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1.
Phys Chem Chem Phys ; 21(35): 19327-19341, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31453592

RESUMO

The mechanisms of plasma in medicine are broadly attributed to plasma-derived reactive oxygen and nitrogen species (RONS). In order to exert any intracellular effects, these plasma-derived RONS must first traverse a major barrier in the cell membrane. The cell membrane lipid composition, and thereby the magnitude of this barrier, is highly variable between cells depending on type and state (e.g. it is widely accepted that healthy and cancerous cells have different membrane lipid compositions). In this study, we investigate how plasma-derived RONS interactions with lipid membrane components can potentially be exploited in the future for treatment of diseases. We couple phospholipid vesicle experiments, used as simple cell models, with molecular dynamics (MD) simulations of the lipid membrane to provide new insights into how the interplay between phospholipids and cholesterol may influence the response of healthy and diseased cell membranes to plasma-derived RONS. We focus on the (i) lipid tail saturation degree, (ii) lipid head group type, and (iii) membrane cholesterol fraction. Using encapsulated molecular probes, we study the influence of the above membrane components on the ingress of RONS into the vesicles, and subsequent DNA damage. Our results indicate that all of the above membrane components can enhance or suppress RONS uptake, depending on their relative concentration within the membrane. Further, we show that higher RONS uptake into the vesicles does not always correlate with increased DNA damage, which is attributed to ROS reactivity and lifetime. The MD simulations indicate the multifactorial chemical and physical processes at play, including (i) lipid oxidation, (ii) lipid packing, and (iii) lipid rafts formation. The methods and findings presented here provide a platform of knowledge that could be leveraged in the development of therapies relying on the action of plasma, in which the cell membrane and oxidative stress response in cells is targeted.


Assuntos
Dano ao DNA , Lipídeos de Membrana/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Colesterol/química , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Fosfolipídeos/química , Espécies Reativas de Nitrogênio/sangue , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/sangue , Vesículas Transportadoras/química
2.
BMC Plant Biol ; 19(1): 304, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291882

RESUMO

BACKGROUND: In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Communication between these compartments is supposed to be tightly regulated at their interfaces. Here, we characterize the deposition pattern of an apoplastic lipid barrier between the maternal inner integument and fertilization products in Arabidopsis thaliana seeds. RESULTS: We demonstrate that an apoplastic lipid barrier is first deposited by the ovule inner integument and undergoes de novo cutin deposition following central cell fertilization and relief of the FERTILIZATION INDEPENDENT SEED Polycomb group repressive mechanism. In addition, we show that the WIP zinc-finger TRANSPARENT TESTA 1 and the MADS-Box TRANSPARENT TESTA 16 transcription factors act maternally to promote its deposition by regulating cuticle biosynthetic pathways. Finally, mutant analyses indicate that this apoplastic barrier allows correct embryo sliding along the seed coat. CONCLUSIONS: Our results revealed that the deposition of a cutin apoplastic barrier between seed maternal and zygotic tissues is part of the seed coat developmental program.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Lipídeos de Membrana/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Endosperma/metabolismo , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
3.
Food Chem ; 295: 26-35, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174758

RESUMO

Bell peppers are susceptible to chilling injury (CI). To uncover the metabolism of membrane lipid fatty acids (FAs) accompanying CI, a gas chromatography-mass spectrometry (GC-MS)-based approach was used to quantitatively profile major membrane lipid FAs in bell peppers. RT-qPCR was performed to investigate the expression of the key genes that regulate the synthesis of unsaturated FAs. Additionally, we used microstructural, organoleptic, and physicochemical investigations to monitor the primary physiological metabolism of bell peppers. The study revealed that CI symptoms mostly resulted from the destabilization of the cytomembrane, which was induced by decreasing FA desaturation. Moreover, three times lower level of the double bond index in chilled fruits, than the control, further proved that membrane FA unsaturation can be considered a key factor during CI. In conclusion, this study revealed that the metabolism of membrane lipid FAs is involved in responses to CI.


