RESUMO
Melting of brain sphingomyelin (bSM) manifests as a broad feature in the DSC curve that encompasses the temperature range of 25 - 45 °C, with two distinguished maxima originating from the phase transitions of two the most abundant components: C24:1 (Tm,1) and C18:0 (Tm,2). While C24:1/C18:0 sphingomyelin transforms from the gel/ripple phase to the fluid/fluid phase, the dynamics of water molecules in the interfacial layer remain completely unknown. Therefore, we carried out a calorimetric (DSC), spectroscopic (temperature-dependent UV-Vis and fluorescence) and MD simulation study of bSM in the absence/presence of Laurdan® (bSM ± L) suspended in Britton-Robinson buffer with three different pH values, 4 (BRB4), 7 (BRB7) and 9 (BRB9), and of comparable ionic strength (I = 100â¯mM). According to DSC, TÌ m, 1 (≈ 34.5 °C/≈ 32.1 °C) and TÌ m, 2 (≈ 38.0 °C/≈ 37.2 °C) of bSM suspended in BRB4, BRB7, and BRB9 in the absence/presence of Laurdan® are found to be practically pH-independent. Turbidity-based data (UV-Vis) detected both qualitative and quantitative differences in the response of bSM suspended in BRB4/BRB7/BRB9 (TÌ m: â¼ 35 °C/32.0 ± 0.2 °C/36.4 ± 0.4), suggesting an intricate interplay of weakening of van der Waals forces between their hydrocarbon chains and of increased hydration in the polar headgroups region during melting. The temperature-dependent response of Laurdan® reported a discontinuous, pH-dependent change in the reorientation of interfacial water molecules that coincides with the melting of C24:1 lipids (on average, TÌ m (LTC/HTC): ≈ 31.8 °C/30.6 °C/30.5 °C). MD simulations elucidated the impact of Laurdan® on a change in the physicochemical properties of bSM lipids and characterized the hydrogen bond network at the interface at 20 °C and 50 °C.
Assuntos
Encéfalo , Simulação de Dinâmica Molecular , Transição de Fase , Esfingomielinas , Água , Esfingomielinas/química , Água/química , Encéfalo/metabolismo , Varredura Diferencial de Calorimetria , Concentração de Íons de Hidrogênio , Lauratos/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/químicaRESUMO
ConspectusLight is ubiquitously available to probe the structure and dynamics of biomolecules and biological tissues. Generally, this cannot be done directly with visible light, because of the absence of absorption by those biomolecules. This problem can be overcome by incorporating organic molecules (chromophores) that show an optical response in the vicinity of those biomolecules. Since those optical properties are strongly dependent on the chromophore's environment, time-resolved spectroscopic studies can provide a wealth of information on biosystems at the molecular scale in a nondestructive way. In this work, we give an overview on the multiscale computational strategy developed by us in the last eight years and prove that theoretical studies and simulations are needed to explain, guide, and predict observations in fluorescence experiments. As we challenge the accepted views on existing probes, we discover unexplored abilities that can discriminate surrounding lipid bilayers and their temperature-dependent as well as solvent-dependent properties. We focus on three archetypal chromophores: diphenylhexatriene (DPH), Laurdan, and azobenzene. Our method shows that conformational changes should not be neglected for the prototype rod-shaped molecule DPH. They determine its position and orientation in a liquid-ordered (Lo) sphingomyelin/cholesterol (SM/Chol) bilayer and are responsible for a strong differentiation of its absorption spectra and fluorescence decay times in dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) membranes, which are at room temperature in liquid-disordered (Ld) and solid-gel (So) phases, respectively. Thanks to its pronounced first excited state dipole moment, Laurdan has long been known as a solvatochromic probe. Since this molecule has however two conformers, we prove that they exhibit different properties in different lipid membrane phases. We see that the two conformers are only blocked in one phase but not in another. Supported by fluorescence anisotropy decay simulations, Laurdan can therefore be regarded as a molecular rotor. Finally, the conformational versatility of azobenzene in saturated Ld lipid bilayers is simulated, along with its photoisomerization pathways. By means of nonadiabatic QM/MM surface hopping analyses (QM/MM-SH), a dual mechanism is found with a torsional mechanism and a slow conversion for trans-to-cis. For cis-to-trans, simulations show a much higher quantum yield and a so-called "pedal-like" mechanism. The differences are related to the different potential energy surfaces as well as the interactions with the surrounding alkyl chains. When tails of increased length are attached to this probe, cis is pushed toward the polar surface, while trans is pulled toward the center of the membrane.
