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1.
Eur Biophys J ; 51(4-5): 401-412, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35716178

RESUMO

We investigated the effects of sugar concentration on the electroporation, size distribution and average size of giant unilamellar vesicles (GUVs). GUVs were prepared from 40 mol% of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 60 mol% of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. Pulsed electric field was applied to the 40%DOPG/60%DOPC-GUVs and it induced lateral electric tension (σc) in the membranes of vesicles. The σc-induced probability of rupture (Ppore) and the rate constant of rupture (kp) of GUVs under the sugar concentration, c = 40, 100 and 300 mM, were determined. Both the Ppore and kp increased with the increase of σc, but higher tension was required to generate the same values of Ppore and kp with increasing c. We also investigated average sizes of GUVs from the size distribution of vesicles under various sugar concentrations. With the increase of c, the peak of the size distribution histograms shifted to the region of smaller vesicles. The average size decreased 1.6-fold when c increased from 10 to 300 mM. These investigations help to understand various biomedical, biophysical, and biochemical processes in vesicles and cells. Electroporation, size distribution and average size of charged GUVs were investigated under various sugar concentrations. The sugar concentration influences the electroporation of vesicles and the average size of GUVs.


Assuntos
Fosfatidilcolinas , Lipossomas Unilamelares , Eletricidade , Eletroporação , Açúcares
2.
J Phys Chem B ; 126(24): 4491-4500, 2022 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-35687481

RESUMO

The intrinsically disordered C-terminus of the prominent oncogenic protein KRAS-4B (KRAS) selectively interacts and clusters with phosphatidylserine (PS) lipids in the plasma membrane (PM). This 11-residue segment, called tK, contains a polybasic domain (PBD) of six contiguous lysine residues and a farnesylated cysteine. Previous molecular dynamics (MD) simulation studies of tK in phosphatidylcholine (PC)/PS bilayers have suggested that backbone conformational dynamics modulate tK-PS interactions. These simulations have been conducted in symmetric membranes whereas the PM is compositionally asymmetric, with the inner leaflet, where KRAS is localized, being enriched with PS and phosphatidylethanolamine (PE) lipids. To examine if bilayer asymmetry affects tK conformational dynamics and interaction with lipids, we conducted two 10 µs long MD simulations of tK bound to a PC/PS and a PC/PS/PE bilayer in which the PS and PE lipids are distributed in one leaflet. We found that, first, these compositional asymmetries caused differences in acyl chain dynamics between leaflets, but the equilibrium structural and dynamic properties of the two asymmetric bilayers are similar; second, in both systems tK is highly dynamic and samples at least two distinct conformational states; third, PS-tK hydrogen-bonding interactions vary with peptide backbone conformations, and lysine side chains in the PBD predominantly interact with the serine oxygens of PS. These results are in good agreement with previous observations of tK in symmetric membranes. The effects of POPS asymmetry or the presence of POPE on tK are limited to modulating the relative contribution of individual side chains to interactions with lipids and redistributing conformational substates. Additional observations include the larger flexibility of tK in the current simulations, which we attribute to the longer duration of the simulations and the use of the CHARMM36m force field, which more accurately models intrinsically disordered peptides such as tK.


Assuntos
Fosfatidiletanolaminas , Fosfatidilserinas , Bicamadas Lipídicas/química , Lisina , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
3.
J Chem Phys ; 156(23): 234706, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35732527

RESUMO

Biomembrane hydration is crucial for understanding processes at biological interfaces. While the effect of the lipid headgroup has been studied extensively, the effect (if any) of the acyl chain chemical structure on lipid-bound interfacial water has remained elusive. We study model membranes composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids, the most abundant lipids in biomembranes. We explore the extent to which the lipid headgroup packing and associated water organization are affected by the lipid acyl tail unsaturation and chain length. To this end, we employ a combination of surface-sensitive techniques, including sum-frequency generation spectroscopy, surface pressure measurements, and Brewster angle microscopy imaging. Our results reveal that the acyl tail structure critically affects the headgroup phosphate orientational distribution and lipid-associated water molecules, for both PE and PC lipid monolayers at the air/water interface. These insights reveal the importance of acyl chain chemistry in determining not only membrane fluidity but also membrane hydration.


