89 results on '"Rodrigo F.M. de Almeida"'
Search Results
2. Ruthenium(II) polypyridyl complexes with benzothiophene and benzimidazole derivatives: Synthesis, antitumor activity, solution studies and biospeciation
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Orsolya Dömötör, Ricardo G. Teixeira, Gabriella Spengler, Fernando Avecilla, Fernanda Marques, Oscar A. Lenis-Rojas, Cristina P. Matos, Rodrigo F.M. de Almeida, Éva A. Enyedy, and Ana Isabel Tomaz
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Inorganic Chemistry ,Ovarian Neoplasms ,Coordination Complexes ,Cell Line, Tumor ,01.04. Kémiai tudományok ,Humans ,Female ,Antineoplastic Agents ,Benzimidazoles ,Thiophenes ,Biochemistry ,Ruthenium - Abstract
With the aim to incorporate pharmacophore motifs into the Ru(II)-polypyridyl framework, compounds [Ru(II)(1,10-phenantroline)
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- 2022
3. C-Glucosylation as a tool for the prevention of PAINS-induced membrane dipole potential alterations
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Maria Conceição Oliveira, Rodrigo F.M. de Almeida, Carla Sousa, Amélia P. Rauter, Maria Teresa Blázquez-Sánchez, and Ana M. Matos
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0301 basic medicine ,Phloretin ,Science ,Biophysics ,Article ,Cell membrane ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Lipid raft ,Multidisciplinary ,Natural product ,food and beverages ,Transmembrane protein ,Chemistry ,Dipole ,030104 developmental biology ,medicine.anatomical_structure ,Membrane ,chemistry ,030220 oncology & carcinogenesis ,Medicine ,Function (biology) - Abstract
The concept of Pan-Assay Interference Compounds (PAINS) is regarded as a threat to the recognition of the broad bioactivity of natural products. Based on the established relationship between altered membrane dipole potential and transmembrane protein conformation and function, we investigate here polyphenols' ability to induce changes in cell membrane dipole potential. Ultimately, we are interested in finding a tool to prevent polyphenol PAINS-type behavior and produce compounds less prone to untargeted and promiscuous interactions with the cell membrane. Di-8-ANEPPS fluorescence ratiometric measurements suggest that planar lipophilic polyphenols—phloretin, genistein and resveratrol—act by decreasing membrane dipole potential, especially in cholesterol-rich domains such as lipid rafts, which play a role in important cellular processes. These results provide a mechanism for their labelling as PAINS through their ability to disrupt cell membrane homeostasis. Aiming to explore the role of C-glucosylation in PAINS membrane-interfering behavior, we disclose herein the first synthesis of 4-glucosylresveratrol, starting from 5-hydroxymethylbenzene-1,3-diol, via C-glucosylation, oxidation and Horner-Wadsworth-Emmons olefination, and resynthesize phloretin and genistein C-glucosides. We show that C-glucosylation generates compounds which are no longer able to modify membrane dipole potential. Therefore, it can be devised as a strategy to generate bioactive natural product derivatives that no longer act as membrane dipole potential modifiers. Our results offer a new technology towards rescuing bioactive polyphenols from their PAINS danger label through C–C ligation of sugars.
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- 2021
4. Sphingolipid‐enriched domains in fungi
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Joaquim T. Marquês, Filipa C. Santos, Andreia Bento-Oliveira, and Rodrigo F.M. de Almeida
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Antifungal ,medicine.drug_class ,Saccharomyces cerevisiae ,Biophysics ,Biochemistry ,Neurospora crassa ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Genetics ,medicine ,Molecular Biology ,030304 developmental biology ,Sphingolipids ,0303 health sciences ,Ergosterol ,biology ,Chemistry ,Cell Membrane ,030302 biochemistry & molecular biology ,Fungi ,Cell Biology ,Protein composition ,biology.organism_classification ,Sphingolipid ,Multicellular organism ,Membrane ,Carbohydrate Sequence ,lipids (amino acids, peptides, and proteins) - Abstract
Plasma membrane carries out multiple physiological functions that require its dynamic and tightly regulated organization into specialized domains of different size, stability, and lipid/protein composition. Sphingolipids are a group of lipids in which the plasma membrane is particularly enriched, thus being crucial for its structure and function. A specific type of sphingolipid-enriched plasma membrane domains, where ergosterol is depleted and lipids are tightly packed in a rigid gel phase, has recently been found in several fungal species, including yeasts and moulds. After presenting the main biophysical features of gel domains and the experimental method for their detection in the fungal plasma membrane, we review these sphingolipid-enriched gel domains and illustrate their importance to both unicellular and multicellular fungi. First, the biophysical properties of the fungal sphingolipid-enriched domains will be analysed taking into consideration the plasma membrane sphingolipidome. Next, their possible biological roles will be summarized, including their relations with plasma membrane compartments and involvement in stress responses. Moreover, since the plasma membrane is a target for several antifungal compounds, a biophysical connection between sphingolipid-enriched domains and antifungal action will be explored.