Assuntos
Capsicum/metabolismo , Ácidos Graxos/metabolismo , Conservação de Alimentos/métodos , Lipídeos de Membrana/metabolismo , Capsicum/química , Capsicum/genética , Ácidos Graxos/análise , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Frutas/química , Frutas/genética , Frutas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Lipoxigenase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Temperatura Ambiente
4.
Food Chem ; 297: 124955, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253342

RESUMO

This study aimed to investigate the effect of hydrogen peroxide (H2O2) on membrane lipids metabolism and its relation to pulp breakdown development of longan fruit during postharvest storage. Compared to the control longans, H2O2-treated longans showed higher pulp breakdown index, cell membrane permeability, and activities of phospholipase D (PLD), lipase and lipoxygenase (LOX). Moreover, H2O2-treated longans maintained higher levels of pulp phosphatidic acid (PA) and saturated fatty acids (SFA). However, H2O2-treated longans exhibited lower levels of pulp phosphatidylcholine (PC), phosphatidylinositol (PI) and unsaturated fatty acids (USFA), lower index of unsaturated fatty acids (IUFA), and lower ratio of USFA to SFA (U/S). These findings demonstrated that H2O2 caused the increased activities of enzymes involving in membrane lipids degradation and the accelerated decompositions of membrane USFA and phospholipids in longan pulp, which eventually triggered the destruction of the pulp cell membrane structure and the development of pulp breakdown in longans during storage.


Assuntos
Enzimas/metabolismo , Frutas/química , Peróxido de Hidrogênio/farmacologia , Lipídeos de Membrana/metabolismo , Sapindaceae/química , Enzimas/química , Ácidos Graxos/análise , Ácidos Graxos/química , Armazenamento de Alimentos , Frutas/efeitos dos fármacos , Frutas/metabolismo , Peróxido de Hidrogênio/química , Lipase/química , Lipase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoxigenase/química , Lipoxigenase/metabolismo , Lipídeos de Membrana/química , Fosfolipase D/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sapindaceae/efeitos dos fármacos , Sapindaceae/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-31176865

RESUMO

Antarctic notothenioid fishes are highly stenothermal, yet their tolerance for warming is species-dependent. Because a body of literature points to the loss of cardiac function as underlying thermal limits in ectothermic animals, we investigated potential relationships among properties of ventricular mitochondrial membranes in notothenioids with known differences in both cardiac mitochondrial metabolism and organismal thermal tolerance. Fluidity of mitochondrial membranes was quantified by fluorescence depolarization for the white-blooded Chaenocephalus aceratus and the red-blooded Notothenia coriiceps. In these same membranes, lipid compositions and products of lipid peroxidation, the latter of which can disrupt membrane order, were analyzed in both species and in a second icefish, Pseudochaenichthys georgianus. Mitochondrial membranes from C. aceratus were significantly more fluid than those of the more thermotolerant species N. coriiceps (P < .0001). Consistent with this, ratios of total phosphatidylethanolamine (PE) to total phosphatidylcholine (PC) were lower in membranes from both species of icefishes, compared to those of N. coriiceps (P < .05). However, membranes of N. coriiceps displayed a greater unsaturation index (P < .0001). No differences among species were found in membrane products of lipid peroxidation. With rising temperatures, greater contents of PC in mitochondrial membranes from ventricles of icefishes are likely to promote membrane hyperfluidization at a lower temperature than for cardiac mitochondrial membranes from the red-blooded notothenioid. We propose that physical and chemical properties of the mitochondrial membranes may contribute to some of the observed differences in thermal sensitivity of physiological function among these species.