Assuntos
Compostos Azo , Bicamadas Lipídicas , Bicamadas Lipídicas/química , Compostos Azo/química , Difenilexatrieno/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Lauratos/química , Simulação de Dinâmica MolecularRESUMO
The widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers' optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations.
Assuntos
1,2-Dipalmitoilfosfatidilcolina , 2-Naftilamina , Lauratos , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Espectrometria de Fluorescência , Temperatura , Água , 1,2-Dipalmitoilfosfatidilcolina/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Lauratos/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Água/química , Polarização de FluorescênciaRESUMO
Hyperspectral imaging is a technique that captures a three-dimensional array of spectral information at each spatial location within a sample, enabling precise characterization and discrimination of biological structures, materials, and chemicals, based on their unique spectral features. Nowadays most commercially available confocal microscopes allow hyperspectral imaging measurements, providing a valuable source of spatially resolved spectroscopic data. Spectral phasor analysis quantitatively and graphically transforms the fluorescence spectra at each pixel of a hyperspectral image into points in a polar plot, offering a visual representation of the spectral characteristics of fluorophores within the sample. Combining the use of environmentally sensitive dyes with phasor analysis of hyperspectral images provides a powerful tool for measuring small changes in lateral membrane heterogeneity. Here, we focus on applications of spectral phasor analysis for the probe LAURDAN on model membranes to resolve packing and hydration. The method is broadly applicable to other dyes and to complex systems such as cell membranes.
Assuntos
Corantes Fluorescentes , Espectrometria de Fluorescência , Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodos , Microscopia Confocal/métodos , Lauratos/química , Membrana Celular/química , Membrana Celular/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Bicamadas Lipídicas/químicaRESUMO
2-Hydroxyoleic acid (2-OHOA) has gained attention as a membrane lipid therapy (MLT) anti-cancer drug. However, in the viewpoint of anti-cancer drug, 2-OHOA shows poor water solubility and its effectiveness still has space for improvement. Thus, this study aimed to overcome the problems by formulating 2-OHOA into liposome dosage form. Furthermore, in the context of MLT reagents, the influence of 2-OHOA on the biophysical properties of the cytoplasmic membrane remains largely unexplored. To bridge this gap, our study specifically focused the alterations in cancer cell membrane fluidity and lipid packing characteristics before and after treatment. By using a two-photon microscope and the Laurdan fluorescence probe, we noted that liposomes incorporating 2-OHOA induced a more significant reduction in cancer cell membrane fluidity, accompanied by a heightened rate of cellular apoptosis when compared to the non-formulated 2-OHOA. Importantly, the enhanced efficacy of 2-OHOA within the liposomal formulation demonstrated a correlation with its endocytic uptake mechanism. In conclusion, our findings underscore the significant influence of 2-OHOA on the biophysical properties of cancer plasma membranes, emphasizing the potential of liposomes as an optimized delivery system for 2-OHOA in anti-cancer therapy.
