RESUMEN
Phytoplankton replace phosphorus-containing lipids (P-lipids) with non-P analogues, boosting growth in P-limited oceans. In the model diatom Thalassiosira pseudonana, the substitution dynamics of lipid headgroups are well described, but those of the individual lipids, differing in fatty acid composition, are unknown. Moreover, the behavior of lipids outside the common headgroup classes and the relationship between lipid substitution and cellular particulate organic P (POP) have yet to be reported. We investigated these through the mass spectrometric lipidomics of P-replete (P+) and P-depleted (P-) T. pseudonana cultures. Nonlipidic POP was depleted rapidly by the initiation of P stress, followed by the cessation of P-lipid biosynthesis and per-cell reductions in the P-lipid levels of successive generations. Minor P-lipid degradative breakdown was observed, releasing P for other processes, but most P-lipids remained intact. This may confer an advantage on efficient heterotrophic lipid consumers in P-limited oceans. Glycerophosphatidylcholine (PC), the predominant P-lipid, was similar in composition to its betaine substitute lipid. During substitution, PC was less abundant per cell and was more highly unsaturated in composition. This may reflect underlying biosynthetic processes or the regulation of membrane biophysical properties subject to lipid substitution. Finally, levels of several diglycosylceramide lipids increased as much as 10-fold under P stress. These represent novel substitute lipids and potential biomarkers for the study of P limitation in situ, contributing to growing evidence highlighting the importance of sphingolipids in phycology. These findings contribute much to our understanding of P-lipid substitution, a powerful and widespread adaptation to P limitation in the oligotrophic ocean.IMPORTANCE Unicellular organisms replace phosphorus (P)-containing membrane lipids with non-P substitutes when P is scarce, allowing greater growth of populations. Previous research with the model diatom species Thalassiosira pseudonana grouped lipids by polar headgroups in their chemical structures. The significance of the research reported here is threefold. (i) We described the individual lipids within the headgroups during P-lipid substitution, revealing the relationships between lipid headgroups and hinting at the underlying biochemical processes. (ii) We measured total cellular P, placing P-lipid substitution in the context of the broader response to P stress and yielding insight into the implications of substitution in the marine environment. (iii) We identified lipids previously unknown in this system, revealing a new type of non-P substitute lipid, which is potentially useful as a biomarker for the investigation of P limitation in the ocean.
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Diatomeas/metabolismo , Fósforo/metabolismo , Estrés Fisiológico , Adaptación Fisiológica , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Espectrometría de Masas , Lípidos de la Membrana/metabolismo , Océano Pacífico , Fosfolípidos/metabolismo , Fósforo/deficiencia , Agua de Mar/químicaRESUMEN
Oxylipins, or fatty aldehydes, are a class of molecules produced from membrane lipids as a result of oxidative stress or enzyme-mediated peroxidation. Here we report the effects of two biologically important fatty aldehydes, trans,trans-2,4-decanedienal (DD) and cis-11-hexadecenal (HD), on the phase behavior of the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in water. We compare the phase behavior of DD/DOPE and HD/DOPE mixtures to the phase behavior of oleic acid/DOPE mixtures and show that DD, HD, and oleic acid have similar effects on the phase diagrams of DOPE. Notably, both DD and HD, like oleic acid, induce the formation of Fd3m inverse micellar cubic phases in DOPE/water mixtures. This is the first time that Fd3m phases in fatty aldehyde-containing mixtures have been reported. We assess the effects of DD, HD, and oleic acid on DOPE in terms of lipid spontaneous curvatures and propose a method to predict the formation of Fd3m phases from the curvature power of amphiphiles. This methodology predicts that Fd3m phases will become stable if the spontaneous curvature of a lipid mixture is -0.48 ± 0.05 nm-1 or less.
