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1.
Langmuir ; 37(38): 11195-11202, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34528800

RESUMO

Numerous research studies have been done for exosomes, particularly focusing on membrane proteins and included nucleic acids, and the volume of the knowledge about the lipids in the exosomal membrane has been increasing. However, the dynamic property of the exosomal membrane is hardly studied. By employing milk exosome as an example, herein the exosomal membrane was characterized focusing on the membrane fluidity and polarity. The lipid composition and phase state of milk exosome (exosome from bovine milk) were estimated. The milk exosome contained enriched Chol (43.6 mol % in total lipid extracts), which made the membrane in the liquid-ordered (lo) phase by interacting with phospholipids. To suggest a model of exosomal vesicle cargo, the liposome compositions that mimic milk exosome were studied: liposomes were made of cholesterol (Chol), milk sphingomyelin (milk SM), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By using fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-2-dimethylaminonaphthalene, the microenvironments of submicron-sized membranes of exosome and model liposomes were investigated. The membrane fluidity of milk exosome was slightly higher than those of Chol/milk SM/POPC liposomes with a similar content of Chol, suggesting the presence of enriched unsaturated lipids. The most purposeful membrane property was obtained by the liposome composition of Chol/milk SM/POPC = 40/15/45. From the above, it is concluded that Chol is a fundamental component of the milk exosomal membrane to construct the enriched lo phase, which could increase the membrane rigidity and contribute to the function of exosome.


Assuntos
Fluidez de Membrana , Fosfatidilcolinas , Animais , Bovinos , Colesterol , Bicamadas Lipídicas , Lipossomos , Fosfolipídeos , Esfingomielinas
2.
Biomolecules ; 11(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34439820

RESUMO

Cold-loving microorganisms of all three domains of life have unique and special abilities that allow them to live in harsh environments. They have acquired structural and molecular mechanisms of adaptation to the cold that include the production of anti-freeze proteins, carbohydrate-based extracellular polymeric substances and lipids which serve as cryo- and osmoprotectants by maintaining the fluidity of their membranes. They also produce a wide diversity of pigmented molecules to obtain energy, carry out photosynthesis, increase their resistance to stress and provide them with ultraviolet light protection. Recently developed analytical techniques have been applied as high-throughoutput technologies for function discovery and for reconstructing functional networks in psychrophiles. Among them, omics deserve special mention, such as genomics, transcriptomics, proteomics, glycomics, lipidomics and metabolomics. These techniques have allowed the identification of microorganisms and the study of their biogeochemical activities. They have also made it possible to infer their metabolic capacities and identify the biomolecules that are parts of their structures or that they secrete into the environment, which can be useful in various fields of biotechnology. This Review summarizes current knowledge on psychrophiles as sources of biomolecules and the metabolic pathways for their production. New strategies and next-generation approaches are needed to increase the chances of discovering new biomolecules.


Assuntos
Adaptação Fisiológica/genética , Antibacterianos/biossíntese , Proteínas Anticongelantes/biossíntese , Bactérias/metabolismo , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Redes e Vias Metabólicas/genética , Proteínas Anticongelantes/genética , Regiões Árticas , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Biotecnologia/métodos , Clorófitas/genética , Clorófitas/crescimento & desenvolvimento , Clorófitas/metabolismo , Temperatura Baixa , Biologia Computacional/métodos , Diatomáceas/genética , Diatomáceas/crescimento & desenvolvimento , Diatomáceas/metabolismo , Matriz Extracelular de Substâncias Poliméricas/genética , Fungos/genética , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Humanos , Lipídeos/biossíntese , Lipídeos/genética , Fluidez de Membrana , Metagenoma , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/genética
3.
Langmuir ; 37(29): 8840-8846, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34264682