Assuntos
Fosfatidilcolinas , Fosfolipídeos , Bicamadas Lipídicas/química , Fluidez de Membrana , Fosfatidilcolinas/química , Fosfolipídeos/química , Água/química
4.
Anal Chim Acta ; 1215: 339979, 2022 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-35680341

RESUMO

Metabolomics-based precision medicine is facing several obstacles including cross-platform data comparison issue and the lack of metabolome benchmark values of healthy population, one of main reasons is the shortage of comprehensive metabolome quantitation methods. Here, we developed an alternate reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) method to quantitatively determine metabolites and lipids. Assisted by a wide set of reference standards and real samples, up to 397 multiple reaction monitoring (MRM) transitions (239 for positive and 158 for negative ion modes) and 1080 MRM transitions (607 for positive and 473 for negative ion modes) were defined respectively in the metabolomic and lipidomic analyses with more than 1000 metabolites and lipids being quantified. Among them, 144 analytes including amines, amino acids, benzenoids, peptides, nucleobases and related, bile acids, carboxylic acids, fatty acids, hormones, indoles and others were absolutely quantified, while carnitines, lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, free fatty acids, sphingomyelins, phosphatidylcholines (PCs), alkyl and alkenyl substituted PCs, phosphatidylethanolamines (PEs), alkyl and alkenyl substituted PEs and triacylglycerols were semiquantified. The developed method was validated to have good analytical characteristics. Analytical results of standard reference material 1950 human plasma had a good agreement with literature data. As a proof of application, this method was used to study serum metabolic pattern changes of patients with hyperuricemia and nonalcoholic fatty liver. This alternate RPLC-MS method for quantitative metabolites and lipids analysis can further be used to provide technology and large-scale data support for precision medicine and life sciences.


Assuntos
Lipidômica , Fosfatidiletanolaminas , Aminas , Humanos , Metaboloma , Metabolômica/métodos , Fosfatidilcolinas
5.
Eur Phys J E Soft Matter ; 45(6): 55, 2022 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-35748998

RESUMO

The effects of the hydrocarbon chain of lipids on the size distribution of giant unilamellar vesicles (GUVs), kinetics of average size, bending modulus, and elastic modulus of membranes have been investigated. 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 (Δ9-Cis) PC (DOPC)), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16:1 (Δ9-Cis) PC), and 1,2-ditridecanoyl-sn-glycerol-3-phosphocholine (13:0 PC (DTPC)) lipids were considered. The number of hydrocarbons in a chain of the corresponding lipid was 18, 16, and 13. GUVs were prepared using the natural swelling method under incubation times of 20, 40, 60, 90, 120, and 180 min. The size distribution of vesicles was fitted using the lognormal distribution. The average sizes of DOPC, 16:1 (Δ9-Cis) PC, and DTPC-GUVs increased with the incubation time until 120 min, and then remained steady at 16.7 ± 0.2, 15.2 ± 0.4 and 12.0 ± 0.3 µm for the corresponding lipids. The average size at equilibrium state increased with the number of hydrocarbons. The incubation time-dependent average size was fitted with an exponential growth equation, and then the kinetic constants of 0.028 ± 0.004, 0.036 ± 0.007, and 0.083 ± 0.009 min-1 for DOPC, 16:1 (Δ9-Cis) PC, and DTPC-GUVs, respectively, were obtained. The equilibrium size distribution was fitted by the theoretical equation, and the bending modulus for DOPC, 16:1 (Δ9-Cis) PC, and DTPC membranes were 19.5 ± 0.2, 18.5 ± 0.1 and 14.3 ± 0.1 kBT, respectively. The bending modulus increased with the number of hydrocarbons. The elastic modulus of these membranes was 261 mN/m with a 4% fluctuation. The correlation between the average size and the square root of the bending modulus was supported by theoretical analysis.