Assuntos
Lipídeos de Membrana/metabolismo , Miocárdio/metabolismo , Perciformes/metabolismo , Fosfolipídeos/metabolismo , Animais , Regiões Antárticas , Aquecimento Global , Peroxidação de Lipídeos , Fluidez de Membrana , Lipídeos de Membrana/química , Membranas Mitocondriais/metabolismo , Fosfolipídeos/química , Especificidade da Espécie , Termotolerância
6.
Plant Mol Biol ; 101(1-2): 81-93, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31201686

RESUMO

KEY MESSAGE: Here we show that accumulation of galactose-containing lipids in plastid membranes in shoots and the other membranes in roots maintains Arabidopsis growth under acidic stress and acidic phosphate deficiency. Soil acidification and phosphate deficiency are closely related to each other in natural environments. In addition to the toxicity of high proton concentrations, acid soil can lead to imbalances of ion availability and nutritional deficiencies, including inorganic phosphate (Pi). Among plants, activation of non-phosphorus-containing galactolipid, digalactosyldiacylglycerol (DGDG), synthesis concomitant with phospholipid degradation, namely membrane lipid remodeling, is crucial for coping with Pi starvation. However, regulation mechanisms of membrane lipid composition during acidic stress have not been clarified. Here, we investigated lipid metabolism in Arabidopsis thaliana grown under acidic stress with or without Pi. Under Pi-sufficient acidic conditions, DGDG was increased in shoot membranes, and some Pi starvation-responsive genes that are involved in lipid remodeling were upregulated without reducing Pi content in leaves. In contrast, under acidic Pi deficiency, membrane lipid remodeling in roots was partially repressed at a lower external pH. Nevertheless, phenotypic comparison between wild type and the double mutant of MGD2/3, which are responsible for DGDG accumulation during Pi starvation, indicated that the complete absence of lipid remodeling in roots resulted in a loss of tolerance to Pi deficiency rather specifically under acidic conditions. This result suggested important physiological roles of galactolipid-enriched membranes under acidic Pi deficiency.


Assuntos
Arabidopsis/fisiologia , Galactolipídeos/metabolismo , Metabolismo dos Lipídeos , Lipídeos de Membrana/metabolismo , Fosfatos/deficiência , Fosfolipídeos/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Fenótipo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plastídeos/metabolismo , Estresse Fisiológico
7.
Nat Commun ; 10(1): 2413, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160571

RESUMO

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx. Here we report the cryo-electron microscopy structure of the Syt1-SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg2+)-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the 'primary' interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1.


Assuntos
Membrana Celular/ultraestrutura , Lipídeos de Membrana/metabolismo , Proteínas SNARE/ultraestrutura , Sinaptotagmina I/ultraestrutura , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Magnésio/metabolismo , Fusão de Membrana , Neurotransmissores/metabolismo , Ligação Proteica , Ratos , Proteínas SNARE/metabolismo , Transmissão Sináptica , Sinaptotagmina I/metabolismo
8.
Folia Histochem Cytobiol ; 57(2): 43-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31099889

RESUMO

Biological membranes are organized in various microdomains, one of the best known being called membrane rafts. The major function of these is thought to organize signaling partners into functional complexes. An important protein found in membrane raft microdomains of erythroid and other blood cells is MPP1 (membrane palmitoylated protein 1)/p55. MPP1 (p55) belongs to the MAGUK (membrane-associated guanylate kinase homolog) family and it is a major target of palmitoylation in the red blood cells (RBCs) membrane. The well-known function of this protein is to participate in formation of the junctional complex of the erythrocyte mem-brane skeleton. However, its function as a "raft organizer" is not well understood. In this review we focus on recent reports concerning MPP1 participation in membrane rafts organization in erythroid cells, including its role in signal transduction. Currently it is not known whether MPP1 could have a similar role in cell types other than erythroid lineage. We present also preliminary data regarding the expression level of MPP1 gene in several non-erythroid cell lines.