Assuntos
Membrana Celular , Lipossomos , Fluidez de Membrana , Lipossomos/química , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Apoptose/efeitos dos fármacos , Lauratos/química , Microscopia de Fluorescência por Excitação Multifotônica , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Ácidos Oleicos/química , Corantes Fluorescentes/químicaRESUMO
Organelles feature characteristic lipid compositions that lead to differences in membrane properties. In cells, membrane ordering and fluidity are commonly measured using the solvatochromic dye Laurdan, whose fluorescence is sensitive to lipid packing. As a general lipophilic dye, Laurdan stains all hydrophobic environments in cells; therefore, it is challenging to characterize membrane properties in specific organelles or assess their responses to pharmacological treatments in intact cells. Here, we describe the synthesis and application of Laurdan-derived probes that read out the membrane packing of individual cellular organelles. The set of organelle-targeted Laurdans (OTL) localizes to the ER, mitochondria, lysosomes, and Golgi compartments with high specificity while retaining the spectral resolution needed to detect biological changes in membrane ordering. We show that ratiometric imaging with OTLs can resolve membrane heterogeneity within organelles as well as changes in lipid packing resulting from inhibition of trafficking or bioenergetic processes. We apply these probes to characterize organelle-specific responses to saturated lipid stress. While the ER and lysosomal membrane fluidity is sensitive to exogenous saturated fatty acids, that of mitochondrial membranes is protected. We then use differences in ER membrane fluidity to sort populations of cells based on their fatty acid diet, highlighting the ability of organelle-localized solvatochromic probes to distinguish between cells based on their metabolic state. These results expand the repertoire of targeted membrane probes and demonstrate their application in interrogating lipid dysregulation.
Assuntos
2-Naftilamina , Lauratos , Fluidez de Membrana , Organelas , Humanos , Lauratos/química , Lauratos/farmacologia , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Fluidez de Membrana/efeitos dos fármacos , Organelas/metabolismo , Organelas/efeitos dos fármacos , Corantes Fluorescentes/química , Ácidos Graxos/metabolismo , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacosRESUMO
While edible algae might seem low in fat, the lipids they contain are crucial for good health and preventing chronic diseases. This study introduces a binary matrix to analyze all the polar lipids in both macroalgae (Wakame-Undaria pinnatifida, Dulse-Palmaria palmata, and Nori-Porphyra spp.) and microalgae (Spirulina-Arthrospira platensis, and Chlorella-Chlorella vulgaris) using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The key lies in a new dual matrix made by combining equimolar amounts of 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). This combination solves the limitations of single matrices: 9AA is suitable for sulfur-containing lipids and acidic phospholipids, while DAN excels as an electron-transfer secondary reaction matrix for intact chlorophylls and their derivatives. By employing the equimolar binary matrix, a wider range of algal lipids, including free fatty acids, phospholipids, glycolipids, pigments, and even rare arsenosugarphospholipids were successfully detected, overcoming drawbacks related to ion suppression from readily ionizable lipids. The resulting mass spectra exhibited a good signal-to-noise ratio at a lower laser fluence and minimized background noise. This improvement stems from the binary matrix's ability to mitigate in-source decay effects, a phenomenon often encountered for certain matrices. Consequently, the data obtained are more reliable, facilitating a faster and more comprehensive exploration of algal lipidomes using high-throughput MALDI-MS/MS analysis.