Assuntos
Hidrocarbonetos , Lipossomas Unilamelares , Módulo de Elasticidade , Cinética , Lipídeos , Fosfatidilcolinas
6.
Nutrients ; 14(12)2022 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-35745225

RESUMO

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole-linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18-104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.


Assuntos
Córtex Motor , Fosfolipídeos , Envelhecimento , Cerebelo , Humanos , Fosfatidilcolinas , Fosfatidilserinas
7.
Artigo em Inglês | MEDLINE | ID: mdl-35737517

RESUMO

A new actinobacterium strain, designated BMG 823T, was isolated from a limestone sample collected in Tunisia. Its taxonomic position was scrutinized using a polyphasic approach. Colonies of strain BMG 823T were pink orange-coloured, regular and had a moist surface. Cells are Gram-stain-positive, catalase-negative and oxidase-negative. The strain grew at pH 5.5-9, 10-40 °C and in presence of up to 4 % NaCl (w/v). Chemotaxonomically, strain BMG 823T was characterized by cell-wall type III containing meso-diaminopimelic acid as diamino acid, glucose, ribose and rhamnose as whole-cell sugars, MK-9(H4) as predominant menaquinone, and phosphatidylcholine, diphosphadidylglycerol, phosphatidethanolamine, phosphatidylcholine, phosphatidylinositol, unidentified glycolipid, unidentified aminophospholipids and unidentified glycophospholipid as major polar lipids. The fatty acid profile consisted of iso-C16 : 0 and iso-C17 : 1 ω9. Phylogenetic trees based on 16S rRNA gene and genome sequences placed strain BMG 823T within the genus Blastococcus and separated it from all type strains of validly published species. Comparison of 16S rRNA gene sequence similarity, digital DNA-DNA hybridization and average nucleotide identity indicated that strain BMG 823T was most closely related to Blastococcus litoris DSM 106127T and Blastococcus colisei BMG 822T with pairwise values well below the species differentiation thresholds. The distinct phenotypic and genotypic features of strain BMG 823T (=DSM 46838T=CECT 8881T) within the genus Blastococcus warrant its recognition as the type strain for the new species for which we propose the name Blastococcus tunisiensis sp. nov.


Assuntos
Actinomycetales , Carbonato de Cálcio , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Fosfatidilcolinas , Fosfolipídeos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Tunísia
8.
Int J Mol Sci ; 23(12)2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35743227

RESUMO

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated "omics" approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the "lysophosphatidylcholines to phosphatidylcholines" and "cholesteryl ester to free cholesterol" ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated "omics" approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.


Assuntos
Resistência à Insulina , Síndrome Metabólica , Colesterol/metabolismo , HDL-Colesterol , Humanos , Lipidômica , Lipoproteínas , Síndrome Metabólica/complicações , Síndrome Metabólica/diagnóstico , Obesidade/complicações , Obesidade Abdominal/complicações , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Fosfatidilcolinas
9.
FASEB J ; 36(7): e22371, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35704337

RESUMO

Untargeted metabolomics of blood samples has become widely applied to study metabolic alterations underpinning disease and to identify biomarkers. However, understanding the relevance of a blood metabolite marker can be challenging if it is unknown whether it reflects the concentration in relevant tissues. To explore this field, metabolomic and lipidomic profiles of plasma, four sites of adipose tissues (ATs) from peripheral or central depot, two sites of muscle tissue, and liver tissue from a group of nondiabetic women with obesity who were scheduled to undergo bariatric surgery (n = 21) or other upper GI surgery (n = 5), were measured by liquid chromatography coupled with mass spectrometry. Relationships between plasma and tissue profiles were examined using Pearson correlation analysis subject to Benjamini-Hochberg correction. Plasma metabolites and lipids showed the highest number of significantly positive correlations with their corresponding concentrations in liver tissue, including lipid species of ceramide, mono- and di-hexosylceramide, sphingomyelin, phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine, dimethyl phosphatidylethanolamine, ether-linked PC, ether-linked PE, free fatty acid, cholesteryl ester, diacylglycerol and triacylglycerol, and polar metabolites linked to several metabolic functions and gut microbial metabolism. Plasma also showed significantly positive correlations with muscle for several phospholipid species and polar metabolites linked to metabolic functions and gut microbial metabolism, and with AT for several triacylglycerol species. In conclusion, plasma metabolomic and lipidomic profiles were reflective more of the liver profile than any of the muscle or AT sites examined in the present study. Our findings highlighted the importance of taking into consideration the metabolomic relationship of various tissues with plasma when postulating plasma metabolites marker to underlying mechanisms occurring in a specific tissue.