RESUMEN
Recently we reported a method for estimating the spontaneous curvatures of lipids from temperature-dependent changes in the lattice parameter of inverse hexagonal liquid crystal phases of binary lipid mixtures. This method makes use of 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) as a host lipid, which preferentially forms an inverse hexagonal phase to which a guest lipid of unknown spontaneous curvature is added. The lattice parameters of these binary lipid mixtures are determined by small-angle X-ray diffraction at a range of temperatures and the spontaneous curvature of the guest lipid is determined from these data. Here we report the use of this method on a wide range of lipids under different ionic conditions. We demonstrate that our method provides spontaneous curvature values for DOPE, cholesterol, and monoolein that are within the range of values reported in the literature. Anionic lipids 1,2-dioleoyl-sn-glycerol-3-phosphatidic acid (DOPA) and 1,2-dioleoyl-sn-glycerol-3-phosphoserine (DOPS) were found to exhibit spontaneous curvatures that depend on the concentration of divalent cations present in the mixtures. We show that the range of curvatures estimated experimentally for DOPA and DOPS can be explained by a series of equilibria arising from lipid-cation exchange reactions. Our data indicate a universal relationship between the spontaneous curvature of a lipid and the extent to which it affects the lattice parameter of the hexagonal phase of DOPE when it is part of a binary mixture. This universal relationship affords a rapid way of estimating the spontaneous curvatures of lipids that are expensive, only available in small amounts, or are of limited chemical stability.
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This study deals with the build-up of biomaterials consisting of biopolymers, namely DNA, and soft particles, poly(amido amine) (PAMAM) dendrimers, and how to model their interactions. We adopted and applied an analytical model to provide further insight into the complexation between DNA (4331 bp) and positively charged PAMAM dendrimers of generations 1, 2, 4, 6 and 8, previously studied experimentally. The theoretical models applied describe the DNA as a semiflexible polyelectrolyte that interacts with dendrimers considered as either hard (impenetrable) spheres or as penetrable and soft spheres. We found that the number of DNA turns around one dendrimer, thus forming a complex, increases with the dendrimer size or generation. The DNA penetration required for the complex to become charge neutral depends on dendrimer generation, where lower generation dendrimers require little penetration to give charge neutral complexes. High generation dendrimers display charge inversion for all considered dendrimer sizes and degrees of penetration. Consistent with the morphologies observed experimentally for dendrimer/DNA aggregates, where highly ordered rods and toroids are found for low generation dendrimers, the DNA wraps less than one turn around the dendrimer. Disordered globular structures appear for high generation dendrimers, where the DNA wraps several turns around the dendrimer. Particularly noteworthy is that the dendrimer generation 4 complexes, where the DNA wraps about one turn around the dendrimers, are borderline cases and can form all types of morphologies. The net-charges of the aggregate have been estimated using zeta potential measurements and are discussed within the theoretical framework.
Asunto(s)
ADN/química , ADN/ultraestructura , Dendrímeros/química , Modelos Químicos , Modelos Moleculares , Sitios de Unión , Simulación por Computador , Sustancias Macromoleculares/química , Conformación de Ácido Nucleico , Electricidad EstáticaRESUMEN
The addition of saturated fatty acids (FA) to phosphatidylcholine lipids (PC) that have saturated acyl chains has been shown to promote the formation of lyotropic liquid-crystalline phases with negative mean curvature. PC/FA mixtures may exhibit inverse bicontinuous cubic phases (Im3m, Pn3m) or inverse topology hexagonal phases (HII), depending on the length of the acyl chains/fatty acid. Here we report a detailed study of the phase behavior of binary mixtures of dioleoylphosphatidylcholine (DOPC)/oleic acid (OA) and dioleoylphosphatidylethanolamine (DOPE)/oleic acid at limiting hydration, constructed using small-angle X-ray diffraction (SAXD) data. The phase diagrams of both systems show a succession of phases with increasing negative mean curvature with increasing OA content. At high OA concentrations, we have observed the occurrence of an inverse micellar Fd3m phase in both systems. Hitherto, this phase had not been reported for phosphatidylethanolamine/fatty acid mixtures, and as such it highlights an additional route through which fatty acids may increase the propensity of bilayer lipid membranes to curve. We also propose a method that uses the temperature dependence of the lattice parameters of the HII phases to estimate the spontaneous radii of curvature (R0) of the binary mixtures and of the component lipids. Using this method, we calculated the R0 values of the complexes comprising one phospholipid molecule and two fatty acid molecules, which have been postulated to drive the formation of inverse phases in PL/FA mixtures. These are -1.8 nm (±0.4 nm) for DOPC(OA)2 and -1.1 nm (±0.1 nm) for DOPE(OA)2. R0 values estimated in this way allow the quantification of the contribution that different lipid species make to membrane curvature elastic properties and hence of their effect on the function of membrane-bound proteins.
