Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 46
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Chem Pharm Bull (Tokyo) ; 67(10): 1099-1103, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582629

RESUMO

Cubosomes are lipidic nanoparticles containing bicontinuous cubic structures. Their unique architecture and potential as drug delivery vehicles have attracted researchers' attention. However, cubosome systems that are more robust in the presence of plasma components are being sought after for applications in intravenous administration. In this study, we prepared cubosomes consisting of 1,2-dioleoyl-sn-glycero-3-hexylphosphocholine (hexyl-DOPC) and compared their interaction with bovine serum albumin (BSA), the most abundant protein in plasma, with that of conventional cubosome systems consisting of several bicontinuous cubic phase-forming lipids, including 1-monoolein (MO), 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCO C22), or 1-O-(5,9,13,17-tetramethyloctadecyl)-ß-D-xylopyranoside (ß-XP). The average number of lipids bound to each BSA molecule was between 1.2-4.0 for MO, EROCO C22, and ß-XP. On the other hand, hexyl-DOPC exhibited negligible binding to BSA. This result suggests that hexyl-DOPC, which was shown to resist removal from particles by BSA, can be used as a new lipid component of cubosomes, and has higher plasma stability than the other cubic phase-forming lipids.


Assuntos
Nanopartículas/química , Fosfatidilcolinas/química , Soroalbumina Bovina/química , Animais , Bovinos , Estrutura Molecular , Fosfatidilcolinas/isolamento & purificação
2.
J Phys Chem B ; 119(30): 9764-71, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26186093

RESUMO

We investigated interparticle transfer of cholesterol (Chol) between large unilamellar vesicles (LUVs) and phospholipid bilayer nanodiscs. The Chol transfer rate from LUVs to nanodiscs was decreased by an increase in the Chol content or incorporation of sphingomyelin in donor phosphatidylcholine/Chol LUVs but was not influenced by the lipid composition of acceptor particles. These results suggest that Chol dissociation from the lipid bilayer into aqueous phase is the rate-limiting step of the transfer and that the process depends on the fluidity of the donor membranes. The Chol dissociation rate from nanodiscs was faster than that from LUVs with similar lipid composition. Chol preferably partitioned to LUVs rather than nanodiscs, which is consistent with the faster dissociation rate from nanodiscs. The activation energy of Chol dissociation from nanodiscs was 1.7 kJ/mol lower than that from LUV, which was brought by increased (less negative) activation entropy and enthalpy. In addition, fluorescence lifetime and anisotropy data revealed that the lipid bilayer of nanodiscs is more tightly packed than that of LUVs. These results suggest that the tighter lipid packing in nanodiscs destabilizes the Chol-containing bilayer by reducing the entropy, which facilitates Chol dissociation.


Assuntos
Colesterol/química , Nanoestruturas/química , Fosfolipídeos/química , Lipossomas Unilamelares/química , Entropia , Cinética , Bicamadas Lipídicas/química , Modelos Moleculares , Conformação Molecular , Temperatura , Termodinâmica
3.
Biochim Biophys Acta ; 1851(5): 598-604, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25661161

RESUMO

Chylomicron remnants, which carry dietary fats and cholesterol, play a role in promoting atherosclerosis. Chylomicron remnants are characterized by high cholesterol content at the surface, different from low-density lipoproteins (LDLs) containing high amounts of esterified cholesterol (CE) in the core. We prepared cholesterol-rich emulsions (TO-PC/cholesterol emulsions) as models for chylomicron remnants and compared their effects on J774 macrophages with acetylated-LDL (ac-LDL). Internalization of TO-PC/cholesterol emulsions into macrophages reduced cell viability, whereas ac-LDL did not. Surprisingly, there was no difference in intracellular free cholesterol content between cells incubated with TO-PC/cholesterol emulsions and with ac-LDL. Furthermore, cholesterol in TO-PC/cholesterol emulsions and ac-LDL both were internalized into J774 macrophages; however, incubation with TO-PC/cholesterol emulsions induced leakage of lysosomal protease, cathepsin-L, to cytosol, which was not observed for incubation with ac-LDL. Inhibition of the activity of cathepsin-L recovered the viability of macrophages that ingested TO-PC/cholesterol emulsions. We suggest an alternative fate of cholesterol-rich emulsions taken up by macrophages, which is different from other atherogenic lipoproteins rich in CE; internalization of TO-PC/cholesterol emulsions into macrophages induces rapid free cholesterol accumulation in lysosomes and cell death due to lysosomal destabilization.