Assuntos
Proteínas Sanguíneas/metabolismo , Membrana Celular/metabolismo , Eritrócitos/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Sanguíneas/genética , Colesterol/metabolismo , Humanos , Fluidez de Membrana/fisiologia , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/genética , Ligação Proteica
9.
Int J Mol Sci ; 20(9)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31052427

RESUMO

Biological membranes are key elements for the maintenance of cell architecture and physiology. Beyond a pure barrier separating the inner space of the cell from the outer, the plasma membrane is a scaffold and player in cell-to-cell communication and the initiation of intracellular signals among other functions. Critical to this function is the plasma membrane compartmentalization in lipid microdomains that control the localization and productive interactions of proteins involved in cell signal propagation. In addition, cells are divided into compartments limited by other membranes whose integrity and homeostasis are finely controlled, and which determine the identity and function of the different organelles. Here, we review current knowledge on membrane lipid composition in the plasma membrane and endomembrane compartments, emphasizing its role in sustaining organelle structure and function. The correct composition and structure of cell membranes define key pathophysiological aspects of cells. Therefore, we explore the therapeutic potential of manipulating membrane lipid composition with approaches like membrane lipid therapy, aiming to normalize cell functions through the modification of membrane lipid bilayers.


Assuntos
Membrana Celular/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Lipídeos de Membrana/química , Doenças Metabólicas/tratamento farmacológico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Animais , Compartimento Celular , Membrana Celular/química , Membrana Celular/metabolismo , Ácidos Graxos Insaturados/uso terapêutico , Humanos , Lipídeos de Membrana/metabolismo
10.
BMC Plant Biol ; 19(1): 221, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138155

RESUMO

BACKGROUND: Drought stress negatively affects plant growth and productivity. Plants sense soil drought at the root level but the underlying mechanisms remain unclear. At the cell level, we aim to reveal the short-term root perception of drought stress through membrane dynamics. RESULTS: In our study, 15 Medicago truncatula accessions were exposed to a polyethylene glycol (PEG)-induced drought stress, leading to contrasted ecophysiological responses, in particular related to root architecture plasticity. In the reference accession Jemalong A17, identified as drought susceptible, we analyzed lateral roots by imaging of membrane-localized fluorescent probes using confocal microscopy. We found that PEG stimulated endocytosis especially in cells belonging to the growth differentiation zone (GDZ). The mapping of membrane lipid order in cells along the root apex showed that membranes of root cap cells were more ordered than those of more differentiated cells. Moreover, PEG triggered a significant increase in membrane lipid order of rhizodermal cells from the GDZ. We initiated the membrane analysis in the drought resistant accession HM298, which did not reveal such membrane modifications in response to PEG. CONCLUSIONS: Our data demonstrated that the plasma membranes of root cells from a susceptible genotype perceived drought stress by modulating their physical state both via a stimulation of endocytosis and a modification of the degree of lipid order, which could be proposed as mechanisms required for signal transduction.


Assuntos
Secas , Endocitose , Medicago truncatula/fisiologia , Lipídeos de Membrana/metabolismo , Genótipo , Medicago truncatula/genética , Células Vegetais/metabolismo , Células Vegetais/fisiologia , Polietilenoglicóis/administração & dosagem , Rizoma/metabolismo , Rizoma/fisiologia , Estresse Fisiológico
11.
Phys Chem Chem Phys ; 21(22): 11554-11563, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31134261

RESUMO

Biological membranes are characterized by lateral inhomogeneities, termed as membrane domains, which are regions enriched with specific types of lipids and proteins. While the functional consequences of membrane domains are well understood, the physicochemical study of domains has proved to be elusive, mainly due to varying spatiotemporal scales associated with them. In this perspective, we provide an overview of representative experimental approaches based on dynamic fluorescence microscopy to analyze organization and dynamics of membrane lipids and proteins. We further elucidate variation of dynamics as a function of area of observation, a unique feature of biological membranes, and its modulation with membrane components such as cholesterol and the actin cytoskeleton. In terms of spatial resolution, we provide examples from super resolution techniques that overcome the diffraction limit encountered in conventional optical microscopes. We conclude that judicious use of a combination of approaches of varying spatiotemporal resolutions, commensurate with spatiotemporal scales of a given membrane process, would provide a comprehensive dynamic model of the biological membrane in terms of membrane organization, dynamics and function.