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Ácido Oléico/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/químicaRESUMEN
One of the most developed theories of phospholipid homeostasis is the intrinsic curvature hypothesis, which, in broad terms, postulates that cells regulate their lipid composition so as to keep constant the membrane stored curvature elastic energy. The implication of this hypothesis is that lipid composition is determined by a ratio control function consisting of the weighted sum of concentrations of type II lipids in the numerator and the weighted sum of concentrations of Type 0 lipids in the denominator. In previous work we used a data-driven approach, based on lipidomic data from asynchronous cell cultures, to determine a criterion that allows the different lipid species to be assigned to the set of type 0 or of type II lipids, and hence construct a ratio control function that serves as a proxy for the lipid contribution to total membrane stored curvature elastic energy in vivo. Here we apply the curvature elastic energy proxy to the analysis of lipid composition data from synchronous HeLa cells as they traverse the cell cycle. Our analysis suggests HeLa cells modify their membrane stored elastic energy through the cell cycle. In S-phase type 0 lipids are the most abundant, whilst in G2 type II lipids are most abundant. Changes in our proxy for membrane stored elastic energy correlate with membrane curvature dependent processes in the HeLa cell around division, providing some insights into the interplay between the individual lipid and protein contributions to membrane free energy.
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Ciclo Celular , Membrana Celular/química , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Membrana Celular/metabolismo , Elasticidad , Células HeLa , Humanos , Fase SRESUMEN
While it is widely accepted that the lipid composition of eukaryotic membranes is under homeostatic control, the mechanisms through which cells sense lipid composition are still the subject of debate. It has been postulated that membrane curvature elastic energy is the membrane property that is regulated by cells, and that lipid composition is maintained by a ratio control function derived from the concentrations of type II and type 0 lipids, weighted appropriately. We assess this proposal by seeking a signature of ratio control in quantified lipid composition data obtained by electrospray ionization mass spectrometry from over 40 independent asynchronous cell populations. Our approach revealed the existence of a universal 'pivot' lipid, which marks the boundary between type 0 lipids and type II lipids, and which is invariant between different cell types or cells grown under different conditions. The presence of such a pivot species is a distinctive signature of the operation in vivo, in human cell lines, of a control function that is consistent with the hypothesis that membrane elastic energy is homeostatically controlled.
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Membrana Celular/fisiología , Elasticidad/fisiología , Homeostasis/fisiología , Metabolismo de los Lípidos/fisiología , Fosfolípidos/metabolismo , Células HeLa , Humanos , Propiedades de SuperficieRESUMEN
Neuropeptide Y (NPY) is widely expressed in the central and peripheral nervous systems and is proliferative for a range of cells types in vitro. NPY plays a key role in regulating adult hippocampal neurogenesis in vivo under both basal and pathological conditions, although the underlying mechanisms are largely unknown. We have investigated the role of nitric oxide (NO) on the neurogenic effects of NPY. Using postnatal rat hippocampal cultures, we show that the proliferative effect of NPY on nestin(+) precursor cells is NO-dependent. As well as the involvement of neuronal nitric-oxide synthase, the proliferative effect is mediated via an NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) and extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. We show that NPY-mediated intracellular NO signaling results in an increase in neuroproliferation. By contrast, extracellular NO had an opposite, inhibitory effect on proliferation. The importance of the NO-cGMP-PKG signaling pathway in ERK1/2 activation was confirmed using Western blotting. This work unites two significant modulators of hippocampal neurogenesis within a common signaling framework and provides a mechanism for the independent extra- and intracellular regulation of postnatal neural precursors by NO.
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Proliferación Celular , Hipocampo/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Células-Madre Neurales/metabolismo , Neuropéptido Y/metabolismo , Óxido Nítrico/metabolismo , Animales , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Hipocampo/citología , Proteínas de Filamentos Intermediarios/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Células-Madre Neurales/citología , Neuropéptido Y/farmacología , Ratas , Ratas WistarRESUMEN
6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V(max), depending on the lipid concentration and vesicle composition, but no change in K(m). Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.