Assuntos
Colesterol/metabolismo , Remanescentes de Quilomícrons/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Animais , Transporte Biológico , Morte Celular , Linhagem Celular , Emulsões , Lipoproteínas LDL/metabolismo , Macrófagos/patologia , Camundongos , Fosfatidilcolinas/metabolismo , Trioleína/metabolismo
4.
Biochim Biophys Acta ; 1828(5): 1340-6, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23357357

RESUMO

Discoidal high-density lipoprotein (HDL) particles are known to fractionalize into several discrete populations. Factors regulating their size are, however, less understood. To reveal the effect of lipid composition on their formation and characteristics, we prepared several reconstituted HDLs (rHDLs) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and sphingomyelin at phospholipid to apolipoprotein A-I ratios of 100 and 25. When reconstitution was conducted at 37°C, the efficiency of rHDL formation from POPC was decreased as compared with that conducted at 4°C. Moreover, large rHDLs with a Stokes diameter of 9.6nm became dominant over small rHDL with a diameter of 7.9nm, which was distinctly observed at 4°C. The aminophospholipids POPS and POPE promoted the formation of small rHDLs at 37°C, but fluorescence experiments revealed that they did so in a different fashion: Fluorescence lifetime data suggested that the head group of POPS reduces hydrophobic hydration, especially in small rHDLs, suggesting that this lipid stabilizes the saddle-shaped bilayer structure in small rHDLs. Fluorescence lifetime and anisotropy data showed that incorporation of POPE increases acyl chain order and water penetration into the head group region in large rHDLs, suggesting that POPE destabilizes the planar bilayer structure. These results imply that these aminophospholipids contribute to the formation of small rHDLs under biological conditions.


Assuntos
Bicamadas Lipídicas/química , Lipoproteínas HDL/química , Fosfolipídeos/química , Algoritmos , Anisotropia , Apolipoproteína A-I/química , Cinética , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Espectrometria de Fluorescência , Esfingomielinas/química , Temperatura
5.
Colloids Surf B Biointerfaces ; 104: 128-32, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23298597

RESUMO

In this work, the changes in the lateral pressure in mixed membranes of egg yolk phosphatidylcholine (EPC) and a nonlamellar-forming lipid diolein (DO) were investigated with respect to increasing DO content. Several fluorescence techniques were employed to probe transitions of EPC/DO lipid mixtures from lamellar to inverted hexagonal via bicontinuous cubic phases. Excimer fluorescence of dipyrenyl phospholipids revealed that the lateral pressure in the acyl chain region of the EPC/DO mixed membrane increased with the mole fraction of DO (X(DO)) in the lamellar phase and further increased almost linearly up to X(DO)=0.2 through the lamellar-to-cubic phase transition. Water penetration into the acyl chain region, as determined by fluorescence lifetime experiments, decreased linearly with increasing X(DO) until the cubic phase was formed. These results suggest that the acyl chain packing becomes tighter by the incorporation of DO in the lamellar phase and that it does not alter during the lamellar-to-cubic phase transition. Only the packing of the headgroup region was found to increase during the transition to the cubic phase, as detected by the fluorescence lifetime of dansyl phosphatidylethanolamine, which localizes in this region. Through the cubic-to-hexagonal transition, headgroup packing was increased further, and the acyl chain packing was in turn loosened. These results suggest that the acyl chain packing stress induced by the incorporation of DO is released not by the lamellar-to-cubic phase transition but by the cubic-to-hexagonal transition.