Assuntos
Lipídeos , Microalgas , Alga Marinha , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Lipídeos/química , Lipídeos/análise , Alga Marinha/química , Microalgas/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Aminacrina/química , Pigmentos Biológicos/análise , Pigmentos Biológicos/química , Spirulina/químicaRESUMO
Exposure to tobacco smoke is highly correlated to the incidence of different types of cancer due to various carcinogenic compounds present in such smoke. Aromatic amines, such as 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA), are produced in tobacco burning and are linked to bladder cancer. Miniaturized solid phase extraction techniques, such as microporous membrane solid phase extraction (MMSPE), have shown potential for the extraction of aromatic compounds. In this study, a bioanalytical method for the determination of 1-NA and 2-NA in human urine was developed using polypropylene microporous membranes as a sorptive phase for MMSPE. Urine samples were hydrolyzed with HCl for 1 h at 80 °C, after which pH was adjusted to 10. Ultrasound-assisted MMSPE procedure was optimized by factorial design as follows. To each sample, 750 µL of methanol was added, and ultrasound-assisted MMSPE was conducted for 1 h with four devices containing seven 2 mm polypropylene membrane segments. After extraction, the segments were transferred to 400 µL of hexane, and desorption was conducted for 30 min. Extracts were submitted to a simple and fast microwave-assisted derivatization procedure, by the addition of 10 µL of PFPA and heating at 480 W for 3 min, followed by clean-up with phosphate buffer pH 8.0 and GC-MS/MS analysis. Adequate linearity was obtained for both analytes in a range from 25 to 500 µg L-1, while the multiple reaction monitoring approach provided satisfactory selectivity and specificity. Intra-day (n = 6) and inter-day (n = 5) precision and accuracy were satisfactory, below 15 % and between 85 and 115 %, respectively. Recovery rates found were 91.9 and 58.4 % for 1-NA and 2-NA, respectively, with adequate precision. 1-NA was found in first-hand smokers' urine samples in a concentration range from 20.98 to 89.09 µg in 24 h, while it could be detected in second-hand smoker's urine samples, and 2-NA detected in all first and second-hand smokers' urine samples. The proposed method expands the applicability of low cost MMSPE devices to aromatic amines and biological fluids.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Limite de Detecção , Polipropilenos , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Humanos , Polipropilenos/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Extração em Fase Sólida/métodos , Carcinógenos/análise , Carcinógenos/isolamento & purificação , Reprodutibilidade dos Testes , 1-Naftilamina/análogos & derivados , 1-Naftilamina/química , Membranas Artificiais , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Porosidade , FumantesRESUMO
Monitoring active membrane cholesterol and lipid raft cholesterol in the inner leaflet of the plasma membrane is significant for understanding the membrane function and cellular physiopathological processes. Limited by existing methods, it is difficult to differentiate active membrane cholesterol and lipid raft cholesterol. A novel dual-monomer solvatochromic probe system (DSPS) that targets two types of cholesterol was developed. Acrylodan-BG/SNAP-D4 composed of SNAP-D4 cholesterol-recognizing monomers and solvatochromic acrylodan-BG-sensing monomers exhibits excellent cholesterol detecting properties in terms of selectivity, accuracy, convenience and economic benefits. Cell imaging revealed that lipid raft cholesterol emitted blue fluorescence, whereas active membrane cholesterol (which partially bobbed in aqueous cytosol) displayed green fluorescence; both the fluorescence emissions increased or decreased in a cholesterol-dependent manner. This system provides a new technology for the determination of two types of cholesterol, which is beneficial for the further study of membrane function, intracellular cholesterol trafficking, and cell signaling.
Assuntos
2-Naftilamina/análogos & derivados , Colesterol , Microdomínios da Membrana , Membrana Celular/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismoRESUMO