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- 2020
5. New anticandidal Cu(<scp>i</scp>) complexes with neocuproine and ketoconazole derived diphenyl(aminomethyl)phosphane: luminescence properties for detection in fungal cells
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Agata Białońska, Aneta Jezierska, Rodrigo F.M. de Almeida, Jakub Suchodolski, Ida Szmigiel, M. Puchalska, Jarosław J. Panek, Radosław Starosta, and Anna Krasowska
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Adult ,Antifungal Agents ,Cell Survival ,Phosphines ,Iodide ,Microbial Sensitivity Tests ,Redox ,Inorganic Chemistry ,Neocuproine ,chemistry.chemical_compound ,Cytochrome P-450 Enzyme System ,Coordination Complexes ,Candida albicans ,Cytochrome P-450 Enzyme Inhibitors ,Humans ,Moiety ,Molecule ,Cells, Cultured ,chemistry.chemical_classification ,Aqueous solution ,Molecular Structure ,Optical Imaging ,Combinatorial chemistry ,Molecular Docking Simulation ,Ketoconazole ,chemistry ,Docking (molecular) ,Luminescent Measurements ,Phosphorescence ,Copper ,Phenanthrolines - Abstract
The search for new antifungals is very important because the large genetic variation of pathogenic organisms has resulted in the development of increasingly effective defense mechanisms by microorganisms. Metal complexes as potential drugs are nowadays gaining interest, because they are characterized by accessible redox states of metal centers and a plethora of easily modifiable geometries. In this work we present two new copper(i) iodide or thiocyanide complexes with 2,9-dimethyl-1,10-phenanthroline (dmp) and a diphenylphosphane derivative of ketoconazole (KeP), where a ketoconazole acetyl group is replaced by the -CH2PPh2 unit, [CuI(dmp)KeP] (1-KeP) and [CuNCS(dmp)KeP] (2-KeP) - their synthesis and structural characteristics. The analysis of the intrinsic fluorescence of the ketoconazole moiety in the coordinated KeP molecule revealed that the copper(i) central atom does not act as a quencher and the observed decrease of fluorescence intensity is a result of a strong inner filter effect caused by the presence of the CuXdmp unit. Moreover, the complexes exhibit a remarkable MLCT (metal-ligand charge transfer) based phosphorescence with the emission maximum at 600-615 nm in aqueous media, which probably results from the formation of dimers and higher order oligomers in the most polar solutions. Both complexes proved to be promising antifungal agents towards Candida albicans, showing a relatively high efficiency towards the fluconazole resistant strains with -CDR1 and CDR2 or MDR1 efflux pump overexpression, which suggests that they overcome at least partially these defense mechanisms. Simulations of docking to the cytochrome P450 14α-demethylase (the azoles' primary molecular target) suggested that the compounds studied were rather incapable of competitively inhibiting this enzyme, unlike ketoconazole and the KeP ligand. On the other hand, the phosphorescence in aqueous solutions allowed recording the confocal micrographs of the complexes which showed that both of them are situated in spherical structures inside the cells, most likely in the vacuoles.