RESUMO

Conjugating polymers to peptides has become a new strategy of designing functional antitumor agents for their improved stability and enhanced activity. In this paper, a novel peptide-polymer conjugate PEPc-PMAA with pH responsiveness was designed and synthesized. The isoelectric point of PEPc was studied by dynamic light scattering for the targeting effect. Also, the transmittances of PMAA at different pHs were measured using an ultraviolet-visible spectrophotometer for determining the triggering pH of the disrupting effect. The results showed that PEPc-PMAA was hydrophilic under neutral conditions and changed to be amphiphilic composed of positively charged PEPc and hydrophobic PMAA under acidic conditions. The interactions between PEPc-PMAA and mimic cells were investigated by the measurements of membrane fluidity and cargo leakage from 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine and 1,2-dipalmitoyl-sn-glycerol-3-phospho-(1-rac-glycerol) (DPPG) liposomes. It proved that PEPc-PMAA caused a distinct membrane disturbance of the DPPG liposome at pH 5.5, resulting in more serious cargo leakage. Because of its targeting and disrupting effects on negatively charged biomembranes under acidic conditions, PEPc-PMAA showed its good potential as an antitumor agent.


Assuntos
Lipossomos , Polímeros , Glicerol/análogos & derivados , Fluidez de Membrana , Peptídeos , Fosforilcolina/análogos & derivados , Polímeros/toxicidade
4.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200063

RESUMO

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.


Assuntos
Archaea/fisiologia , Membrana Celular/fisiologia , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fluidez de Membrana , Esqualeno/análogos & derivados , Archaea/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Esqualeno/farmacologia , Temperatura
5.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199294

RESUMO

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.


Assuntos
Aclimatação/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Membrana Celular/enzimologia , Temperatura Baixa , Proteínas Quinases Ativadas por Mitógeno/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Congelamento , Cinética , Fluidez de Membrana , Biossíntese de Proteínas , Transcrição Genética
6.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207189

RESUMO

Studying the interactions between lipid membranes and various bioactive molecules (e.g., polyphenols) is important for determining the effects they can have on the functionality of lipid bilayers. This knowledge allows us to use the chosen compounds as potential inhibitors of bacterial and cancer cells, for elimination of viruses, or simply for keeping our healthy cells in good condition. As studying those effect can be exceedingly difficult on living cells, model lipid membranes, such as liposomes, can be used instead. Liposomal bilayer systems represent the most basic platform for studying those interactions, as they are simple, quite easy to prepare and relatively stable. They are especially useful for investigating the effects of bioactive compounds on the structure and kinetics of simple lipid membranes. In this review, we have described the most basic methods available for preparation of liposomes, as well as the essential techniques for studying the effects of bioactive compounds on those liposomes. Additionally, we have provided details for an easy laboratory implementation of some of the described methods, which should prove useful especially to those relatively new on this research field.


Assuntos
Bicamadas Lipídicas/química , Lipossomos/química , Polifenóis/química , Fluidez de Membrana , Análise Espectral/métodos
7.
Methods Mol Biol ; 2341: 1-7, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264454

RESUMO

Many S. aureus strains produce membrane-associated carotenoid pigments, advantageous secondary metabolites that can alter membrane fluidity, resistance to antimicrobial peptides (AMPs) and act as antioxidants, properties that can impact resistance against aspects of the host innate immune system. Several studies have reported connections between mutations in both regulatory (i.e., alternative sigma factor B) and metabolic (purine biosynthesis, oxidative phosphorylation) genes, and noticeable differences in carotenoid pigmentation. This chapter outlines a simple protocol to quantify cellular pigments using a methanol extraction method.


Assuntos
Carotenoides/isolamento & purificação , Metanol/química , Staphylococcus aureus/crescimento & desenvolvimento , Carotenoides/química , Fracionamento Químico , Regulação Bacteriana da Expressão Gênica , Fluidez de Membrana , Espectrofotometria , Staphylococcus aureus/metabolismo
8.
Methods Mol Biol ; 2341: 55-68, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264461

RESUMO

Fluorescence polarization is a method to determine membrane fluidity using a hydrophobic fluorescent dye that intercalates into the fatty acid bilayer. A spectrofluorometer is used to polarize UV light as a vertical excitation beam which passes through the dye-labeled membrane where the dye fluoresces. The beams perpendicular and horizontal to the excitation light are then collected and analyzed. Membrane structural properties are largely due to the packing of the fatty acids in the lipid bilayer that determines the membrane biophysical parameters. Staphylococcus aureus contains straight-chain (SCFAs) and branched-chain (BCFAs) fatty acids in the membrane and alters the proportion of membrane fluidizing BCFAs and stabilizing SCFAs as a response to a variety of stresses. Herein, we describe a method for determination of membrane fluidity in S. aureus using diphenylhexatriene, one of the most used fluorescent dyes for this purpose.


