Добірка наукової літератури з теми "Membrane destabilization"

SUMMARYThe ability ofTrypanosoma cruzito induce erythrocyte membrane destabilizationin vitrowas studied. Epimastigote forms adhered to human erythrocytes and caused fusion or lysis of the red cells, depending on the conditions of the interaction. Red cells were fused in the presence of calcium, while haemolysis was induced in the absence of the cation. Dextran 60 C facilitated fusion but delayed lysis. Optimum pH and temperature for fusion were 7·4 and 37 °C, respectively. Lipid alterations were produced in the plasma membrane of the red cell during the interaction with the parasite. A Ca2+-independent increase of lysophospholipids and free fatty acids was common to both the lysis and fusion processes. A relative increase of 1, 2-diacylglycerides was unique to the fusion process and these changes were dependent on Ca2+. The transfer of free fatty acids and lysophospholipids fromT. cruzito erythrocyte membranes was demonstrated using parasites pre-labelled with radioactive phospholipids. Pre-treatment of parasites with exogenous phospholipase A2abolished the fusogenicity, while lysis was increased. Neither fusion nor haemolysis occurred when the parasites were pre-treated with fatty acid free albumin, phospholipase A2inhibitors or when these compounds were present in the medium during the parasite-erythrocyte interaction. Our results suggest thatT. cruziinduces erythrocyte membrane destabilizationin vitroby transfer of lipid material in a calcium independent manner and that this ion is necessary for other membrane alterations that lead to erythrocyte fusion.

PINK1 is a mitochondrial kinase mutated in some familial cases of Parkinson’s disease. It has been found to work in the same pathway as the E3 ligase Parkin in the maintenance of flight muscles and dopaminergic neurons in Drosophila melanogaster and to recruit cytosolic Parkin to mitochondria to mediate mitophagy in mammalian cells. Although PINK1 has a predicted mitochondrial import sequence, its cellular and submitochondrial localization remains unclear in part because it is rapidly degraded. In this study, we report that the mitochondrial inner membrane rhomboid protease presenilin-associated rhomboid-like protein (PARL) mediates cleavage of PINK1 dependent on mitochondrial membrane potential. In the absence of PARL, the constitutive degradation of PINK1 is inhibited, stabilizing a 60-kD form inside mitochondria. When mitochondrial membrane potential is dissipated, PINK1 accumulates as a 63-kD full-length form on the outer mitochondrial membrane, where it can recruit Parkin to impaired mitochondria. Thus, differential localization to the inner and outer mitochondrial membranes appears to regulate PINK1 stability and function.

Key Points The common HE mutation αL260P reduces spectrin tetramer links between junctional complexes in red cell membranes by favoring closed dimers. Favoring closed spectrin dimer formation is a new mechanism of red cell membrane destabilization by hereditary anemia mutations.