Assuntos
Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Linhagem Celular , Membrana Celular/química , Difusão , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana , Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/química , Espectrometria de Fluorescência/métodos , Fatores de Tempo
12.
Nat Commun ; 10(1): 2234, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110175

RESUMO

The ß2 adrenergic receptor (ß2AR) signals through both Gs and Gi in cardiac myocytes, and the Gi pathway counteracts the Gs pathway. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study ß2AR-Gi interactions. Here we investigate the role of phospholipid composition on Gs and Gi coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the ß2AR, we find that they impair coupling to Gi3 and facilitate coupling to Gs. Positively charged Ca2+ and Mg2+, known to interact with the negative charge on phospholipids, facilitates Gi3 coupling. Mutational analysis suggests that Ca2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of Gi3. Taken together, our observations suggest that local membrane charge modulates the interaction between ß2AR and competing G protein subtypes.


Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Lipídeos de Membrana/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Motivos de Aminoácidos , Animais , Cátions/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Lipídeos de Membrana/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Eletricidade Estática
13.
J Mol Model ; 25(5): 121, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31020452

RESUMO

Heroin, or diamorphine (C21H23NO5), is an opium product used for various pharmaceutical and euphoric purposes. In this work, the molecular dynamics simulation study of the heroin inside the two lipid bilayers, dipalmitoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) are presented. The whole study was conducted at three different temperatures. The location of the heroin drug, the nature of the diffusion, rotational correlation function and structural variation inside both lipid bilayers is studied. Moreover, the free energy of the solvation of the drug inside both lipid bilayers is calculated. It is found that during the whole molecular dynamics study, the drug locates at the center of both lipid membranes. The effect of the temperature is not seen at the drug location. The nature of the diffusion of the heroin drug is anomalous. The radius of gyration is calculated to study the structural variations of the heroin molecule inside both lipid bilayers. It is found that the heroin molecule does not change its structure at three temperatures. From the rotational correlation function, it is seen that the drug is more hindered for rotation inside the DPPC lipid bilayer as compared to the DMPC lipid bilayer. It is applicable for all three temperatures. The rotational correlation time of the drug is decreased while the temperature of the system is increased. In the case of DMPC, there is an abrupt change in rotational correlation time while the phase is changed.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , Heroína/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Difusão , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Heroína/toxicidade , Humanos , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Termodinâmica
14.
Virus Res ; 265: 143-149, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30928427

RESUMO

En Bloc transmission of viruses allow multiple genomes to collectivelly penetrate and initiate infection in the same cell, often resulting in enhanced infectivity. Given the quasispecies (mutant cloud) nature of RNA viruses and many DNA viruses, en bloc transmission of multiple genomes provides different starting points in sequence space to initiate adaptive walks, and has implications for modulation of viral fitness and for the response of viral populations to lethal mutagenesis. Mechanisms that can enable multiple viral genomes to be transported en bloc among hosts has only recently been gaining attention. A growing body of research suggests that extracellular vesicles (EV) are highly prevalent and robust vehicles for en bloc delivery of viral particles and naked infectious genomes among organisms. Both RNA and DNA viruses appear to exploit these vesicles to increase their multiplicity of infection and enhance virulence.