Assuntos
Metaboloma , Fosfatidiletanolaminas , Biomarcadores/metabolismo , Éteres/metabolismo , Feminino , Humanos , Fígado/metabolismo , Metabolômica/métodos , Músculos/metabolismo , Obesidade/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Triglicerídeos/metabolismo
10.
Cell Rep ; 39(11): 110899, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35705027

RESUMO

B1 B cells reactive to phosphatidyl choline (PtC) exhibit restricted immunoglobulin heavy chain (HC) and light chain (LC) combinations, exemplified by VH12/Vκ4/5H. Two checkpoints are thought to focus PtC+ B cell maturation in VH12-transgenic mice (VH12 mice): V-J rearrangements encoding a "permissive" LC capable of VH12 HC pairing are selected first, followed by positive selection based on PtC binding, often requiring LC receptor editing to salvage PtC- B cells and acquire PtC reactivity. However, evidence obtained from breeding VH12 mice to editing-defective dnRAG1 mice and analyzing LC sequences from PtC+ and PtC- B cell subsets instead suggests that receptor editing functions after initial positive selection to remove PtC+ B cells in VH12 mice. This offers a mechanism to constrain natural, polyreactive B cells to limit their frequency. Sequencing also reveals occasional in-frame hybrid LC genes, reminiscent of type 2 gene replacement, that, testing suggests, arise via a recombination-activating gene (RAG)-independent mechanism.


Assuntos
Região Variável de Imunoglobulina , Fosfatidilcolinas , Animais , Linfócitos B , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Transgênicos , Baço
11.
Langmuir ; 38(24): 7545-7557, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35671406

RESUMO

Molecular dynamics (MD) simulations in the MARTINI model are used to study the assembly of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules under spatial confinement, such as during solvent evaporation from ultrasmall (femtoliter quantity) droplets. The impact of surface polarity on molecular assembly is discussed in detail. To the best of our knowledge, this work represents the first of its kind. Our results reveal that solvent evaporation gives rise to the formation of well-defined stacks of lipid bilayers in a smectic alignment. These smectic mesophases form on both polar and nonpolar surfaces but with a notable distinction. On polar surfaces, the director of the stack is oriented perpendicular to the support surface. By contrast, the stacks orient at an angle on the nonpolar surfaces. The packing of head groups on surfaces and lipid molecular mobility exhibits significant differences as surface polarity changes. The role of glycerol in the assembly and stability is also revealed. The insights revealed from the simulation have a significant impact on additive manufacturing, biomaterials, model membranes, and engineering protocells. For example, POPC assemblies via evaporation of ultrasmall droplets were produced and characterized. The trends compare well with the bilayer stack models. The surface polarity influences the local morphology and structures at the interfaces, which could be rationalized via the molecule-surface interactions observed from simulations.


Assuntos
Bicamadas Lipídicas , Fosfatidilcolinas , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Solventes
12.
J Org Chem ; 87(12): 8194-8197, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35649118

RESUMO

Lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) are important membrane constituents implicated in signaling and immune regulation. Synthesis of LPCs is challenging due to rapid acyl migration, e.g., induced by chromatography. We here report a highly regioselective synthesis of LPC and mixed PC via an intermediate allowing specific terminal acyl introduction, yielding the pure LPC without chromatography by an exceedingly mild TBS deprotection, using 1 equiv of TFA in aqueous solution. The method enabled the synthesis of glycerol-, acyl-, and choline-labeled LPC.