Assuntos
Diglicerídeos/química , Fosfatidilcolinas/química , Gema de Ovo/química , Fluorescência , Transição de Fase , Pressão
6.
Colloids Surf B Biointerfaces ; 94: 1-6, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22391322

RESUMO

Phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PC) in large unilamellar vesicles (LUVs) consisting of PC and either glycerol monooleate (GMO) or methyl oleate (MeO) were monitored in situ and in real time by using a choline oxidase-immobilized oxygen electrode. This technique revealed reaction differences between 2 bacterial PLDs. PLD from Streptomyces chromofuscus, which is closely homologous to bacterial alkaline phosphatase, hydrolyzed only 6% of surface PC owing to product inhibition. The catalytic activity of this enzyme was not sensitive to the addition of GMO. On the other hand, typical bacterial PLD from Streptomyces sp. was found to hydrolyze all the PC molecules at the outer surface of LUVs suggesting that this enzyme is free from product inhibition. Introduction of GMO or MeO into the bilayer increased exposure of the PC headgroup and facilitated PC hydrolysis mediated by PLD from Streptomyces sp. GMO and MeO have the same lipophilic tail but the latter lacks hydroxyl groups on its polar head. From kinetic analysis by using the Michaelis-Menten model extended to the reaction at the interface, these compounds were found to activate PLD from Streptomyces sp. in different ways, i.e., MeO increased the protein binding to membranes and GMO stimulated the enzyme-substrate complex formation at membrane surface.


Assuntos
Proteínas de Bactérias/metabolismo , Glicerídeos/metabolismo , Ácidos Oleicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipase D/metabolismo , Streptomyces/enzimologia , Oxirredutases do Álcool/química , Eletrodos , Ativação Enzimática , Enzimas Imobilizadas , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/metabolismo , Cinética , Oxigênio/química , Ligação Proteica , Streptomyces/química , Streptomyces/metabolismo , Especificidade por Substrato , Lipossomas Unilamelares
7.
J Phys Chem B ; 116(1): 476-82, 2012 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-22148179

RESUMO

Plasma triglyceride-rich lipoproteins vary in their lipid composition during metabolism. We investigated the effects of cholesterol (Chol) on the surface properties of lipid emulsions and on the interactions with two amphipathic peptides, acetyl-DWLKAFYDKVAEKLKEAF-amide (Ac-18A-NH(2)) and acetyl-KWLDAFYDEVAEKLKKAF-amide (Ac-18G*-NH(2)), which differ in charge distribution. The fluorescence lifetimes of N-dansyl phosphatidylethanolamine (dansyl-PE) and n-(9-anthroyloxy)stearic acid (n-AS, n = 2, 6, and 12) were used to assess the water penetration into the headgroup and acyl chain regions of phosphatidylcholine (PC), respectively. Steady-state fluorescence anisotropy of n-AS was also performed to evaluate the acyl chain fluidity in emulsion surface monolayers. Chol decreased the fluorescence lifetime of dansyl-PE and increased the lifetimes and anisotropy values of n-AS. These results demonstrated that Chol alters the surface properties of emulsions, i.e., induces PC headgroup separation and acyl chain condensation. The two peptides showed different responses to Chol in several experiments: Addition of Chol to emulsions decreased and increased the dissociation constants of Ac-18A-NH(2) and Ac-18G*-NH(2), respectively. Furthermore, the α-helical content of Ac-18A-NH(2) was decreased by Chol, whereas that of Ac-18G*-NH(2) was unchanged. The higher reduction in helicity for Ac-18A-NH(2) is probably due to its deeper penetration than Ac-18G*-NH(2) into the hydrocarbon region of surface monolayers in the absence of Chol, which was demonstrated by Trp quenching experiments with n-AS. From these results, the charge distribution of the amphipathic helices is suggested to be a determining factor in their response to Chol enrichment in emulsions.


