUFA. These type of dietary habits, by leading to endogenous changes in FA and in lipid metabolism, may, ultimately, lead to changes in the proportions of the different FA in body tissues. The lipid composition of the cell membranes are important determinants in the rheological and physico-chemical properties of the cells, influencing the activity of membrane channels, pumps, transporters and receptors. Thus, it is important in the modulation of cell signalling and in several biological functions. The lipids of the red blood cell membrane are in constant equilibrium with plasmatic lipids and lipoproteins. While the levels of lipids in plasma are highly influenced by the fasting status, the levels and type of lipids of the erythrocyte membrane reflect the lipid balance of longer periods. The red blood cell (RBC), as a non-nucleated cell, exhibits a very limited biosynthesis capacity and poor repair mechanisms. Therefore, when exposed to physical and/or chemical stress, during their lifespan, the erythrocyte suffers and accumulates the damage imposed by such stress. In oxidative stress conditions the erythrocyte membrane, may suffer oxidative damage in membrane lipids and proteins. Thus, the erythrocyte is a good model to study the oxidative damage of lipids and proteins occurring in pro-inflammatory and oxidative conditions and may also provide an important model to study the impact of dietary habits in the lipid and protein composition of the cell membranes. The objective of this study was to analyse the impact of obesity in the lipid profile, glucose homeostasis and inflammation, as well as the impact of obesity and the associated changes in the RBC membrane composition. We studied 34 obese children and adolescents [15 (44.1%); mean age of 14.1 years (8-17)] from Hospital S. João and the Children’s Hospital Maria Pia. The total group was divided according to the body mass index (BMI) percentile in 17 obese, 8 overweight and 9 controls. Obesity was defined as a BMI higher than the 95th percentile, for age and gender, according to the “2000 Centre for Disease Control and Prevention (CDC) growth charts”. Overweight was considered for BMI percentiles equal or higher than 85 and lower than 95; and control subjects BMI were lower than the 85th percentile, adjusted for gender and age. The three groups were matched for age, gender and tanner stage. It were determined the circulating levels of triglycerides, cholesterol, high density lipoprotein cholesterol, Low density lipoprotein cholesterol, lipoprotein (a), apolipoprotein A, apolipoprotein B, C-reactive protein, glucose, and insulin. A basic hematologic study was also performed. The erythrocyte membrane was studied with the determination of erythrocyte damage markers: membrane bound haemoglobin, proteic carbonylation, lipid peroxidation and band 3 profile - high molecular weight aggregates, monomers and proteolytic fragments. The membrane fatty acid profile was determined. We found that the obese individuals, presented risk changes in the lipid profile, increased insulin resistance and inflammation, when compared to their lean counterparts. Thus, several changes in cardiovascular disease (CVD) risk markers were observed. No significant changes were found in the erythrogram and in the erythrocyte damage markers. Concerning the FA membrane profile, the unsaturated FA showed a trend to increase, while saturated FA showed a trend to decrease, with obesity. Despite that, behenic acid (22:0) presented a significantly increase in obese, in comparison with controls. An increasing proportion of 20:0, 18:3n3, 20:3n6 and 22:4n6 were found for overweight and obese individuals, as compared to control. These FA increased with growing obesity, and presented the most significant associations with the studied CVD markers associated with obesity. Further studies are needed to clarify the associations between membrane FA changes, and CVD risk markers. A larger study, with a higher number of individuals could also clarify some observed trends. Regarding the lipid membrane analysis, some interesting approaches could be made, such as to separate and analyze the different phospholipids and cholesterol ester and their associated FA, and to perform a separate study of inner and outer membrane sheets. Furthermore, optimization of the separation technique and consequent identification of FA would be crucial, as in this study we could not evaluate important FA, e.g. EPA (20:5n3) and GLA (18:3n6), due to technical limitations.

Biological membranes are vital components of living cells as they function to maintain the structural integrity of the cells. Red blood cell (RBC) membrane comprises the lipid bilayer and the cytoskeleton network. The lipid bilayer consists of phospholipids, integral membrane proteins, peripheral proteins and cholesterol. It behaves as a 2D fluid. The cytoskeleton is a network of spectrin tetramers linked at the actin junctions. It is connected to the lipid bilayer primarily via Band-3 and ankyrin proteins. In this paper, we introduce a coarse-grained model with high computational efficiency for simulating a variety of dynamic and topological problems involving erythrocyte membranes. Coarse-grained agents are used to represent a cluster of lipid molecules and proteins with a diameter on the order of lipid bilayer thickness and carry both translational and rotational freedom. The membrane cytoskeleton is modeled as a canonical exagonal network of entropic springs that behave as Worm-Like-Chains (WLC). By simultaneously invoking these characteristics, the proposed model facilitates simulations that span large length-scales (∼ μm) and time-scales (∼ ms). The behavior of the model under shearing at different rates is studied. At low strain rates, the resulted shear stress is mainly due to the spectrin network and it shows the characteristic non-linear behavior of entropic networks, while the viscosity of the fluid-like lipid bilayer contributes to the resulting shear stress at higher strain rates. The apparent ease of this model in combining the spectrin network with the lipid bilayer presents a major advantage over conventional continuum methods such as finite element or finite difference methods for cell membranes.

The hemolytic disorders of hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) affect the lives of millions of individuals worldwide. In HS and HE, connections in the vertical and horizontal directions between components of the RBC membrane (see Fig. 1(a)), are disrupted due to defective proteins, leading to loss of the structural and functional integrity of the membrane (1–2). Moreover, disruptions of either the vertical interactions or horizontal interactions affect the lateral diffusivity of the mobile band 3 proteins, as the motion of band 3 in the RBC membrane is confined by the cytoskeleton (3). Although a number of coarse-grained molecular dynamics (CGMD) RBC membrane models have been developed in the past two decades, very few RBC membrane models have been used to study the disordered band 3 diffusion and membrane vesiculation in HS and HE. The implicit representations of either the lipid bilayer or the cytoskeleton in these membrane models limit their applications in the membrane instability problems in HS and HE. In this extended abstract, we develop a two-component CGMD human RBC membrane model that explicitly comprises both the lipid bilayer and the cytoskeleton. In this way, the interactions between the cytoskeleton and the proteins embedded in the lipid bilayer can be simulated. The proposed model allows us to measure the band 3 lateral mobility and simulate the process of membrane vesiculation in the membrane with protein defects.