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Geobacillus stearothermophilus/metabolismo , Lípidos/química , Fosfofructoquinasa-1/metabolismo , Biocatálisis , Cinética , Liposomas/química , Ácido Oléico , FosfatidilcolinasAsunto(s)
Materiales Biocompatibles/química , Hidrogeles/química , Animales , Cápsulas/química , Células Cultivadas , Humanos , Ratones , Nanopartículas/química , Neovascularización Fisiológica , Silicatos/química , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The interaction of nucleic acids with the nanoarchitectures formed by lipidic systems is a new area of research that may offer insights into the functioning of genetic materials in vivo. Here we report that ssRNA has a strong preference to reside in isotropic solution rather than in inverse hexagonal (HII) liquid crystalline phases. This is in contrast to dsDNA, which becomes localized in the pores of the HII phase. The RNA that does associate with the external surfaces of the HII phase appears to form an accretion layer, tens of molecules thick, but this layer still allows the transcription of dsDNA that resides within the pores of the phase.
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Lípidos/química , Cristales Líquidos/química , ARN/química , ADN/químicaRESUMEN
Recently, we reported that DNA associated with inverse hexagonal (H(II)) lyotropic liquid crystal phases of the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was actively transcribed by T7 RNA polymerase. Our findings suggested that key components of the transcription process, probably the T7 RNA polymerase and the DNA, remained associated with the monolithic H(II) phase throughout transcription. Here, we investigate the partitioning of DNA between an H(II) lyotropic liquid crystal phase and an isotropic supernatant phase in order to develop insights into the localization of DNA in liquid crystalline environments. Our results show that linear double stranded DNA (dsDNA) molecules partition spontaneously into monolithic preformed H(II) liquid crystal phases of DOPE. We propose that this process is driven by the increase in entropy due to the release of counterions from the DNA when it inserts into the aqueous pores of the H(II) phase.
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ADN/química , Fosfatidiletanolaminas/química , Calibración , Cristalización , ARN Polimerasas Dirigidas por ADN/química , Microscopía Fluorescente , Proteínas Virales/químicaRESUMEN
Mixtures of dsDNA and lipids, so-called lipoplexes, are widely used as less toxic alternatives to viral vectors in transfection studies. However, the transfection efficiency achieved by lipoplexes is significantly lower than that of viral vectors and is a barrier to their use in the clinic. There is now significant evidence suggesting that the molecular organization and structure (nanoarchitecture) of lipoplexes might correlate with biological activity. As a consequence, the ability to predict quantitatively the nanoarchitecture of new systems, and how these might change intracellularly, would be a major tool in the development of rational discovery strategies for more efficient lipoplex formulations. Here we report the use of a coarse-grain molecular dynamics simulation to predict the phases formed by two lipoplex systems: dsDNA-DOPE and dsDNA-DOPE-DOTAP. The predictions of the simulations show excellent agreement with experimental data from polarized light microscopy and small-angle X-ray diffraction (SAXS); the simulations predicted the formation of phases with d-spacings that were comparable to those measured by SAXS. More significantly, the simulations were able to reproduce for the first time the experimentally observed change from a fluid lamellar to an inverse hexagonal phase in the dsDNA-DOPE-DOTAP system as a function of changes in lipid composition. Our studies indicate that coarse-grain MD simulations could provide a powerful tool to understand, and hence design, new lipoplex systems.
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ADN/química , ADN/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Simulación de Dinámica Molecular , Ácidos Grasos Monoinsaturados/química , Ácidos Grasos Monoinsaturados/metabolismo , Conformación Molecular , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo , Dispersión del Ángulo Pequeño , Agua/química , Difracción de Rayos XRESUMEN
Recently we proposed that the antineoplastic properties observed in vivo for alkyl-lysophospholipid and alkylphosphocholine analogues are a direct consequence of the reduction of membrane stored elastic stress induced by these amphiphiles. Here we report similar behavior for a wide range of cationic surfactant analogues. Our systematic structure-activity studies show that the cytotoxic properties of cationic surfactants follow the same pattern of activity we observed previously for alkyl-lysophospholipid analogues, indicating a common mechanism of action that is consistent with the theory that these amphiphiles reduce membrane stored elastic stress. We note that several of the cationic surfactant compounds we have evaluated are also potent antibacterial and antifungal agents. The similarity of structure-activity relationships for cationic surfactants against microorganisms and those we have observed in eukaryotic cell lines leads us to suggest the possibility that the antibacterial and antifungal properties of cationic surfactants may also be due to modulation of membrane stored elastic stress.