Several aromatic amines (AA) are classified as human carcinogens, and tobacco smoke is one of the main sources of exposure. Once in the human body, they undergo different metabolic pathways which lead to either their excretion or ultimately to the formation of DNA and protein adducts. The aim of this study was to investigate AA in 68 urine samples (aged 29-79, 47% female), including 10 smokers (S), 28 past-smokers (PS) and 30 never-smokers (NS), and to study if there was a relation between the smoking status and the amount of the AA present. GCxGC-MS was used to analyze AA in complex urine samples due to its high peak capacity and the fact that it provides two sets of retention times and structural information, which facilitates the separation and identification of the target analytes. First, a qualitative comparison of an example set of a NS, PS and S sample was carried out, in which 38, 45 and 46 AA, respectively, could be tentatively identified. Afterwards, seven AA were successfully quantified in the samples. Of these, 4-ethylaniline (4EA, p = 0.015), 2,4,6-trimethylaniline (2,4,6TMA, p = 0.030), 2-naphthylamine (2NA, p = 0.014) and the sum of 2,4- and 2,6-dimethylaniline (DMA, p = 0.017) were found in significantly different (α = 0.05) concentrations for the S, 29 ± 14, 87 ± 49, 41 ± 26, and 105 ± 57 ng/L respectively, compared to the NS, 15 ± 6, 42 ± 30, 16 ± 6, and 48 ± 28 ng/L. And 2,4,6TMA (39 ± 26, p = 0.022), 2NA (18 ± 9, p = 0.025) and DMA (53 ± 46, p = 0.030), were also found at significantly higher concentrations in samples from S when compared to PS. However, some samples had AA concentrations outside the calibration curve and could not be taken into account, especially for 2-methylaniline (2MA). Therefore, all the samples were evaluated using a quantitative screening approach, by which the intensities of 4EA (p = 0.019), 2,4,6TMA (p = 0.048), 2NA (p = 0.016), DMA (p = 0.019) and 2MA (p = 0.006) in S were found to be significantly (α = 0.05) higher than in the NS, and 2MA (p = 0.019) and 4EA (p = 0.023) in S were found to be significantly higher than in the PS. An association between the smoking status and the amount of certain AA present could therefore be found. This information could be used to study the relation between the smoking status, the amount of AA present, and smoking related diseases like bladder cancer.
Assuntos
Aminas , Fumar , Humanos , Feminino , Masculino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Aminas/química , Aminas/urina , Carcinógenos , 2-Naftilamina/análiseRESUMO
Objective: To investigate the role and related mechanisms of the LiaSR two-component system in acid tolerance and biofilm formation abilities of Streptococcus mutans (Sm) 593. Methods: The growth curves of various Sm strains in pH=5.5 brian heart infusion (BHI) medium were analyzed. And colony forming unit (CFU) was also performed to evaluate the acid tolerance of Sm. Laurdan probe, H+-K+adenosine triphosphate (ATP)ase activity analysis kit, proton permeability assay and real-time fluorescence quantitative PCR (RT-qPCR) were conducted to detect the acid tolerant mechanisms of LiaSR two-component system in Sm. Crystal violet staining, CFU, SYTOX probe and anthrone-sulfuric method were used to analyze the properties and structures of the Sm biofilms. RT-qPCR was conducted to detect the expression levels of underlying regulated genes. Results: The growth of mutants in acidic BHI were inhibited (P<0.05). The acid tolerance of mutants significantly decreased compared to the wild-type strain (P<0.05). In mutants, the activity of H+-ATPase (917.06±59.53 and 469.53±47.65) were elevated by 7.22-folds and 3.70-folds compared to the wild-type strain (127.00±50.71) (P<0.001, P<0.001) and the encoded gene atpD (3.39±0.21 and 1.94±0.17) were also elevated by 3.39-folds and 1.94-folds compared to the wild-type strain (1.00±0.15) (P<0.001, P=0.001). The Laurdan generalized polarization of mutants (0.18±0.04 and 0.18±0.05) increased significantly compared to the wild-type strain (0.08±0.05) (P=0.006, P=0.003) and the expression levels of fabM gene were decreased in mutants (0.52±0.11 and 0.57±0.05) by 1/2 (P=0.014, P=0.022). In liaR deletion mutant, the reduced terminal pH (4.76±0.01) can also be observed (P<0.001). The total amount of the biofilms of three Sm didn't show significant differences (P>0.05). But the number of viable bacteria of mutants' biofilms were decreased [Sm 593: (12.00±2.80)×107 CFU/ml; Sm ΔliaS: (2.95±1.13)×107 CFU/ml; Sm ΔliaR: (7.25±1.60)×107 CFU/ml] (P=0.001, P=0.024). The extracellular DNA were increased by 18.00-folds and 6.50-folds in mutants' biofilms (128.73±15.65 and 46.38±5.52) compared to the wild-type strain (7.16±3.62) (P<0.001, P=0.003). Water-soluble exopolysaccharides could be found up-regulated in liaS deletion mutant [(138.73±10.12) µg/ml] (P=0.003) along with the expression level of gtfC gene (1.65±0.39) (P=0.014). The expression level of gtfD were elevated by 47.43-folds and 16.90-folds in mutants (P<0.001, P=0.010). Conclusions: The LiaSR two-component system can promote the expression of fabM gene and increase the fluidity of Sm which contributes to acid tolerance. The LiaR can also decrease the proton permeability and restrict the entrance of H+. The LiaSR two-component system can negatively regulate the production of the extracellular matrix in Sm biofilm.