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- 2020
6. New diphenylphosphane derivatives of ketoconazole are promising antifungal agents
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Filipa C. Santos, Aneta Jezierska, Michalina Alicka, Anna Krasowska, Krzysztof Marycz, Radosław Starosta, Jarosław J. Panek, Jakub Suchodolski, and Rodrigo F.M. de Almeida
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0301 basic medicine ,Antifungal Agents ,genetic structures ,030106 microbiology ,lcsh:Medicine ,Apoptosis ,Saccharomyces cerevisiae ,behavioral disciplines and activities ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Candida albicans ,medicine ,Humans ,Drug discovery and development ,lcsh:Science ,Ergosterol ,Multidisciplinary ,biology ,Stem Cells ,Lanosterol ,Biphenyl Compounds ,lcsh:R ,Cytochrome P450 ,Drug Synergism ,biology.organism_classification ,Corpus albicans ,Ketoconazole ,030104 developmental biology ,Adipose Tissue ,nervous system ,Mechanism of action ,Biochemistry ,chemistry ,Docking (molecular) ,biology.protein ,lcsh:Q ,medicine.symptom ,psychological phenomena and processes ,medicine.drug - Abstract
Four new derivatives of ketoconazole (Ke) were synthesized: diphenylphosphane (KeP), and phosphane chalcogenides: oxide (KeOP), sulphide (KeSP) and selenide (KeSeP). These compounds proved to be promising antifungal compounds towards Saccharomyces cerevisiae and Candida albicans, especially in synergy with fluconazole. Simulations of docking to the cytochrome P450 14α-demethylase (azoles’ primary molecular target) proved that the new Ke derivatives are capable of inhibiting this enzyme by binding to the active site. Cytotoxicity towards hACSs (human adipose-derived stromal cells) of the individual compounds was studied and the IC50 values were higher than the MIC50 for C. albicans and S. cerevisiae. KeP and KeOP increased the level of the p21 gene transcript but did not change the level of p53 gene transcript, a major regulator of apoptosis, and decreased the mitochondrial membrane potential. Taken together, the results advocate that the new ketoconazole derivatives have a similar mechanism of action and block the lanosterol 14α-demethylase and thus inhibit the production of ergosterol in C. albicans membranes.
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- 2019
7. Experience report on the recovery of a person infected with covid-19 with admission to the Intensive Care Unit (ICU): physical education and return to activities of daily living / Relatório de experiência sobre a recuperação de uma pessoa infectada pela covid-19 com admissão na Unidade de Terapia Intensiva (UTI): educação física e retorno às atividades da vida diária
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Carlos Nazareno Ferreira Borges, Hudson Renato de Paula Oliveira, Rodrigo F.M. de Almeida, Walk Loureiro, and Leonardo Perovano-Camargo
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Activities of daily living ,Coronavirus disease 2019 (COVID-19) ,Pandemic ,business.industry ,General Medicine ,Physical rehabilitation ,Intensive care unit ,law.invention ,Physical education ,Virus ,Nursing ,law ,Medicine ,Experience report ,Pandemic, Virus ,PE ,business - Abstract
Faced with the challenge to global health experience with the respiratory disease called Coronavirus Disease 2019 (Covid-19) - discovered in December 2019 and declared a pandemic in March 2020 -, the recovery process of a hospitalized person with severe symptomsof COVID-19 is reported, who had physical limitations, and returned to activities of daily living after the immediate rehabilitation process. The text organization is in the form of an experience report. The investigated subject had the covid-19 infection confirmed by a nasal swab exam collected on February 25, 2021. The onset of symptoms reported was four days earlier, presenting dyspnea, dry cough, headache, and fever after being hospitalized after laboratory, imaging, and clinical examinations. After medical discharge (held on March 19, 2021), he was released with the recommendation of respiratory physiotherapy activities. After four weeks of intervention with a physical education professional in resistance activities, stretching, and aerobic exercises, the individual was able to perform the activities of daily living that he did before the infection. His only current complaint is related to intense activities, in which he still feels uncomfortable, requiring longitudinal monitoring for the security of his full recovery.