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Cationes , Fosforilcolina/química , Algoritmos , Antifúngicos/química , Antifúngicos/farmacología , Antineoplásicos/farmacología , Elasticidad , Células HL-60 , Homeostasis , Humanos , Lípidos/química , Cristales Líquidos , Lisofosfolípidos/química , Membranas/química , Modelos Químicos , Tensoactivos/químicaRESUMEN
We report that a 4.3 kbp linearised T7 DNA plasmid is actively transcribed when it is dispersed in the hexagonal liquid crystalline phase of dioleoylphosphoethanolamine (DOPE).
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ADN Viral/química , Fosfatidiletanolaminas/química , Plásmidos/química , Bacteriófago T7/enzimología , Bacteriófago T7/genética , Cristalización , ADN Viral/genética , Ácidos Grasos Monoinsaturados/química , Luciferasas/genética , Plásmidos/genética , Compuestos de Amonio Cuaternario/química , Transcripción GenéticaRESUMEN
The homeostatic control of membrane lipid composition appears to be of central importance for cell functioning and survival. However, while lipid biosynthetic reaction networks have been mapped in detail, the underlying control architecture which underpins these networks remains elusive. A key problem in determining the control architectures of lipid biosynthetic pathways, and the mechanisms through which control is achieved, is that the compositional complexity of lipid membranes makes it difficult to determine which membrane parameter is under homeostatic control. Recently, we reported that membrane stored elastic energy provides a physical feedback signal which modulates the activity in vitro of CTP:phosphocholine cytidylyltransferase (CCT), an extrinsic membrane enzyme which catalyses a key step in the synthesis of phosphatidylcholine lipids in the Kennedy pathway (Kennedy 1953 J. Am. Chem. Soc. 75, 249-250). We postulate that stored elastic energy may be the main property of membranes that is under homeostatic control. Here we report the results of simulations based on this postulate, which reveal a possible control architecture for lipid biosynthesis networks in vivo.
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Membrana Celular/química , Simulación por Computador , Retroalimentación Fisiológica , Lípidos de la Membrana/biosíntesis , Modelos Biológicos , AlgoritmosRESUMEN
Recent advances in electrospray ionisation mass spectrometry (ESI-MS) have greatly facilitated the analysis of phospholipid molecular species in a growing diversity of biological and clinical settings. The combination of ESI-MS and metabolic labelling employing substrates labelled with stable isotopes is especially exciting, permitting studies of phospholipid synthesis and turnover in vivo. This review will first describe the methodology involved and will then detail dynamic lipidomic studies that have applied the stable isotope incorporation approach. Finally, it will summarise the increasing number of studies that have used ESI-MS to characterise structural and signalling phospholipid molecular species in development and disease.
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Fosfolípidos/análisis , Fosfolípidos/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Diferenciación Celular , Membrana Celular/metabolismo , Enfermedad , Salud , Humanos , Fosfolípidos/químicaAsunto(s)
Carbono/química , Microscopía de Fuerza Atómica/instrumentación , Microscopía de Fuerza Atómica/métodos , Nanotecnología/métodos , Nanotubos de Carbono/química , Ingeniería Biomédica/métodos , Hierro/química , Microscopía Electrónica , Modelos Moleculares , Nanotecnología/instrumentación , Porosidad , Análisis de Secuencia de ADN , Programas Informáticos , Propiedades de Superficie , Titanio/químicaRESUMEN
A major drawback of subunit vaccines is their inability to generate cytolytic T lymphocytes (CTL), a deficit attributed to segregation of the class I and class II antigen-processing pathways. We sought to understand processes involved in CTL induction by three proprietary adjuvants: Tomatine, PROVAX, and a synthesized glycolipid (Glc-N-(8/16), Glycolipid). We used in vivo models to investigate antigen uptake, macrophage involvement, TAP-independent processing, and costimulatory molecule dependencies. Glycolipid required splenic and lymph node macrophages, whereas Tomatine generated CTL independently of either macrophage population. In contrast, PROVAX showed partial macrophage requirements. Immunized TAP knockout mice revealed that ovalbumin (OVA)-Tomatine and OVA-PROVAX, but not OVA-Glycolipid, generate class I-peptide complexes. All three immunostimulants also elicited CD86-dependent TH1 cytokine responses.