Assuntos
2-Naftilamina/análogos & derivados , Lauratos , Prótons , Streptococcus mutans , Streptococcus mutans/genética , BiofilmesRESUMO
The solvatochromic dye Laurdan is widely used in sensing the lipid packing of both model and biological membranes. The fluorescence emission maximum shifts from about 440 nm (blue channel) in condensed membranes (So) to about 490 nm (green channel) in the liquid-crystalline phase (Lα). Although the fluorescence intensity based generalized polarization (GP) is widely used to characterize lipid membranes, the fluorescence lifetime of Laurdan, in the blue and the green channel, is less used for that purpose. Here we explore the correlation between GP and fluorescence lifetimes by spectroscopic measurements on the So and Lα phases of large unilamellar vesicles of DMPC and DPPC. A positive correlation between GP and the lifetimes is observed in each of the optical channels for the two lipid phases. Microfluorimetric determinations on giant unilamellar vesicles of DPPC and DOPC at room temperature are performed under linearly polarized two-photon excitation to disentangle possible subpopulations of Laurdan at a scale below the optical resolution. Fluorescence intensities, GP and fluorescence lifetimes depend on the angle between the orientation of the linear polarization of the excitation light and the local normal to the membrane of the optical cross-section. This angular variation depends on the lipid phase and the emission channel. GP and fluorescence intensities in the blue and green channel in So and in the blue channel in Lα exhibit a minimum near 90o. Surprisingly, the intensity in the green channel in Lα reaches a maximum near 90o. The fluorescence lifetimes in the two optical channels also reach a pronounced minimum near 90o in So and Lα, apart from the lifetime in the blue channel in Lα where the lifetime is short with minimal angular variation. To our knowledge, these experimental observations are the first to demonstrate the existence of a bent conformation of Laurdan in lipid membranes, as previously suggested by molecular dynamics calculations.
Assuntos
Lauratos , Lipossomas Unilamelares , Membrana Celular , Lauratos/análise , Lauratos/química , 2-Naftilamina/química , Corantes Fluorescentes/química , Polarização de FluorescênciaRESUMO
Laurdan and Prodan were designed for the evaluation of the surrounding hydration state. When inserted into lipid bilayer systems, both probes are located at different positions and their fluorescence properties are drastically varied, depending on their surrounding environment. In this study, a novel method using the above fluorescence probes was proposed on the basis of fluorescence lifetime (τ) and emission peak (λ), called as τ vs. λ plot, determined by global analysis of their multiple fluorescence decays and deconvolution of these decay-associated spectra. According to the evaluation of τ vs. λ plot, the existence of multiple fluorescence components in the membrane was revealed. In addition, their fluorescence distribution properties, described on τ vs. λ plot, of each probe tended to correspond to the phase state and vertical direction of the lipid membrane. To assess the contribution of environmental effect to each distribution, we defined the region in the τ vs. λ plot, which was modeled from a series of solvent mixtures (hexane, acetone, ethanol and water) to emulate the complex environment in the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer system. The distributions of fluorescence components of Laurdan and Prodan in lipid membranes were classified into each solvent species, and Prodan partition into bulk water was distinguished. The sensitivity of Prodan to the phase pretransition of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer system was also observed in increasing the temperature. Noticeably, most of the fluorescence components was assigned to the solvent model, except for a single component that has longer lifetime and shorter emission wavelength. This component was dominant in solid-ordered phase; hence, it is assumed to be a specific component in lipid membranes that cannot be represented by solvents. Although these are still qualitative analytical methods, the unique approach proposed in this study provides novel insights into the multi-focal property of the membrane.