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- 2021
8. Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes
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Joaquim T. Marquês, Maria da Soledade Santos, Amélia P. Rauter, Rodrigo F.M. de Almeida, Ricardo Dias, João P. Pais, Alice Martins, Patrícia Serra, Rogério Tenreiro, Andreia Almeida, Marialessandra Contino, Catarina Dias, Ana S. Viana, Rafael Nunes, Maria Cristina Oliveira, Maria-Teresa Blázquez-Sánchez, Nuno M. Xavier, Nicola Antonio Colabufo, Miguel Machuqueiro, Diogo Vila-Viçosa, and Ana Pelerito
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0301 basic medicine ,Lipid Bilayers ,Bacillus cereus ,General Physics and Astronomy ,01 natural sciences ,Bacterial cell structure ,Cell membrane ,chemistry.chemical_compound ,Cell Wall ,Carbohydrate Conformation ,Glycosides ,Lipid bilayer ,lcsh:Science ,Multidisciplinary ,biology ,Chemistry ,3. Good health ,Bacillus anthracis ,Anti-Bacterial Agents ,medicine.anatomical_structure ,Biochemistry ,medicine.symptom ,Cell Survival ,Science ,Microbial Sensitivity Tests ,010402 general chemistry ,Article ,General Biochemistry, Genetics and Molecular Biology ,Phase Transition ,03 medical and health sciences ,Structure-Activity Relationship ,medicine ,Humans ,Phosphatidylethanolamine ,Microbial Viability ,Phosphatidylethanolamines ,Cell Membrane ,fungi ,General Chemistry ,biology.organism_classification ,0104 chemical sciences ,Kinetics ,030104 developmental biology ,Mechanism of action ,lcsh:Q ,Caco-2 Cells ,Bacteria - Abstract
Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials., Bacillus anthracis causes the infectious disease anthrax. Here, the authors synthesized deoxy glycosides that are effective against B. anthracis and related bacteria and found that these amphiphilic compounds kill bacteria via an unusual mechanism of action.
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- 2018
9. Glucosylpolyphenols as Inhibitors of Aβ-Induced Fyn Kinase Activation and Tau Phosphorylation: Synthesis, Membrane Permeability, and Exploratory Target Assessment within the Scope of Type 2 Diabetes and Alzheimer's Disease
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Claire J. Garwood, M Teresa Blázquez-Sánchez, Andreia Bento-Oliveira, Filipa Marcelo, Amélia P. Rauter, Joana S. Cristóvão, Pedro Lopes, José G. Fernández-Bolaños, Nicola Antonio Colabufo, Ke Ning, Ana Diniz, Cláudio M. Gomes, Ana M. Matos, Rafael Nunes, M Conceição Oliveira, Imane Ghafir El Idrissi, Beat Ernst, Cleide dos Santos Souza, Beining Chen, Rodrigo F.M. de Almeida, Philipp Dätwyler, Miguel Machuqueiro, and Óscar López
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Cell Membrane Permeability ,Amyloid ,Membrane permeability ,Glycoside Hydrolases ,Induced Pluripotent Stem Cells ,Context (language use) ,tau Proteins ,Pharmacology ,Proto-Oncogene Proteins c-fyn ,01 natural sciences ,03 medical and health sciences ,FYN ,Glucosides ,Alzheimer Disease ,Drug Discovery ,medicine ,Dementia ,Cholinesterases ,Humans ,Phosphorylation ,Butyrylcholinesterase ,030304 developmental biology ,0303 health sciences ,Amyloid beta-Peptides ,Molecular Structure ,Drug discovery ,Chemistry ,Polyphenols ,medicine.disease ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,HEK293 Cells ,Diabetes Mellitus, Type 2 ,Molecular Medicine - Abstract
Despite the rapidly increasing number of patients suffering from type 2 diabetes, Alzheimer's disease, and diabetes-induced dementia, there are no disease-modifying therapies that are able to prevent or block disease progress. In this work, we investigate the potential of nature-inspired glucosylpolyphenols against relevant targets, including islet amyloid polypeptide, glucosidases, and cholinesterases. Moreover, with the premise of Fyn kinase as a paradigm-shifting target in Alzheimer's drug discovery, we explore glucosylpolyphenols as blockers of Aβ-induced Fyn kinase activation while looking into downstream effects leading to Tau hyperphosphorylation. Several compounds inhibit Aβ-induced Fyn kinase activation and decrease pTau levels at 10 μM concentration, particularly the per-O-methylated glucosylacetophloroglucinol and the 4-glucosylcatechol dibenzoate, the latter inhibiting also butyrylcholinesterase and β-glucosidase. Both compounds are nontoxic with ideal pharmacokinetic properties for further development. This work ultimately highlights the multitarget nature, fine structural tuning capacity, and valuable therapeutic significance of glucosylpolyphenols in the context of these metabolic and neurodegenerative disorders.