Assuntos
Difenilexatrieno/química , Ácidos Graxos/análise , Corantes Fluorescentes/química , Staphylococcus aureus/crescimento & desenvolvimento , Membrana Externa Bacteriana/química , Ácidos Graxos/química , Polarização de Fluorescência , Bicamadas Lipídicas/química , Fluidez de Membrana , Espectrometria de Fluorescência , Staphylococcus aureus/química
9.
Phys Chem Chem Phys ; 23(28): 15127-15137, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34254086

RESUMO

Cations play a critical role in the stability and morphology of lipid-A aggregates by neutralizing, hydrating and cross-linking these glycolipid molecules. Monophosphorylated lipid-A is the major immunostimulatory principle in commercially available adjuvants containing Al3+ such as adjuvant system 04 (AS04). The antagonist/agonist immunomodulatory properties of lipid-A are associated with chemical variations (e.g. the number of acyl chains and phosphate groups) and their aggregate arrangements (e.g. lamellar, nonlamellar or mixed). Therefore, the identification of the active form of lipid-A can provide valuable guidance in the development of vaccine adjuvants capable of boosting the immune system with decreased reactogenicity. Although the effect of mono and divalent cations on the structural polymorphism and endotoxicity of LPS has been previously investigated, much less is known about the effect of trivalent cations. We have investigated the effect of NaCl and AlCl3 salt solutions on the structural dynamics and stability of mono and diphosphorylated lipid-A membranes via atomistic MD simulations. The Al3+ ion exerts two major effects on the structural dynamics of lipid-A membranes. It acts as an efficient cross-linker of mono or diphosphorylated lipid-A molecules, thus stabilizing the lamellar arrangement of these glycolipids. It also alters the lipid-A packing and membrane fluidity, inducing disorder → order structural transitions of the membrane. This effect is promptly reversed upon the addition of NaCl solution, which promotes a nearly threefold increase in the amount of water in the carbohydrate moiety of the Al3+-containing lipid-A membranes. The exchange dynamics and residence times of cation-coordinated water molecules in these membranes provide insights into the molecular mechanism for the Na+-induced transition from a densely packed ordered phase to a disordered one. Al3+ counter-ions favor ordered lamellar aggregates, which has been previously associated with the lack of endotoxic activity and cytokine-inducing action. The resulting microscopic understanding of the structure and dynamics of lipid-A aggregates in the presence of Al3+ and Na+ salts can provide valuable guidance in the development of vaccine adjuvants capable of boosting the immune system with decreased reactogenicity.


Assuntos
Alumínio/química , Reagentes para Ligações Cruzadas/química , Lipídeo A/química , Bicamadas Lipídicas/química , Sódio/química , Cátions/química , Cristalização , Cinética , Fluidez de Membrana , Conformação Molecular , Simulação de Dinâmica Molecular , Transição de Fase , Relação Estrutura-Atividade , Água/química
10.
ACS Appl Mater Interfaces ; 13(25): 29936-29948, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34143617

RESUMO

Alzheimer's disease (AD) is a major cause of dementia characterized by the overexpression of transmembrane amyloid precursor protein and its neurotoxic byproduct amyloid beta (Aß). A small peptide of considerable hydrophobicity, Aß is aggregation prone catalyzed by the presence of cell membranes, among other environmental factors. Accordingly, current AD mitigation strategies often aim at breaking down the Aß-membrane communication, yet no data is available concerning the cohesive interplay of the three key entities of the cell membrane, Aß, and its inhibitor. Using a lipophilic Laurdan dye and confocal fluorescence microscopy, we observed cell membrane perturbation and actin reorganization induced by Aß oligomers but not by Aß monomers or amyloid fibrils. We further revealed recovery of membrane fluidity by ultrasmall MoS2 quantum dots, also shown in this study as a potent inhibitor of Aß amyloid aggregation. Using discrete molecular dynamics simulations, we uncovered the binding of MoS2 and Aß monomers as mediated by hydrophilic interactions between the quantum dots and the peptide N-terminus. In contrast, Aß oligomers and fibrils were surface-coated by the ultrasmall quantum dots in distinct testudo-like, reverse protein-corona formations to prevent their further association with the cell membrane and adverse effects downstream. This study offers a crucial new insight and a viable strategy for regulating the amyloid aggregation and membrane-axis of AD pathology with multifunctional nanomedicine.