Assuntos
2-Naftilamina , Bicamadas Lipídicas , Solventes , Água , Corantes Fluorescentes , Espectrometria de FluorescênciaRESUMO
Dissolution dynamic nuclear polarization (dDNP) increases the sensitivity of magnetic resonance imaging by more than 10,000 times, enabling in vivo metabolic imaging to be performed noninvasively in real time. Here, we are developing a group of dDNP polarized tracers based on nicotinamide (NAM). We synthesized 1-15N-NAM and 1-15N nicotinic acid and hyperpolarized them with dDNP, reaching (13.0 ± 1.9)% 15N polarization. We found that the lifetime of hyperpolarized 1-15N-NAM is strongly field- and pH-dependent, with T1 being as long as 41 s at a pH of 12 and 1 T while as short as a few seconds at neutral pH and fields below 1 T. The remarkably short 1-15N lifetime at low magnetic fields and neutral pH drove us to establish a unique pH neutralization procedure. Using 15N dDNP and an inexpensive rodent imaging probe designed in-house, we acquired a 15N MRI of 1-15N-NAM (previously hyperpolarized for more than an hour) in less than 1 s.
Assuntos
2-Naftilamina , Niacinamida , Niacinamida/farmacologia , Isótopos de NitrogênioRESUMO
Hyperpolarization of 13C by dissolution dynamic nuclear polarization (dDNP) boosts the sensitivity of magnetic resonance spectroscopy (MRS), making possible the monitoring in vivo and in real time of the biochemical reactions of exogenously infused 13C-labeled metabolic tracers. The preparation of a hyperpolarized substrate requires the use of free radicals as polarizing agents. Although added at very low doses, these radicals are not biologically inert. Here, we demonstrate that the presence of the nitroxyl radical TEMPOL influences significantly the cerebral metabolic readouts of a hyperpolarized [1-13C] lactate bolus injection in a mouse model of ischemic stroke with reperfusion. Thus, the choice of the polarizing agent in the design of dDNP hyperpolarized MRS experiments is of great importance and should be taken into account to prevent or to consider significant effects that could act as confounding factors.
Assuntos
Fenômenos Bioquímicos , AVC Isquêmico , Animais , Camundongos , 2-NaftilaminaRESUMO
Influenza A virus is a highly mutable pathogenic pathogen that could cause a global pandemic. It is necessary to find new anti-influenza drugs to resist influenza epidemics due to the seasonal popularity of a certain area every year. Naphthalene derivatives had potential antiviral activity. A series of naphthalene derivatives were synthesized via the metal-free intramolecular hydroarylation reactions of alkynes. Evaluation of their biological efficacy showed that compound 2-aminonaphthalene 4d had better antiviral activity in vitro than ribavirin. By studying the mechanism of action of 2-aminonaphthalene 4din vivo and in vitro, we found that 4d had antiviral activity to three different subtype influenza viruses of A/Weiss/43 (H1N1), A/Virginia/ATCC2/2009 (H1N1) and A/California/2/2014 (H3N2). Compound 4d had the best effect after viral adsorption, and mainly played in the early stage of virus replication. 2-Aminonaphthalene 4d could reduce the replication of virus by inhibiting the NP and M proteins of virus. Compound 4d cut down ROS accumulation, autophagy and apoptosis induced by influenza virus. Inflammatory response mediated by RIG-1 pathway were suppressed in the cell and mice. In addition, the pathological changes of lung tissue and virus titer in mice were reduced by the administration of 4d. Therefore, naphthalene derivative 4d is a potential drug for the treatment of influenza A virus infection.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Animais , Camundongos , Humanos , Vírus da Influenza A Subtipo H3N2 , 2-Naftilamina/metabolismo , 2-Naftilamina/farmacologia , 2-Naftilamina/uso terapêutico , Antivirais/uso terapêutico , Influenza Humana/tratamento farmacológico , Replicação ViralRESUMO
Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from â¼0.60 to â¼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.