Thesis (MEng.)--University of Stellenbosch, 2000.<br>ENGLISH ABSTRACT: The aim of the thesis was to investigate the use of flow destabilization methods, combined with permeate backflushing (BIF) or on their own, on flux recovery and maintenance in capillary UF membrane systems under cross-flow (XF) and dead-end (DE) operating conditions. Various hydraulic and mechanical methods have been used to remove the accumulated cake layer and improve steady state process flux. Permeate backflushing (B/F) is the most widely used but the drawbacks are loss of product and extensive down-time. In a pilot plant study for ultrafiltration of surface waters containing high NOM, turbidity and cation loads, the use of flow destabilization, or feed flow reversal (FFR) combined with cross-flow B/F was able to improve the normalised flux by 10.7 ± 3.4 %, compared with 3.2 ± 1.6 % improvement for BIF without FFR. When a second B/F included FFR, the flux improvement was 7.0 ± 2.0 % compared with 4.3 ± 2.5 % for a B/F without FFR. The hypothesis was proposed that the flow destabilization caused slight lifting of the oriented cake layer, while the cross-flow B/F was able to sweep the lifted cake out of the lumen. If the flow destabilization may be effected by a simple but effective and low-cost method, and if this flow destabilization may be combined with reverse flow for short durations, the "lift-and-sweep" approach will be the ideal method of maintaining process flux and increasing membrane life. Such a flow destabilization method, now named "reversepressure pulsing" (RIP), was developed. The method involves circulation of feed water in a recycle loop for 2 s to gain momentum, followed by closure of a fast-action valve upstream of the modules. The momentum of the water in the concentrate loop carries it into an air-filled feed accumulator, while concentrate and reverse-flow permeate (which also lifts the fouling layer) are discharged to the atmosphere using the recycle pump for 15 s. When the valve opens again, the air in the accumulator forces the water under pressure through the membrane lumens, causing a pressure pulse and flow perturbations that lift, shift and break up the fouling layer. During 3 such "lift-and-sweep" events, the cake is lifted and the debris is swept out of the lumen. Experimental results for uninterrupted dead-end filtration at a UF pilot plant using RIP only on a severely fouled membrane, indicated that the RIP increased the flux by 18.4 % and decreased the dP by 8.2 % over a 7.2 h period. The method is effective in removing the cake layer intermittently and no long-term flux decline occurred for a period of 555 h since the previous chemical cleaning.<br>AFRIKAANSE OPSOMMING: Die doel van die tesis was om die gebruik van vloei-destabiliserings metodes, alleen of gekombineer met permeaat-terugwas, op vloed-herwinning en instandhouding in kapillêre UF membraan-stelsels tydens kruisvloei en doodloop bedryf, te ondersoek. Verskeie meganiese en hidrouliese metodes word gebruik in membraan stelsels om die koeklaag op die membraan se oppervlak te verwyder en die gestadigde-toestand vloed te verbeter. Vanhierdie metodes word permeaat-terugwas die meeste gebruik, maar het sy nadele insluitend verlies van produk en produksietyd. In 'n loodsstudie vir die ultrafiltrasie van oppervlakwaters wat hoë beladings NOM, turbiditeit en katione bevat, is die waarneming gemaak dat kruisvloei terugwas met vloeidestabilisering (voerrigting-verandering) die genormaliseerde vloed met 10.7 ± 3.4 % kon verbeter, vergeleke met 'n 3.2 ± l.6 % verbetering sonder voerrigting-verandering. Vir 'n tweede terugwas was die verbetering 7.0 ± 2.0 % vergeleke met 4.3 ± 2.5 % sonder voerrigtingverandering. Die hipotese was voorgestel dat die vloei-destabilisering die geoiënteerde koeklaag van die oppervlak gelig het, en die kruisvloei terugwas die geligde koeklaag uit die lumen kon vee. Indien hierdie vloei-destabilisering bewerk kan word deur 'n eenvoudige maar effektiewe manier, en indien dit gekombineer kan word met terugvloei van produk vir kort tydperke, sal hierdie "lig-en-vee" benadering die ideale metode wees om die membrane se vloed te verbeter en leeftyd te verleng. So 'n vloei-destabiliseringsmetode, nou genoem "terugdruk-pulsering", is ontwikkel. Die metode behels die sirkuiering van voer-water vir 2 s in 'n hersirkulasielus om momentum op te bou, gevolg deur die toemaak van 'n snel-aksie klep stroom-op van die modules. Die water in die konsentraat-lus se momentum dra dit vorentoe tot in In lug-gevulde voer-akkumulator, terwyl konsentraat en terug-vloei permeaat (wat ook tot 'n mate die koeklaag lig) ook na die atmosfeer gewend word vir 15 s deur die hersirkulasiepomp. As die klep weer oopgaan, ontspan die lug in die akkumulator, en forseer die water daarin onder druk deur die membraan-lumens. Die druk-puls en vloei-perturbasies lig, skuif en breek die koeklaag op. Tydens 3 agtereenvolgende "lig-en-vee" aksies word die koeklaag effektief opgebreek en uit die lumen gevee. Eksperimentele uitslae vir ononderbroke doodloop bedryf op uitermate aangevuilde membrane van 'n ultrafiltrasie loodsaanleg toegerus met terugdruk-pulsering, het getoon dat die vloed met 18.4 % verbeter kon word en die dP met 8.2 % verminder kon word in slegs 7.2 h. Die metode breek die koeklaag effektief op, en geen langtermyn vloed-afname is waargeneem vir meer as 555 h sedert die vorige chemiese was-prosedure nie.