Assuntos
Colesterol/metabolismo , Emulsões/química , Lipídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Colesterol/química , Dicroísmo Circular , Polarização de Fluorescência , Dados de Sequência Molecular , Peptídeos/química , Fosfatidiletanolaminas/química , Ligação Proteica , Estrutura Secundária de Proteína , Propriedades de Superfície
8.
Lipids ; 46(4): 323-32, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21080234

RESUMO

Apolipoprotein C-III (apoC-III) decreases the apolipoprotein E (apoE)-mediated uptake of lipoprotein remnants by the liver, and a high plasma concentration of apoC-III in VLDL is associated with hypertriglyceridemia and the risk of coronary heart disease. In this study, we prepared lipid emulsions containing triolein, phosphatidylcholine and cholesterol as model particles of lipoproteins, and examined the roles of apoC-III in apoE-mediated uptake of emulsions by HepG2 cells. Cholesterol in emulsion particles enhanced the apoE-mediated uptake via heparan sulfate proteoglycan and LDL receptor-related protein pathways. The amount of apoE bound to emulsion particles was increased by the presence of cholesterol at the particle surface, whereas cholesterol had no effect on the binding amount of apoC-III. Surface cholesterol alleviated the inhibitory effect of apoC-III on apoE incorporation into the emulsion surface. However, ApoC-III almost completely inhibited the apoE-mediated uptake of cholesterol-containing emulsions despite sufficient binding of apoE to emulsions. These findings suggest that apoC-III attenuates the binding of apoE to the lipoprotein surface and apoE-mediated cellular uptake of lipoprotein remnants. Furthermore, cholesterol may affect these functions of apoC-III and apoE involved in the clearance of lipoprotein remnants.


Assuntos
Apolipoproteína C-III/metabolismo , Apolipoproteínas E/metabolismo , Células Hep G2/metabolismo , Lipoproteínas VLDL/metabolismo , Apolipoproteínas E/genética , Emulsões/química , Humanos
9.
J Phys Chem B ; 114(38): 12376-82, 2010 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-20812712

RESUMO

The class A amphipathic α-helical peptide 18A is known to form discoidal phospholipid complexes (nanodiscs) similar to that formed by apolipoprotein A-I (apoA-I). To reveal the structural differences in nanodiscs formed with this protein and peptide, we prepared nanodiscs with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and applied fluorescence techniques to these nanoparticles. Fluorescence resonance energy transfer between 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) in nanodiscs revealed that lipid exchange with 18A nanodiscs is mediated by collisions between nanodiscs. The fluorescence lifetime of dansyl phosphatidylethanolamine and excimer fluorescence of 1,2-bis(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine showed that the degree of hydration of the membrane surface and lateral pressure of acyl chains in 18A nanodiscs are independent of the disc size, suggesting that 18A nanodiscs form planar lipid bilayers irrespective of their size, which differs from apoA-I nanodiscs, whose bilayer deforms to a saddle surface with decreasing size. These results suggest that the flexible structure of a chain of helices in apoA-I is crucial for the formation of saddle surfaces in nanodiscs.


Assuntos
Apolipoproteínas/química , Nanoestruturas/química , Peptídeos/química , Termodinâmica , Transferência Ressonante de Energia de Fluorescência , Bicamadas Lipídicas/química , Modelos Moleculares , Tamanho da Partícula , Fosfolipídeos/síntese química , Fosfolipídeos/química
10.
J Phys Chem B ; 114(24): 8228-34, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20518504