Assuntos
Peptídeos beta-Amiloides , Dissulfetos/química , Fluidez de Membrana/fisiologia , Molibdênio/química , Pontos Quânticos/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Actinas/química , Actinas/metabolismo , Doença de Alzheimer , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lauratos/química , Microscopia Confocal , Simulação de Dinâmica Molecular , Nanomedicina
11.
Commun Biol ; 4(1): 653, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079059

RESUMO

It has been proposed that adaptation to high temperature involved the synthesis of monolayer-forming ether phospholipids. Recently, a novel membrane architecture was proposed to explain the membrane stability in polyextremophiles unable to synthesize such lipids, in which apolar polyisoprenoids populate the bilayer midplane and modify its physico-chemistry, extending its stability domain. Here, we have studied the effect of the apolar polyisoprenoid squalane on a model membrane analogue using neutron diffraction, SAXS and fluorescence spectroscopy. We show that squalane resides inside the bilayer midplane, extends its stability domain, reduces its permeability to protons but increases that of water, and induces a negative curvature in the membrane, allowing the transition to novel non-lamellar phases. This membrane architecture can be transposed to early membranes and could help explain their emergence and temperature tolerance if life originated near hydrothermal vents. Transposed to the archaeal bilayer, this membrane architecture could explain the tolerance to high temperature in hyperthermophiles which grow at temperatures over 100 °C while having a membrane bilayer. The induction of a negative curvature to the membrane could also facilitate crucial cell functions that require high bending membranes.


Assuntos
Archaea/química , Archaea/fisiologia , Extremófilos/química , Extremófilos/fisiologia , Lipídeos de Membrana/química , Aclimatação/fisiologia , Ambientes Extremos , Temperatura Alta , Bicamadas Lipídicas/química , Fluidez de Membrana , Lipídeos de Membrana/síntese química , Modelos Moleculares , Estrutura Molecular , Difração de Nêutrons , Permeabilidade , Pressão , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Esqualeno/análogos & derivados , Esqualeno/química , Terpenos/química , Difração de Raios X
12.
J Virol ; 95(17): e0080721, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34106748

RESUMO

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.


Assuntos
Membrana Celular/metabolismo , Fenretinida/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Oxirredutases/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Fusão Celular , Membrana Celular/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Fluidez de Membrana/genética , Oxirredutases/deficiência , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
13.
Aging (Albany NY) ; 13(9): 12817-12832, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33972461

RESUMO

Aging is one of the significant risk factors for Alzheimer's disease (AD). Therefore, this study aimed to propose a new hypothesis "membrane aging" as a critical pathogenesis of AD. The concept of "membrane aging" was reviewed, and the possible mechanisms of membrane aging as the primary culprit of AD were clarified. To further prove this hypothesis, a hydroxyurea-induced "membrane aging" model was established in vitro and in vivo. First, neuronal aging was validated by immunocytochemistry with age-related markers, and membrane aging phenotypes were confirmed. The alterations of membrane fluidity within APP/PS1 mice were re-proved by intracerebroventricular injection of hydroxyurea. Decreased membrane fluidity was found in vitro and in vivo, accompanied by increased total cholesterol concentration in neurons but decreased cholesterol levels within membrane fractions. The Aß level increased considerably after hydroxyurea treatment both in vitro and in vivo. DHA co-treatment ameliorated membrane aging phenotypes and Aß aggregation. The study revealed the AMP-activated protein kinase/acetyl CoA carboxylase/carnitine palmitoyl transferase 1 pathway involved in membrane aging processes. These results strongly supported the idea that membrane aging was a pathogenesis of AD and might serve as a new therapeutic target for AD.


Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Membrana Celular/patologia , Fluidez de Membrana/efeitos dos fármacos , Neurônios/patologia , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/patologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hidroxiureia/administração & dosagem , Hidroxiureia/toxicidade , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Ratos
14.
Artigo em Inglês | MEDLINE | ID: mdl-33991653

RESUMO

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.


Assuntos
Bicamadas Lipídicas/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lítio/farmacologia , Lipídeos de Membrana/metabolismo , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Privação do Sono/metabolismo , Animais , Masculino , Fluidez de Membrana/efeitos dos fármacos , Ratos
15.
Methods Mol Biol ; 2262: 251-258, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977481

RESUMO

Phospholipid fatty acid (FA) composition influences the biophysical properties of the plasma membrane and plays an important role in cellular signaling. Our previous work has demonstrated that plasma membrane fatty acid composition is an important determinant of oncogenic Ras signaling and that dietary (exogenous) modulation of membrane composition may underlie the chemoprotective benefits of long chain n-3 polyunsaturated fatty acids (PUFA). In this chapter, we describe in vitro methods to modulate membrane phospholipid fatty acid composition of cultured cells using fatty acids complexed to bovine serum albumin (BSA). Furthermore, we describe a method to quantify the biophysical properties of plasma membranes in live cells using Di-4-ANEPPDHQ (Di4) and image-based flow cytometry.


Assuntos
Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Fluidez de Membrana , Fosfolipídeos/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Citometria de Fluxo
16.
Sci Total Environ ; 780: 146612, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34030318

RESUMO

Porphyridium cruentum, a cell-wall-free marine Rhodophyta microalga was cultured under a 5-day cold stress at 0 °C and 15 °C, after reaching the late logarithmic growth phase. Compared with the control at 25 °C, the cold stress treatment significantly (p < 0.05) increased the microalgal biomass (1.21-fold); the amounts of total polyunsaturated fatty acids (1.22-fold); individual fatty acids including linoleic acid (1.50-fold) and eicosatrienoic acid (1.85-fold), and a major carotenoid zeaxanthin (1.53-fold). Furthermore, production of biodiesel feedstock including total C16 + C18 fatty acids was significantly enhanced (p < 0.05) by 1.18-fold after the cold stress treatment. Principal component analysis further indicated that the biosynthetic pathways of fatty acids and carotenoids in this microalga were correlated with the cold stress treatment. These results suggested that P. cruentum had adjusted its cellular membrane fluidity via an 'arm-raising and screw-bolt fastening' mechanism mediated by the synergistic roles of cis-unsaturated fatty acids and carotenoids. The insight obtained from the responses to cold stress in P. cruentum could be a novel technological approach to enhance the production of microalgal metabolites and biodiesel feedstock.


Assuntos
Microalgas , Porphyridium , Biocombustíveis , Biomassa , Resposta ao Choque Frio , Ácidos Graxos , Fluidez de Membrana
17.
Phys Chem Chem Phys ; 23(16): 9686-9694, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33908427

RESUMO

The accumulation of toxic soluble oligomers of the amyloid-ß peptide (Aß) is a key step in the pathogenesis of Alzheimer's disease. There are mainly two conformationally distinct oligomers, namely, prefibrillar and fibrillar oligomers, that are recognized by conformation-specific antibodies, anti-amyloid oligomer antibody (A11) and anti-amyloid fibrillar antibody (OC), respectively. Previous studies have shown that the interaction of Aß oligomers with the lipid membrane is one of the key mechanisms of toxicity produced by Aß oligomers. However, the mechanism by which structurally distinct Aß oligomers interact with the lipid membrane remains elusive. In this work, we dissect the molecular mechanism underlying the interaction of structurally distinct Aß42 oligomers with the lipid membrane derived from the brain total lipid extract. Using picosecond time-resolved fluorescence spectroscopy, we show that the A11-positive Aß42 oligomers undergo a membrane-induced conformational change that promotes the deeper immersion of these oligomers into the lipid hydrocarbon region and results in an increase in the membrane micro-viscosity. In sharp contrast, OC-positive Aß42 oligomers interact with the lipid membrane via electrostatic interactions between the negatively-charged lipid headgroup and positively-charged residues of Aß42 without perturbing the membrane dynamics. We show that the two structurally distinct Aß42 oligomers demonstrating different interaction mechanisms with the lipid membrane eventually lead to the formation of typical amyloid fibrils. Our findings provide the mechanistic underpinning of the perturbation of lipid membranes by two conformationally distinct Aß42 oligomers and can be of prime importance in designing anti-Alzheimer's therapeutic agents targeting Aß-membrane interactions.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Lipossomas Unilamelares/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Polarização de Fluorescência , Fluidez de Membrana/efeitos dos fármacos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Lipossomas Unilamelares/química
18.
Arch Microbiol ; 203(6): 3353-3360, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33871675