Assuntos
Vesículas Extracelulares/virologia , Genoma Viral , Viroses/transmissão , Vírus/genética , Animais , Vírus de DNA/genética , Vírus de DNA/patogenicidade , Humanos , Lipídeos de Membrana/metabolismo , Camundongos , Quase-Espécies , Vírus de RNA/genética , Vírus de RNA/patogenicidade , Vírion/genética , Replicação Viral , Vírus/patogenicidade
15.
Plant Cell Physiol ; 60(7): 1567-1580, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31020320

RESUMO

The surface of most aerial plant organs is covered with the cuticle, a membrane consisting of a variety of organic compounds, including waxes, cutin (a polyester) and polysaccharides. The cuticle serves as the multifunctional interface between the plant and the environment, and plays a major role in protecting plants against various environmental stress factors. Characterization of the molecular arrangements in the intact cuticle is critical for the fundamental understanding of its physicochemical properties; however, this analysis remains technically challenging. Here, we describe the nondestructive characterization of the intact cuticle of Brassica oleracea L. leaves using polarization modulation-infrared (IR) reflection-absorption spectroscopy (PM-IRRAS). PM-IRRAS has a probing depth of less than several hundreds of nanometers, and reveals the crystalline structure of the wax covering the cuticle surface (epicuticular wax) and the nonhydrogen-bonding character of cutin. Combined analysis using attenuated total reflection-IR spectra suggested that hemicelluloses xylan and xyloglucan are present in the outer cuticle region close to the epicuticular wax, whereas pectins are dominant in the inner cuticle region (depth of ≤2 µm). PM-IRRAS can also determine the average orientation of the cuticular molecules, as indicated by the positive and negative spectral peaks. This unique advantage reveals the orientational order in the intact cuticle; the hydrocarbon chains of the epicuticular wax and cutin and the backbones of hemicelluloses are oriented perpendicular to the leaf surface. PM-IRRAS is a versatile, informative and easy-to-use technique for studying plant cuticles because it is nondestructive and does not require sample pretreatment and background measurements.


Assuntos
Brassica/metabolismo , Folhas de Planta/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Brassica/química , Glucanos/química , Glucanos/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Epiderme Vegetal/química , Epiderme Vegetal/metabolismo , Folhas de Planta/química , Xilano Endo-1,3-beta-Xilosidase/química , Xilano Endo-1,3-beta-Xilosidase/metabolismo , Xilanos/química , Xilanos/metabolismo
16.
Food Microbiol ; 82: 303-315, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027788

RESUMO

The replacement of the most prevalent Salmonella enterica genotypes has been documented worldwide. Here we tested the hypothesis that the current prevalent sequence type ST213 of serotype Typhimurium in Mexico has a higher resistance to stressful food preservation conditions than the displaced sequence ST19. ST19 showed higher cell viability percentages than ST213 in osmotic (685 mM NaCl) and acidic (pH 3.5) stress conditions and in combination with refrigeration (4 °C) and ambient (≈22 °C) temperatures. Both genotypes showed the same poststress recovery growth. ST213 formed biofilm and filamentous cells (FCs) under stress, whereas ST19 did not. ST213 cells also showed higher motility. The capacity of ST213 to form FCs may explain its lower viability percentages when compared with ST19, i.e., ST213 cells divided less under stress conditions, but FCs had the same recovery capacity of ST19 cells. ST213 presented a higher unsaturated/saturated fatty acids ratio (0.5-0.6) than ST19 (0.2-0.5), which indicates higher membrane fluidity. The transcript levels of the rpoS gene were similar between genotypes under the experimental conditions employed. Biofilm formation, the generation of FCs, cell motility and membrane modification seem to make ST213 more resistant than ST19 to food preservation environments.