Assuntos
Lisofosfatidilcolinas , Fosfatidilcolinas , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/farmacologia , Fosfatidilcolinas/química , Água
13.
J Colloid Interface Sci ; 624: 579-592, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-35690012

RESUMO

Rhamnolipids (RLs) are biosurfactants with significant tensioactive and emulsifying properties. They are mainly composed by mono-RL and di-RL components. Although there are numerous studies concerning their molecular properties, information is scarce regarding the mechanisms by which each of the two components interacts with cell membranes. Herein, we performed phase-contrast and fluorescence microscopy experiments on plasma membrane models represented by giant-unilamellar-vesicles (GUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-[[(E,2S,3R)-1,3-dihydroxy-2-(octadecanoylamino) octadec-4-enyl]peroxy-hydroxyphosphoryl]oxyethyl-trimethylazanium (sphingomyelin, SM) and (3ß)-cholest-5-en-3-ol (cholesterol, CHOL) (1:1:1 M ratio), which present liquid-order (Lo) liquid-disorder (Ld) phase coexistence, in the presence of either mono-RL or di-RL in 0.06-0.25 mM concentration range. A new method has been developed to determine area and volume of GUVs with asymmetrical shape and a kinetic model describing GUV-RL interaction in terms of two mechanisms, RL-insertion and pore formation, has been worked out. Results show that the insertion of mono-RL in the membrane outer leaflet is the dominant process with no pore formation and a negligible effect in modifying membrane permeability, but induces lipid mixing. Conversely, the di-RL-GUV interaction begins with the insertion mechanism and, as the time passes by, the pore formation process occurs. The analyses of di-RL show that the whole process is only relevant in the Ld phase with a higher extent to 0.25 mM than to 0.06 mM.


Assuntos
Esfingomielinas , Lipossomas Unilamelares , Membrana Celular , Decanoatos , Glicolipídeos , Bicamadas Lipídicas , Fosfatidilcolinas , Ramnose/análogos & derivados
14.
Front Immunol ; 13: 830788, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35663973

RESUMO

A strategy adopted to combat human immunodeficiency virus type-1 (HIV-1) infection is based on interfering with virus entry into target cells. In this study, we found that phosphatidylcholine (PC) liposomes reduced the expression of the CD4 receptor in human primary type-1 macrophages but not in CD4+ T cells. The down-regulation was specific to CD4, as any effect was not observed in CCR5 membrane expression. Moreover, the reduction of membrane CD4 expression required the Ca2+-independent protein kinase C (PKC), which in turn mediated serine phosphorylation in the intracytoplasmic tail of the CD4 receptor. Serine phosphorylation of CD4 was also associated with its internalization and degradation in acidic compartments. Finally, the observed CD4 downregulation induced by PC liposomes in human primary macrophages reduced the entry of both single-cycle replication and replication competent R5 tropic HIV-1. Altogether, these results show that PC liposomes reduce HIV entry in human macrophages and may impact HIV pathogenesis by lowering the viral reservoir.


Assuntos
Infecções por HIV , HIV-1 , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , HIV-1/fisiologia , Humanos , Lipossomos , Macrófagos/metabolismo , Fosfatidilcolinas/farmacologia , Serina
15.
Mol Med Rep ; 26(2)2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35686555