Diphtheria toxin translocation (T) domain is a water soluble protein that consists of ten alpha-helices in high pH solution. Experimental studies have determined that T-domain undergoes conformational changes upon decrease of solution pH, which shifts the protein population from its initial water soluble to a membrane-competent in solution. It was hypothesized that conformational changes of the latter state prepare the protein structure for subsequent membrane binding. After binding, refolding of T-domain on the membrane results in the formation of a transmembrane state, which is characterized by the permeation of the lipid bilayer. The function of transmembrane state is to help the translocation of a catalytic domain attached to the protein N-terminal across the lipid bilayer. The first goal of this work is to study the pH-dependent destabilization of T-domain structure in solution and understand the role of protonation of key residues using a variety of computational and experimental methods. The second goal is to study the subsequent membrane binding of T-domain to lipid bilayers and propose a theoretical model of the early steps of T-domain refolding on the membrane interface using a multiscale approach. Modeling of the low pH induced conformational changes and membrane association of T-domain is performed in two stages. In the first stage, protonation of N-terminal histidines triggers conformational changes of the N-terminal helices of T-domain in solution. The role of histidines was confirmed by thermodynamic integration, continuum electrostatic calculations, and microsecond long molecular dynamics (MD) simulations. Two microsecond MD simulations of a low pH model of T-domain sampled similar destabilized protein conformations; however, an N-terminal helix showed dissimilar degree of refolding. To improve the sampling of conformational changes, we proposed and implement a sampling method based on the accelerated molecular dynamics simulations method. Our proposed implementation accelerates the sampling of the conformational landscape of the low pH T-domain model by boosting the direct-space electrostatic interactions of solute-solute atom pairs. In general, this implementation accelerates the sampling of the conformational space of alanine-dipeptide in comparison to the original implementation of accelerated molecular dynamics. In the second stage, a multiscale approach is used to model the membrane association of the low pH destabilized model of T-domain to lipid bilayers of different compositions. Two preferable membrane-bound conformations of the low pH T-domain model are predicted by equilibrium and free energy calculations. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the protein and the lipid headgroups. In contrast, the less frequently observed membrane-bound conformation is stabilized by hydrophobic interactions between the protein and lipid headgroups. These interactions allow for a deeper insertion of T-domain in the membrane interface. The predicted membrane-bound conformations were refined by atomistic molecular dynamics simulations, which show that membrane-bound conformations are stable for several microseconds. Furthermore, atomistic MD simulations suggested that neutralization of glutamate and aspartate sidechains favored a deeper inserted state of T-domain in the membrane interface. This observation is in good agreement with reported pH-dependent insertion of T-domain in the membrane interface. To study the assembly of transmembrane helices, a coarse-grained model based on a residue level of representation and a rigid-body Monte-Carlo sampling method is developed. The scoring energy function is constructed using a knowledge based potential extracted from water soluble protein structures. To compensate the protein interior packing and the solvation differences, an experimentally determined membrane partition scale for all residues was used. This scoring function was tested in a set of three transmembrane homodimers. The proposed scoring function and the associated rigid-body Monte-Carlo sampling method can be applied in the first steps of prediction of near-native structures of transmembrane proteins followed by structural refinement using atomistic MD simulations.