Assuntos
Conservação de Alimentos/métodos , Salmonella typhimurium/fisiologia , Estresse Fisiológico , Adaptação Fisiológica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Temperatura Baixa , Genótipo , Concentração de Íons de Hidrogênio , Lipídeos de Membrana/metabolismo , Viabilidade Microbiana , Salmonella typhimurium/citologia , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Cloreto de Sódio , Estresse Fisiológico/genética
17.
Nat Cell Biol ; 21(4): 452-461, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936472

RESUMO

Particles that bud off from the cell surface, including viruses and microvesicles, typically have a unique membrane protein composition distinct from that of the originating plasma membrane. This selective protein composition enables viruses to evade the immune response and infect other cells. But how membrane proteins sort into budding viruses such as human immunodeficiency virus (HIV) remains unclear. Proteins could passively distribute into HIV-assembly-site membranes producing compositions resembling pre-existing plasma-membrane domains. Here, we demonstrate that proteins instead sort actively into HIV-assembly-site membranes, generating compositions enriched in cholesterol and sphingolipids that undergo continuous remodelling. Proteins are recruited into and removed from the HIV assembly site through lipid-based partitioning, initiated by oligomerization of the HIV structural protein Gag. Changes in membrane curvature at the assembly site further amplify this sorting process. Thus, a lipid-based sorting mechanism, aided by increasing membrane curvature, generates the unique membrane composition of the HIV surface.


Assuntos
HIV/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Vírion/metabolismo , Animais , Antígeno 2 do Estroma da Médula Óssea/metabolismo , Células COS , Membrana Celular/ultraestrutura , Cercopithecus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , Humanos , Vírion/química
18.
Methods Mol Biol ; 1951: 209-216, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825155

RESUMO

Plasma membrane lipid rafts are highly ordered membrane microdomains enriched for glycosphingolipids and cholesterol, which play an important role during T-cell antigen receptor (TCR) signaling. Our previous work has demonstrated that plasma membrane lipid composition is an important determinant of human CD4+ T-cell function and that defects in lipid raft expression contribute to CD4+ dysfunction in patients with autoimmunity. In this chapter we share three flow cytometry-based methods to quantitatively analyze plasma membrane lipid composition in primary human CD4+ T cells. We describe the quantification of glycosphingolipid expression using cholera toxin subunit B, cholesterol expression using filipin staining, and membrane "lipid order" using di-4-ANEPPDHQ. These methods can easily be adapted to analyze different cell types.


Assuntos
Membrana Celular/metabolismo , Citometria de Fluxo , Lipídeos de Membrana/metabolismo , Linfócitos T/metabolismo , Toxina da Cólera/metabolismo , Filipina/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Microdomínios da Membrana/metabolismo , Subpopulações de Linfócitos T/metabolismo
19.
Appl Microbiol Biotechnol ; 103(9): 3863-3874, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30863878

RESUMO

Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.


Assuntos
Aspergillus nidulans/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lipídeos de Membrana/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
J Biol Chem ; 294(16): 6468-6482, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819802

RESUMO

Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and water-related stress. Their suggested functions are in membrane stabilization and cell protection. All dehydrins contain at least one copy of the highly conserved K-segment, proposed to be a membrane-binding motif. The dehydrin Lti30 (Arabidopsis thaliana) is up-regulated during cold and drought stress conditions and comprises six K-segments, each with two adjacent histidines. Lti30 interacts with the membrane electrostatically via pH-dependent protonation of the histidines. In this work, we seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection- fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations. The dependence of the diffusion coefficient explored by ITIR-FCS together with MD simulations yields insights into Lti30 binding, domain partitioning, and aggregation. The effect of Lti30 on membrane lipid diffusion was studied on fluorescently labeled supported lipid bilayers of different lipid compositions at mechanistically important pH conditions. In parallel, we compared the mode of diffusion for short individual K-segment peptides. The results indicate that Lti30 binds the lipid bilayer via electrostatics, which restricts the mobility of lipids and bound protein molecules. At low pH, Lti30 binding induced lipid microdomain formation as well as protein aggregation, which could be correlated with one another. Moreover, at physiological pH, Lti30 forms nanoscale aggregates when proximal to the membrane suggesting that Lti30 may protect the cell by "cross-linking" the membrane lipids.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Lipídeos de Membrana , Simulação de Dinâmica Molecular , Pressão Osmótica , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Domínios Proteicos
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