RESUMO

Formulation of cationic liposomes is a key factor that determine the gene knockdown efficiency by cationic liposomes/siRNA complexes (siRNA lipoplexes). Here, to determine the optimal combination of cationic lipid and phospholipid in cationic liposomes for in vitro and in vivo gene knockdown using siRNA lipoplexes, three types of cationic lipid were used, namely 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB) and 11-[(1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino]-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12). Thereafter, 30 types of cationic liposome composed of each cationic lipid with phosphatidylcholine or phosphatidylethanolamine containing saturated or unsaturated dialkyl chains (C14, C16, or C18) were prepared. The inclusion of phosphatidylethanolamine containing unsaturated and long dialkyl chains with DOTAP- or DDAB-based cationic liposomes induced strong luciferase gene knockdown in human breast cancer MCF-7-Luc cells stably expressing luciferase gene. Furthermore, the inclusion of phosphatidylcholine or phosphatidylethanolamine containing saturated and short dialkyl chains or unsaturated and long dialkyl chains into TC-1-12-based cationic liposomes resulted in high gene knockdown efficacy. When cationic liposomes composed of DDAB/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), TC-1-12/DOPE and TC-1-12/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine were used, significant gene knockdown occurred in the lungs of mice following systemic injection of siRNA lipoplexes. Overall, the present findings indicated that optimal phospholipids in cationic liposome for in vitro and in vivo siRNA transfection were affected by the types of cationic lipid used.


Assuntos
Técnicas de Silenciamento de Genes , Lipossomos , Animais , Cátions , Feminino , Humanos , Luciferases , Pulmão , Células MCF-7 , Camundongos , Fosfatidilcolinas , Fosfatidiletanolaminas , Fosfolipídeos , RNA Interferente Pequeno/genética , Transfecção
16.
Langmuir ; 38(22): 6798-6807, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35608952

RESUMO

Lipid oxidation has significant effects on lipid bilayer properties; these effects can be expected to extend to interactions between the lipid bilayer and integral membrane proteins. Given that G protein-coupled receptor (GPCR) activity is known to depend on the properties of the surrounding lipid bilayer, these proteins represent an intriguing class of molecules in which the impact of lipid oxidation on protein behavior is studied. Here, we study the effects of lipid oxidation on the human serotonin 1A receptor (5-HT1AR). Giant unilamellar vesicles (GUVs) containing integral 5-HT1AR were fabricated by the hydrogel swelling method; these GUVs contained polyunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinPC) and its oxidation product 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) at various ratios. 5-HT1AR-integrated GUVs were also fabricated from lipid mixtures that had been oxidized by extended exposure to the atmosphere. Both types of vesicles were used to evaluate 5-HT1AR activity using an assay to quantify GDP-GTP exchange by the coupled G protein α subunit. Results indicated that 5-HT1AR activity increases significantly in bilayers containing oxidized lipids. This work is an important step in understanding how hyperbaric oxidation can change plasma membrane properties and lead to physiological dysfunction.


Assuntos
Bicamadas Lipídicas , Lipídeos de Membrana , Receptor 5-HT1A de Serotonina , Humanos , Bicamadas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos de Membrana/metabolismo , Oxirredução , Fosfatidilcolinas , Receptor 5-HT1A de Serotonina/metabolismo , Serotonina , Lipossomas Unilamelares/síntese química
17.
Biomolecules ; 12(5)2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35625563

RESUMO

Phospholipase D reacts with alcohols or water, transphosphatidylating or hydrolysing lipids such as phosphatidylcholine, generating phosphatidylalcohols or phosphatidic acid, respectively. The enzyme has been employed in many applications making use of the transphosphatidylation reaction and the enzyme's tolerance for organic solvents in order to synthesize natural and artificial phospholipids. Yet, its catalytic properties with respect to the transphosphatidylation reaction are not well understood. Here, we introduce a novel high-throughput assay, making use of 96-well plates, that employs Fluorescamine for the detection of transphosphatidylated amino alcohols. This assay allowed to monitor the KM and VMax at different temperatures, revealing that the former will be elevated by the temperature, while the latter is increased by a combination of both temperature and alcohol acceptor concentration being elevated, suggesting that increase in temperature may open up a new binding site for the alcohol acceptor.