RESUMO

Nascent high-density lipoproteins (HDLs), which are also known as discoidal HDLs, are formed by the interaction of apolipoprotein A-I (apoA-I) with transmembrane ATP-binding cassette transporter A1 (ABCA1). However, the molecular mechanism governing disc formation is not fully understood. Here, we evaluated the thermodynamic and kinetic stability of disc formation from mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine and apoA-I by quantifying the discs and vesicles produced. Sodium cholate dialysis experiments revealed that the discs are thermodynamically more stable than the vesicle/apoA-I mixture (Delta*G = -52 kJ/disc mol at 37.0 degrees C) because the decrease in enthalpy (Delta*H = -620 kJ/disc mol) exceeds the decrease in entropy (TDelta*S = -570 kJ/disc mol). Circular dichroism spectral measurements ascribed 68% of the decrease in enthalpy during disc formation to the formation of helices in apoA-I. Fluorescence measurements suggested that phospholipids enclosed in the discs are more closely packed than those in the vesicles so that they are entropically destabilized. To determine if the disc could be spontaneously produced from vesicles, we measured the decrease in the turbidity of vesicles in response to the addition of apoA-I. However, the rate of disc formation was very slow, suggesting that the large kinetic barrier against disc formation makes the vesicle/apoA-I mixtures metastable. These results raise the possibility that ABCA1 may act to lower the activation energy, thereby facilitating disc formation.


Assuntos
Apolipoproteína A-I/química , Lipoproteínas HDL/química , Fosfatidilcolinas/química , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Dicroísmo Circular , Cinética , Conformação Proteica , Estabilidade Proteica , Colato de Sódio/química , Espectrometria de Fluorescência , Termodinâmica
11.
Chem Pharm Bull (Tokyo) ; 58(1): 51-5, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20045966

RESUMO

SX-3228, 6-benzyl-3-(5-methoxy-1,3,4-oxadiazol-2-yl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2(1H)-one, is a newly synthesized benzodiazepine receptor agonist intended to be developed as a tablet preparation. However, it was found that the drug substance was remarkably chemically unstable in tablet form compared to the powder mixture for tableting. Chemical destabilization due to compression also occurred in the drug substance alone. After investigating the cause of the destabilization, powder X-ray diffraction analysis showed that the crystallinity of the drug substance decreased depending on the extent of mechanical treatments such as compression and grinding. In thermal analysis it became evident that the exothermic peaks due to degradation clearly broadened and shifted toward lower temperatures by these mechanical treatments. It was then revealed that the degradation temperature decreased and the amount of degradation products after storage increased with decreasing crystallinity, even though there was little change in the amount of degradation products shortly after mechanical treatments. These results demonstrated that the drug substance became chemically unstable with decreasing crystallinity. It was proved that chemical instability of the drug substance in the tablet preparation was due to decreasing crystallinity caused by compression. It would therefore be difficult to produce chemically stable tablets containing this compound using a conventional manufacturing process. Tablets for this compound should be prepared without mechanical treatments such as compression and grinding.


Assuntos
Agonistas GABAérgicos/química , Agonistas de Receptores de GABA-A , Cristalografia por Raios X , Composição de Medicamentos , Estabilidade de Medicamentos , Comprimidos/química
12.
J Pharm Sci ; 99(1): 293-302, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19548311

RESUMO

The solubility of organic compounds in mixtures of water and an organic cosolvent can be reasonably estimated from the solubility values in the neat solvents and the composition of the solvent mixture, by means of the log-linear solubilization model. However, deviations from the model are frequently observed in different degree. Such deviations, which tend to become more pronounced with decreasing polarity of the cosolvent, are to a good extent the result the nonideal mixing of water and cosolvent, due to the interactions between the two solvent components. We present a model that incorporates the effect of water-cosolvent interactions into the log-linear model. The effect of nonideality of mixing takes the form of a pseudoquadratic expression on the cosolvent concentration, obtainable from vapor pressure data of the solute-free water-cosolvent mixture.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Preparações Farmacêuticas/química , Solventes/química , Água/química , Modelos Lineares , Modelos Químicos , Solubilidade , Termodinâmica
13.
Chem Pharm Bull (Tokyo) ; 57(10): 1096-9, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19801864