Alzheimer’s disease is the most common form of dementia, characterized by the aggregation of the amyloid β peptide (Aβ) in the brain and by an impairment of calcium homeostasis caused by excessive activation of glutamatergic receptors, named excitotoxicity. Here, we studied the effects on calcium homeostasis caused by the formation of Aβ oligomeric assemblies, formed with Aβ40 and Aβ42 peptide, and by the model protein HypF-N. We found that these oligomers cause a rapid influx of calcium ions (Ca2+) across the cell membrane by rapidly activating extrasynaptic N-methyl-D-aspartate receptors (NMDAr) and, to a lower extent, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAr), and also by causing a perforation of lipid bilayers. Instead, none of the Ca2+ channels, including those found in previous interactome studies to physically interact with the oligomers, were found to participate to the observed Ca2+ influx. We also observed, however, that misfolded oligomers do not interact directly with NMDAr and AMPAr. Further experiments with lysophosphatidylcholine and arachidonic acid, which cause membrane compression and stretch, respectively, indicated that these receptors are activated through a change in membrane tension induced by the oligomers and transmitted mechanically to the receptors via the lipid bilayer. Indeed, lysophosphatidylcholine is able to neutralise the oligomer-induced activation of the NMDAr, whereas arachidonic acid activates the receptors similarly to the oligomers with no additive effects. An increased rotational freedom observed for a fluorescent probe embedded within the membrane in the presence of the oligomers also indicates a membrane stretch. These results reveal a further mechanism of toxicity of Aβ oligomers in Alzheimer’s disease through the perturbation of the mechanical properties of lipid membranes sensed by NMDAr and AMPAr, in addition to others based on the direct binding to membrane receptors and on permeabilization of lipid membranes.

Some interactions of enveloped viruses with the host cell membrane have a cholesterol-dependent component, which may account for clinical manifestations of the infectious disease and can be used for the development of antiviral drugs. These cholesterol-dependent interactions can be mediated by cholesterol-recognition amino-acid consensus (CRAC) motifs present in viral proteins. The S protein of the SARS-CoV and SARS-CoV2 coronaviruses contains CRAC motifs that can be involved in the process of virus entry into the cell. Besides, during viral envelope formation, CRAC motifs can be responsible for binding of cell membrane cholesterol, leading to depletion of cell membrane cholesterol and subsequent malfunctioning of cellular cholesterol-dependent proteins, destabilization and permeabilization of cell membranes and, ultimately, to the death of infected cells. Understanding the mechanisms of cholesterol-dependent virus–cell interactions and the role of CRAC-containing viral proteins in the pathogenesis of the disease can serve as the basis for the development of new drugs that prevent both coronavirus entry into the cell and the damage of the infected cell during the viral morphogenesis. The target for such drugs can be the S-protein/cholesterol interface. CRAC-containing peptides derived from viral proteins may be among these agents. These peptides can also be used as experimental tools to study cholesterol-dependent virus–cell interactions.

A wealth of evidence accumulated over the last two decades has unambiguously linked lipid rafts to neurodegenerative diseases, in particular to Alzheimer’s disease (AD). These microdomains are highly dynamic membrane platforms with differentiated physicochemical and molecular properties compared to the surrounding membrane microenvironment, and are the locus for a number of central processes in neuronal physiology. Most recent evidence pinpoint to lipid rafts as main players in AD neuropathology. It is now widely accepted that lipid rafts actively participate in the processing of amyloid precursor protein to generate amyloid beta peptides, a main component of amyloid plaques. Current evidence have highlighted the existence of severe alterations in the molecular structure and functionality of lipid rafts in the frontal cortex of human brains affected by Alzheimer’s disease. An exceptionally interesting observation is that lipid raft destabilization can be demonstrated even at the earliest stages of AD neuropathology. In the present review, we will first elaborate on the structure and function of these multifaceted subcellular structures and second to focus on the impact of their alterations in neuronal pathophysiology along the onset and progression of AD continuum.