RESUMO

Listeria monocytogenes is a food-borne pathogen with the ability to grow at low temperatures down to - 0.4 °C. Maintaining cytoplasmic membrane fluidity by changing the lipid membrane composition is important during growth at low temperatures. In Listeria monocytogenes, the dominant adaptation effect is the fluidization of the membrane by shortening of fatty acid chain length. In some strains, however, an additional response is the increase in menaquinone content during growth at low temperatures. The increase of this neutral lipid leads to fluidization of the membrane and thus represents a mechanism that is complementary to the fatty acid-mediated modification of membrane fluidity. This study demonstrated that the reduction of menaquinone content for Listeria monocytogenes strains resulted in significantly lower resistance to temperature stress and lower growth rates compared to unaffected control cultures after growth at 6 °C. Menaquinone content was reduced by supplementation with aromatic amino acids, which led to a feedback inhibition of the menaquinone synthesis. Menaquinone-reduced Listeria monocytogenes strains showed reduced bacterial cell fitness. This confirmed the adaptive function of menaquinones for growth at low temperatures of this pathogen.


Assuntos
Listeria monocytogenes/crescimento & desenvolvimento , Fluidez de Membrana , Vitamina K 2/metabolismo , Aclimatação , Aminoácidos Aromáticos/farmacologia , Temperatura Baixa , Listeria monocytogenes/química , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/metabolismo , Estresse Fisiológico
19.
Biomolecules ; 11(3)2021 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799403

RESUMO

The lipid composition of biomembranes influences the properties of the lipid bilayer and that of the proteins. In this study, the lipidome and the lipid/protein ratio of membranes from High Five™ insect cells overexpressing mouse P-glycoprotein was characterized. This provides a better understanding of the lipid environment in which P-glycoprotein is embedded, and thus of its functional and structural properties. The relative abundance of the distinct phospholipid classes and their acyl chain composition was characterized. A mass ratio of 0.57 ± 0.11 phospholipids to protein was obtained. Phosphatidylethanolamines are the most abundant phospholipids, followed by phosphatidylcholines. Membranes are also enriched in negatively charged lipids (phosphatidylserines, phosphatidylinositols and phosphatidylglycerols), and contain small amounts of sphingomyelins, ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated, with significant amounts of saturated chains. The characterization of the phospholipids by HPLC-MS allowed identification of the combination of acyl chains, with palmitoyl-oleoyl being the most representative for all major phospholipid classes except for phosphatidylserines, which are mostly saturated. A mixture of POPE:POPC:POPS in the ratio 45:35:20 is proposed for the preparation of simple representative model membranes. The adequacy of the model membranes was further evaluated by characterizing their surface potential and fluidity.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Fenômenos Biofísicos , Lipidômica , Animais , Anisotropia , Linhagem Celular , Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Insetos , Fluidez de Membrana , Camundongos , Fosfolipídeos/metabolismo , Espectrometria de Fluorescência
20.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803648

RESUMO

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.


Assuntos
Ácidos Graxos/metabolismo , Corantes Fluorescentes/metabolismo , Espaço Intracelular/metabolismo , Fluidez de Membrana , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Animais , Fluorescência , Lauratos/química , Lisossomos/metabolismo , Células PC12 , Ratos , Solventes , Viscosidade
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