RESUMO

Formaldehyde is a well-known air impurity. The possibility was investigated in this study that pharmaceutical excipients commonly used in oral solid dosage forms might also be sources of formaldehyde. The results showed that formaldehyde is generated by the excipients lactose, D-mannitol, microcrystalline cellulose, low-substituted hydroxypropylcellulose, magnesium stearate and light anhydrous silicic acid. Since the quality and safety of pharmaceutical products can be significantly affected by the presence of formaldehyde, various amines were then investigated for their ability to decrease levels of formaldehyde using an aqueous solution system. Of the four amines investigated, only meglumine proved capable of reducing formaldehyde levels. The reaction product between formaldehyde and meglumine was obtained by fractionation using the preparative HPLC system and the structure was clarified by (1)H-, (13)C-NMR, various types of two-dimensional NMR and mass spectroscopy. The reaction product was determined to be a compound with a 1,3-oxazinane skeleton and containing one more carbon than meglumine. It was presumed that formaldehyde reacted with the secondary amino group in meglumine to form the reaction product via an iminium salt intermediate by cyclization. As meglumine is permitted to be used as a pharmaceutical excipient in both oral and parenteral dosage forms by regulations worldwide, the addition of meglumine to pharmaceutical products can be expected to contribute to the stabilization of many drug substances.


Assuntos
Aminas/química , Excipientes/química , Formaldeído/química , Meglumina/química , Absorção , Cromatografia Líquida de Alta Pressão , Formaldeído/metabolismo
14.
Biochemistry ; 48(32): 7756-63, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19610670

RESUMO

Discoidal high-density lipoprotein (HDL) particles are known to be fractionalized into several discrete populations in plasma and to differ in behavior according to size; however, their structural differences and the factors regulating their size are less understood. In this study, we prepared several reconstituted HDLs (rHDLs) for structural evaluation by gel filtration chromatography and fluorometric analyses. With initial ratios of phospholipid (PL) to apolipoprotein A-I (apoA-I) between 25:1 and 100:1, unsaturated PLs constructed rHDLs with diameters of 9.5-9.6, 8.8-9.0, and 7.8-7.9 nm. Conversely, saturated PLs formed only the largest type of rHDLs (9.5-9.9 nm). While the largest rHDL comprised 23% cholesterol (Chol), the smallest rHDL contained only 13% Chol, which approximates liquid-ordered phase composition. As the size of rHDLs decreased, both the lateral pressure in the lipid bilayer, as determined from the excimer fluorescence of dipyrenylphosphatidylcholine, and the degree of hydration of the membrane surface, which was examined using the mean fluorescence lifetime of dansyl phosphatidylethanolamine, decreased well below the values obtained for large unilamellar vesicles. These results demonstrated that smaller rHDLs form a saddle surface, distinct from the planar bilayer produced by the largest forms.


Assuntos
Bicamadas Lipídicas , Lipoproteínas HDL , Animais , Apolipoproteína A-I/química , Colesterol/química , Humanos , Bicamadas Lipídicas/química , Lipoproteínas HDL/química , Lipoproteínas HDL/ultraestrutura , Tamanho da Partícula , Fosfolipídeos/química , Lipossomas Unilamelares/química
15.
J Am Chem Soc ; 131(23): 8308-12, 2009 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-19456103

RESUMO

Nanodiscs are phospholipid-protein complexes which are relevant to nascent high-density lipoprotein and are applicable as a drug carrier and a tool to immobilize membrane proteins. We evaluated the structure and dynamics of the nanoparticles consisting of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (apoA-I) with small-angle neutron scattering (SANS) and fluorescence methods and compared them with static/dynamic properties for large unilamellar vesicles. SANS revealed that the nanodisc includes a lipid bilayer with a thickness of 44 A and a radius of 37 A, in which each lipid occupies a smaller area than the reported molecular area of DMPC in vesicles. Fluorescence measurements suggested that DMPC possesses a lower entropy in nanodiscs than in vesicles, because apoA-I molecules, which surround the bilayer, force closer lipid packing, but allow water penetration to the acyl chain ends. Time-resolved SANS experiments revealed that nanodiscs represent a 20-fold higher lipid transfer via an entropically favorable process. The results put forward a conjunction of static/dynamic properties of nanodiscs, where the entropic constraints are responsible for the accelerated desorption of lipids.