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- 2020
10. Biophysical Analysis of Lipid Domains in Mammalian and Yeast Membranes by Fluorescence Spectroscopy
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Andreia Bento-Oliveira, Beatriz Mestre, Rodrigo F.M. de Almeida, Carla A. Sousa, Liana C. Silva, and Filipa C. Santos
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0301 basic medicine ,030103 biophysics ,Liposome ,biology ,Chemistry ,Saccharomyces cerevisiae ,biology.organism_classification ,Fluorescence spectroscopy ,Yeast ,03 medical and health sciences ,030104 developmental biology ,Membrane ,Förster resonance energy transfer ,Biophysics ,Fluorescence microscope ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin - Abstract
The use of steady-state and time-resolved fluorescence spectroscopy to study sterol and sphingolipid-enriched lipid domains as diverse as the ones found in mammalian and fungal membranes is herein described. We first address how to prepare liposomes that mimic raft-containing membranes of mammalian cells and how to use fluorescence spectroscopy to characterize the biophysical properties of these membrane model systems. We further illustrate the application of Forster resonance energy transfer (FRET) to study nanodomain reorganization upon interaction with small bioactive molecules, phenolic acids, an important group of phytochemical compounds. This methodology overcomes the resolution limits of conventional fluorescence microscopy allowing for the identification and characterization of lipid domains at the nanoscale.We continue by showing how to use fluorescence spectroscopy in the biophysical analysis of more complex biological systems, namely the plasma membrane of Saccharomyces cerevisiae yeast cells and the necessary adaptations to the filamentous fungus Neurospora crassa , evaluating the global order of the membrane, sphingolipid-enriched domains rigidity and abundance, and ergosterol-dependent properties.
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- 2020
11. Biophysical Analysis of Lipid Domains by Fluorescence Microscopy
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Tânia C B Santos, Ana E. Ventura, Liana C. Silva, Rodrigo F.M. de Almeida, and Joaquim T. Marquês
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0301 basic medicine ,Chemistry ,Vesicle ,Cell ,Synthetic membrane ,Biological membrane ,Context (language use) ,02 engineering and technology ,021001 nanoscience & nanotechnology ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Membrane ,medicine ,Fluorescence microscope ,Biophysics ,lipids (amino acids, peptides, and proteins) ,0210 nano-technology ,Lipid raft - Abstract
The study of the structure and dynamics of membrane domains in vivo is a challenging task. However, major advances could be achieved through the application of microscopic and spectroscopic techniques coupled with the use of model membranes, where the relations between lipid composition and the type, amount and properties of the domains present can be quantitatively studied.This chapter provides protocols to study membrane organization and visualize membrane domains by fluorescence microscopy both in artificial membrane and living cell models of Gaucher Disease (GD ). We describe a bottom-up multiprobe methodology, which enables understanding how the specific lipid interactions established by glucosylceramide, the lipid that accumulates in GD , affect the biophysical properties of model and cell membranes, focusing on its ability to influence the formation, properties and organization of lipid raft domains. In this context, we address the preparation of (1) raft-mimicking giant unilamellar vesicles labeled with a combination of fluorophores that allow for the visualization and comprehensive characterization of those membrane domains and (2) human fibroblasts exhibiting GD phenotype to assess the biophysical properties of biological membrane in living cells using fluorescence microscopy.