Assuntos
Lauratos , Gotículas Lipídicas , Gotículas Lipídicas/metabolismo , Lauratos/análise , Lauratos/metabolismo , Metabolismo dos Lipídeos , 2-Naftilamina/análise , 2-Naftilamina/metabolismoRESUMO
Studies of biological membrane heterogeneity particularly benefit from the use of the environment-sensitive fluorescent probe Laurdan, for which shifts in the emission, produced by any stimulus (e.g., fluidity variations), are ascribed to alterations in hydration near the fluorophore. Ironically, no direct measure of the influence of the membrane hydration level on Laurdan spectra has been available. To address this, we investigated the fluorescence spectrum of Laurdan embedded in solid-supported lipid bilayers as a function of hydration and compared it with the effect of cholesterolâa major membrane fluidity regulator. The effects are illusively similar, and hence the results obtained with this probe should be interpreted with caution. The dominant phenomenon governing the changes in the spectrum is the hindrance of the lipid internal dynamics. Furthermore, we unveiled the intriguing mechanism of dehydration-induced redistribution of cholesterol between domains in the phase-separated membrane, which reflects yet another regulatory function of cholesterol.
Assuntos
Lauratos , Bicamadas Lipídicas , Membrana Celular , 2-Naftilamina , Corantes Fluorescentes , ColesterolRESUMO
Several aromatic amines (AAs) are established by the International Agency for Research on Cancer as carcinogenic (group 1) or probable/possible carcinogens to humans (group 2A/2B). AAs can be found in mainstream and sidestream smoke from combustible tobacco products, as well as in certain environmental pollution and occupational exposure from several chemical industry sectors. Exposure to AAs can be estimated by measuring their concentrations in urine; however, information about the short-term and long-term stabilities of AAs in urine need to be characterized before conducting large-scale population studies on AA exposure and the potentially harmful effects of AA exposure. In this report, the storage stability of o-toluidine, 2,6-dimethylaniline, o-anisidine, 1-aminonaphthalene, 2-aminonaphthalene, and 4-aminobiphenyl fortified in pooled, filtered, non-smokers' urine is analyzed by isotope dilution gas chromatography-triple quadrupole mass spectrometry (ID GC-MS/MS). The six AAs were measured in urine samples stored at ~20 °C (collection temperature), 4 °C and 10 °C (short-term transit temperatures), and -20 °C and -70 °C (long-term storage temperatures) over a 10-day period. All six analytes were stable for 10 days at transit and long-term storage temperatures but showed reduced recovery at 20 °C. The instability of the target AAs at 20 °C suggests that immediate storage of freshly voided urine at low temperatures is needed to attenuate degradation. A subset of the urine samples was analyzed following a longer storage duration at -70 °C: all AAs were stable for up to 14 months at this temperature. The stability of the six AAs in urine samples can be maintained at the various temperature levels and storage times expected in a typical study set.
Assuntos
Aminas , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Aminas/urina , Carcinógenos/análise , 2-Naftilamina/análiseRESUMO
The reactions of 2-naphthylamine and methyl 6-amino-2-naphthoate with formalin and paraformaldehyde were studied experimentally, spectrally, and by quantum chemical calculations. It was found that neither the corresponding aminals nor imines were formed under the described conditions but could be prepared and spectrally characterized at least in situ under modified conditions. Several of the previously undescribed intermediates and by-products were isolated or at least spectrally characterized. First principle density functional theory (DFT) calculations were performed to shed light on the key aspects of the thermochemistry of decomposition and further condensation of the corresponding aminals and imines. The calculations also revealed that the electrophilicity of methanal was significantly greater than that of ordinary oxo-compounds, except for perfluorinated ones. In summary, methanal was not behaving as the simplest aldehyde but as a very electron-deficient oxo-compound.