Assuntos
Apolipoproteína A-I/química , Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Nanoestruturas/ultraestrutura , Apolipoproteína A-I/ultraestrutura , Estrutura Molecular , Nêutrons , Espalhamento a Baixo Ângulo
16.
J Phys Chem B ; 113(19): 6745-8, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19385639

RESUMO

We applied a time-resolved small-angle neutron scattering technique to vesicle systems to determine interparticle transfer and flip-flop of phospholipids. Measurements were performed for large unilamellar vesicles, consisting of dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), or 1-palmitoyl-2-oleoylphosphatidic acid (POPA), which differ either in their acyl chains or headgroup. POPC, which is analogous to naturally occurring phosphatidylcholines, exhibited no transbilayer transfer and very slow interbilayer migration. POPC on the inner leaflet of vesicles did not flop even when phospholipase D converted all POPC molecules on the outer leaflet into POPA, which was shown to exhibit fast flip-flop. From these results, together with the observation that the flip-flop of DMPC was entirely inhibited in the presence of cholesterol, it is deduced that the flip-flop of phosphatidylcholines does not take place spontaneously in cellular plasma membranes rich in cholesterol and that it requires enzymatic activities of energy-dependent and/or -independent flippases/floppases.


Assuntos
Difração de Nêutrons , Fosfolipídeos/metabolismo , Espalhamento a Baixo Ângulo , Lipossomas Unilamelares/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/metabolismo , Hidrólise , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/química , Fatores de Tempo , Lipossomas Unilamelares/química
17.
Langmuir ; 25(8): 4336-8, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19296640

RESUMO

This report introduces modified cellulose polymers as new emulsifiers of cubosomes. We prepared novel nanoparticles containing cubic-phase-forming lipids using hydroxypropyl methylcellulose acetate succinate (HPMCAS). Small-angle X-ray scattering showed a much lower incorporation of HPMCAS into the cubic structure of monoolein than did a conventional emulsifier, Pluronic F127, which is known to modify the cubic structure. Cubosomes prepared with HPMCAS showed roughly equal stability as nanoparticles with Pluronic F127. These results suggest that HPMCAS can be a novel emulsifier of cubosomes, which brings about no internal structure modification.


Assuntos
Celulose/química , Polímeros/química , Físico-Química/métodos , Emulsões , Glicerídeos/química , Lipídeos/química , Metilcelulose/análogos & derivados , Metilcelulose/química , Modelos Químicos , Nanopartículas/química , Nanotecnologia/métodos , Poloxâmero/química , Espalhamento de Radiação , Propriedades de Superfície , Raios X
18.
J Pept Sci ; 15(5): 327-36, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19206072

RESUMO

In an attempt to develop non-ATP-competitive inhibitors of the autophosphorylation of IR, the effects of the synthetic peptides, Ac-DIY(1158)ET-NH(2) and Ac-DY(1162)Y(1163)RK-NH(2), on the phosphorylation of IR were studied in vitro. The peptides were derived from the amino-acid sequence in the activation loop of IR. They inhibited the autophosphorylation of IR to 20.5 and 40.7%, respectively, at 4000 microM. The Asp/Asn- and Glu/Gln-substituted peptides, Ac-NIYQT-NH(2) and Ac-NYYRK-NH(2), more potently inhibited the autophosphorylation than did the corresponding parent peptides. The inhibitory potencies of the substituted peptides were decreased with increasing concentrations of ATP, indicating that these peptides employ an ATP-competitive mechanism in inhibiting the autophosphorylation of IR. In contrast, those of the parent peptides were not affected. Mass spectrometry showed that the parent peptides were phosphorylated by IR, suggesting that they interact with the catalytic loop. Moreover, docking simulations predicted that the substituted peptides would interact with the ATP-binding region of IR, whereas their parent peptides would interact with the catalytic loop of IR. Thus, Ac-DIYET-NH(2) and Ac-DYYRK-NH(2) are expected to be non-ATP-competitive inhibitors. These peptides could contribute to the development of a drug employing a novel mechanism.