Coronary artery disease remains the major cause of morbidity and mortality on a global scale. Intimal thickening and fatty streaks represent early adaptive vascular changes, which are often regressive. Pathological intimal thickening represents the earliest progressive atherosclerotic lesion characterized by a focal accumulation of smooth muscle cells and acellular areas, often associated with lipid pools. Fibroatheroma is characterized by a necrotic core and can be split into early and late fibroatheroma, where macrophage apoptosis and defective efferocytosis play important roles in lesion progression. Intraplaque hypoxia is believed to result in neovascularization with subsequent intraplaque haemorrhage because of immature and leaky microvessels. Due to excessive cholesterol in remnants of erythrocyte membranes, intraplaque haemorrhage may result in rapid progression of necrotic core and plaque destabilization. Healed plaque rupture has been well delineated as an important mechanism of gradual luminal narrowing, where changes in collagen deposition allow recognition of rupture and healing sites. Calcification results from apoptosis of smooth muscle cells and macrophages or may also be caused by active release of cellular vesicles involved in calcium haemostasis. Extracellular matrix changes are associated with progression of atherosclerosis, while integrin signalling has been recognized as an important outside-in transcellular target of the inflammatory response.

Coronary artery disease remains the major cause of morbidity and mortality on a global scale. Intimal thickening and fatty streaks represent early adaptive vascular changes, which are often regressive. Pathological intimal thickening represents the earliest progressive atherosclerotic lesion characterized by a focal accumulation of smooth muscle cells and acellular areas, often associated with lipid pools. Fibroatheroma is characterized by a necrotic core and can be split into early and late fibroatheroma, where macrophage apoptosis and defective efferocytosis play important roles in lesion progression. Intraplaque hypoxia is believed to result in neovascularization with subsequent intraplaque haemorrhage because of immature and leaky microvessels. Due to excessive cholesterol in remnants of erythrocyte membranes, intraplaque haemorrhage may result in rapid progression of necrotic core and plaque destabilization. Healed plaque rupture has been well delineated as an important mechanism of gradual luminal narrowing, where changes in collagen deposition allow recognition of rupture and healing sites. Calcification results from apoptosis of smooth muscle cells and macrophages or may also be caused by active release of cellular vesicles involved in calcium haemostasis. Extracellular matrix changes are associated with progression of atherosclerosis, while integrin signalling has been recognized as an important outside-in transcellular target of the inflammatory response.

The purpose of this work is to develop a carrier system for delivering RNA molecules aimed to downregulate specific functions in T cells. In many forms of cancer, T cells that express the protein Forkhead Box P3 (Foxp3) are associated with cancer progression. These cells can be identified by CD4 and CD25, molecules express on the cell surface. Studies have shown that downregulation of Foxp3 can increase the ability of other immune cells to destroy tumors. A class of RNA molecules, commonly referred to as “siRNA”, bind to and degrade specific messenger RNA (mRNA) in a sequence-dependent manner such that expression of the encoded protein is terminated. Because mRNA molecules are located inside cells, a carrier system is required to facilitate the uptake of siRNA, which does not passively diffuse through the plasma membrane. To this end, nanosized polymeric particles coated with the polycation, ornithinex10-histidinex6 (or O10H6) were used to adsorb siRNA that bind to the mRNA encoding Foxp3. The RNA-loaded particles are spherical and uniform in size (normally distributed, polydispersity index = 0.072). Loading of RNA to the particles was confirmed using gel electrophoresis. RNA complexed with the particles are protected from serum destabilization: 83.1% of RNA were recovered compared to 36.1% in RNA that were not associated with the particles. Association with the particles increased the uptake of the RNA in mouse T cells from 3.2±0.2% (free RNA) to 20.1±3.9%. Specifically, uptake of the RNA in T cells that express CD4 increased from 2.7±0.2% to 27.1±1.3% when particles were employed. These differences are statistically significant in three experiments conducted (p &amp;lt; 0.01). Internalization of the RNA into T cells was confirmed using confocal imaging. Flow cytometric analysis showed that the particle-complexed RNA reduced the percentage of T cells that express both CD4 and CD25 in mice carrying tumors from 24.0% when free RNA molecules were used to 13.5%. In these cells, the level of Foxp3 mRNA was reduced by 30%. In conclusion, the particles facilitate the uptake of siRNA molecules into a population of T cells that is known to promote cancer growth.