Assuntos
Fosfolipase D , Etanol , Fosfatidilcolinas , Fosfolipase D/metabolismo , Solventes , Temperatura
18.
Langmuir ; 38(20): 6411-6424, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35561255

RESUMO

Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine's influence on the cell membrane, including permeability, binding, and rearrangement at the molecular level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as in vitro models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid composition on the membrane interactions of quinacrine. Using electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Our EIS data reveal that the drug is permeable through ternary DOPC:SM:Chol and DOPC-only bilayer compositions. In contrast, the binary cholesterol/DOPC membrane arrested permeation, yet the drug binds or intercalates at this membrane as reflected by an increase in membrane impedance. SERS supported the EIS data, which was utilized to gain structural insights into the drug-membrane interaction. Our SERS data also provides a simple but powerful label-free assessment of drug permeation because a significant SERS enhancement of the drug's Raman signature was observed only if the drug accessed the plasmonic interior of the pore cavity passing through the membrane. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophysical insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug-membrane interactions, making it a powerful tool for in vitro drug screening.


Assuntos
Bicamadas Lipídicas , Quinacrina , Membrana Celular/metabolismo , Colesterol/química , Espectroscopia Dielétrica , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Quinacrina/farmacologia
19.
Adv Exp Med Biol ; 1372: 77-86, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35503176

RESUMO

The sphingomyelin synthase (SMS) gene family has three members: SMS1 and SMS2 have SM synthase activity, while SMS-related protein (SMSr) has no SM synthase activity but has ceramide phosphorylethanolamine (CPE) synthase activity in vitro. Recently, we found that SMS family members are a group of phospholipase Cs (PLC). SMS1 and SMS2 are two phosphatidylcholine (PC)-PLCs and SMSr is a phosphatidylethanolamine (PE)-PLC. SMS family members not only influence SM levels but also influence the levels of diacylglycerol (DAG), PC, PE, and glycosphingolipids, thus influencing cell functions. In this chapter, we will discuss the recent progress in the research field of SMS family and will focus on its impact on metabolic diseases.


Assuntos
Fosfolipases , Esfingomielinas , Fosfatidilcolinas/metabolismo , Esfingomielinas/genética , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Fosfolipases Tipo C/metabolismo
20.
Front Endocrinol (Lausanne) ; 13: 831793, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35498422

RESUMO

Introduction: Diabetic cardiovascular autonomic neuropathy (CAN) is associated with increased mortality and morbidity. To explore metabolic mechanisms associated with CAN we investigated associations between serum metabolites and CAN in persons with type 1 diabetes (T1D). Materials and Methods: Cardiovascular reflex tests (CARTs) (heart rate response to: deep breathing; lying-to-standing test; and the Valsalva maneuver) were used to diagnose CAN in 302 persons with T1D. More than one pathological CARTs defined the CAN diagnosis. Serum metabolomics and lipidomic profiles were analyzed with two complementary non-targeted mass-spectrometry methods. Cross-sectional associations between metabolites and CAN were assessed by linear regression models adjusted for relevant confounders. Results: Participants were median (IQR) aged 55(49, 63) years, 48% males with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had CAN. A total of 75 metabolites and 106 lipids were analyzed. In crude models, the CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4-deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and the amino acid methionine (p<0.05). Upon adjustment, positive associations with the CAN diagnoses were retained for hydroxy fatty acids, tricarboxylic acid (TCA) cycle-based sugar derivates, citric acid, and phenols (P<0.05). Conclusion: Metabolic pathways, including the TCA cycle, hydroxy fatty acids, phosphatidylcholines and sugar derivatives are associated with the CAN diagnosis in T1D. These pathway may be part of the pathogeneses leading to CAN and may be modifiable risk factors for the complication.


Assuntos
Diabetes Mellitus Tipo 1 , Neuropatias Diabéticas , Ácido Cítrico , Estudos Transversais , Diabetes Mellitus Tipo 1/complicações , Neuropatias Diabéticas/complicações , Neuropatias Diabéticas/etiologia , Ácidos Graxos , Feminino , Glucose , Humanos , Masculino , Fenóis , Fosfatidilcolinas , Açúcares
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