Assuntos
Trifosfato de Adenosina/metabolismo , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Receptor de Insulina/metabolismo , Animais , Humanos , Espectrometria de Massas , Oligopeptídeos/síntese química , Oligopeptídeos/química , Ratos
19.
Biochim Biophys Acta ; 1778(11): 2594-603, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18789885

RESUMO

To investigate the effect of apolipoprotein B (apoB) on cell viability, we used lipid-free apoB as a model for denatured apoB. Lipid-free apoB had cytotoxicity to J774 macrophages, CHO cells and HepG2 cells, whereas apoB bound to low density lipoprotein (LDL) and lipid-free apolipoprotein A-I had no effect on cell viability. Lipid-free apoB induced apoptosis in J774 macrophages assessed by caspase-3 activation and annexin V binding. LDL receptor, heparan sulfate proteoglycans, and class A scavenger receptor were involved in the binding/uptake of lipid-free apoB, but lipid-free apoB binding/uptake by the cells did not correlate with cytotoxicity. Lipid-free apoB disrupted the lipid bilayer of large unilamellar vesicles containing calcein. We evaluated the interaction between apoB and cellular membrane by monitoring the change in intracellular Ca(2+) concentration using Fura-2, and found that lipid-free apoB rapidly disrupted the cellular membrane in the absence or presence of the inhibitors for cellular binding/uptake mediated by the receptors. Therefore, it is suggested that lipid-free apoB induces cell death by disturbance of the plasma membrane. In addition to other lipid component in modified LDL, apoB itself has an ability to induce apoptosis and plays a crucial role in the development of atherosclerotic lesions.


Assuntos
Apolipoproteínas B/farmacologia , Animais , Anexina A5/metabolismo , Anticorpos/farmacologia , Apolipoproteínas B/química , Sinalização do Cálcio/efeitos dos fármacos , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Ativação Enzimática/efeitos dos fármacos , Fluoresceínas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , L-Lactato Desidrogenase/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Receptores Depuradores Classe A/metabolismo
20.
J Lipid Res ; 49(11): 2419-26, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18645209

RESUMO

The molecular mechanism by which nascent HDL forms via the interaction of apolipoprotein A-I (apoA-I) and transmembrane ABCA1 is poorly understood. Here, because ABCA1 has been reported to localize to acidic intracellular compartments, including the Golgi and endosome, we studied the interaction of apoA-I with model membranes under acidic conditions. Pure phosphatidylcholine liposomes were persistent against apoA-I at pH levels above 5.0, but were progressively transformed into reconstituted HDLs (rHDLs) by apoA-I at lower pH. Circular dichroism spectral measurements and 8-anilino-1-naphthalenesulfonic acid fluorescence measurements of lipid-free apoA-I ascribed this accelerated rHDL formation to the conformational change of the protein into a rather hydrophobic alpha-helical structure under acidic conditions. The addition of phosphatidylserine (PS) increased acidity at the bilayer surface and enabled the formation of discoidal rHDLs even at the pH of the endosome and slightly lower pH of the Golgi. These results suggest the following new scenario of nascent HDL formation: ABCA1, which colocalizes with apoA-I in acidic intracellular compartments, including the Golgi and endosome, increases acidity at the membrane surface on the luminal side by PS translocase activity and causes apoA-I to form nascent HDL.


Assuntos
Apolipoproteína A-I/química , HDL-Colesterol/biossíntese , Líquido Intracelular/metabolismo , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Animais , Apolipoproteína A-I/metabolismo , Bovinos , Dicroísmo Circular , Lipoproteínas de Alta Densidade Pré-beta/biossíntese , Humanos , Concentração de Íons de Hidrogênio , Lipossomos , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Estrutura Secundária de Proteína
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...