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- 2020
12. Liquid-Ordered Phase Formation by Mammalian and Yeast Sterols: A Common Feature With Organizational Differences
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H. Susana Marinho, Andreia Bento-Oliveira, Rodrigo F.M. de Almeida, Joaquim T. Marquês, Gerson M. da S. Lobo, Andreas Herrmann, Rui Malhó, André E.P. Bastos, Silvia Scolari, Alena Khmelinskaia, and Catarina A.C. Antunes
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0301 basic medicine ,Zymosterol ,zymosterol ,Membrane permeability ,Liquid ordered phase ,570 Biologie ,plasma membrane ,Cell and Developmental Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,ddc:570 ,polycyclic compounds ,lcsh:QH301-705.5 ,Original Research ,fluorescence lifetime imaging microscopy ,Ergosterol ,Plasma membrane organization ,ergosterol ,sterol-rich domain ,cholesterol ,Cell Biology ,fluorescence spectroscopy ,Yeast ,Sterol ,030104 developmental biology ,Membrane ,lcsh:Biology (General) ,chemistry ,030220 oncology & carcinogenesis ,Biophysics ,lipids (amino acids, peptides, and proteins) ,lipid-lipid interactions ,Developmental Biology - Abstract
Here, biophysical properties of membranes enriched in three metabolically related sterols are analyzed both in vitro and in vivo. Unlike cholesterol and ergosterol, the common metabolic precursor zymosterol is unable to induce the formation of a liquid ordered (lo) phase in model lipid membranes and can easily accommodate in a gel phase. As a result, Zym has a marginal ability to modulate the passive membrane permeability of lipid vesicles with different compositions, contrary to cholesterol and ergosterol. Using fluorescence-lifetime imaging microscopy of an aminostyryl dye in living mammalian and yeast cells we established a close parallel between sterol-dependent membrane biophysical properties in vivo and in vitro. This approach unraveled fundamental differences in yeast and mammalian plasma membrane organization. It is often suggested that, in eukaryotes, areas that are sterol-enriched are also rich in sphingolipids, constituting highly ordered membrane regions. Our results support that while cholesterol is able to interact with saturated lipids, ergosterol seems to interact preferentially with monounsaturated phosphatidylcholines. Taken together, we show that different eukaryotic kingdoms developed unique solutions for the formation of a sterol-rich plasma membrane, a common evolutionary trait that accounts for sterol structural diversity.
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- 2020
13. Unveiling the Mechanism of Action of 7α-acetoxy-6β-hydroxyroyleanone on an MRSA/VISA Strain: Membrane and Cell Wall Interactions
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Patrícia Rijo, Catarina Pinto Reis, Teresa A. Figueiredo, Catarina A.C. Antunes, Catarina Garcia, Filipe Pereira, Rodrigo F.M. de Almeida, Lia Ascensão, Rita G. Sobral, UCIBIO - Applied Molecular Biosciences Unit, and DCV - Departamento de Ciências da Vida
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Methicillin-Resistant Staphylococcus aureus ,0301 basic medicine ,Cell Membrane Permeability ,Lysis ,medicine.drug_class ,030106 microbiology ,Antibiotics ,lcsh:QR1-502 ,Microbial Sensitivity Tests ,MRSA ,medicine.disease_cause ,Biochemistry ,Article ,lcsh:Microbiology ,Microbiology ,Cell wall ,03 medical and health sciences ,antibacterial activity ,medicine ,7α-Acetoxy-6β-hydroxyroyleanone ,Humans ,membrane interaction ,Mode of action ,Molecular Biology ,Microbial Viability ,biology ,Chemistry ,Cell Membrane ,biology.organism_classification ,Vancomycin-Resistant Staphylococcus aureus ,3. Good health ,Bacterial Outer Membrane ,Membrane interaction ,030104 developmental biology ,Mechanism of action ,Staphylococcus aureus ,cell wall ,Antibacterial activity ,Diterpenes ,medicine.symptom ,Bacteria - Abstract
The number of cases of failure in the treatment of infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, 7&alpha, Acetoxy-6&beta, hydroxyroyleanone (AHR), a diterpene isolated from different Plectranthus species, showed antibacterial activity, namely against Methicillin-resistant Staphylococcus aureus (MRSA) strains. The high antibacterial activity and low cytotoxicity render this natural compound an interesting alternative against resistant bacteria. The aim of this study is to understand the mechanism of action of AHR on MRSA, using the MRSA/Vancomycin-intermediate S. aureus (VISA) strain CIP 106760, and to study the AHR effect on lipid bilayers and on the cell wall. Although AHR interacted with lipid bilayers, it did not have a significant effect on membrane passive permeability. Alternatively, bacteria treated with this royleanone displayed cell wall disruption, without revealing cell lysis. In conclusion, the results gathered so far point to a yet undescribed mode of action that needs further investigation.
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- 2020
14. Sphingolipid hydroxylation in mammals, yeast and plants – An integrated view
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H. Susana Marinho, Joaquim T. Marquês, and Rodrigo F.M. de Almeida
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0301 basic medicine ,Membrane lipids ,Cell ,Saccharomyces cerevisiae ,Hydroxylation ,Biochemistry ,Membrane Lipids ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,Animals ,Humans ,Mammals ,Sphingolipids ,Molecular Structure ,Chemistry ,Cell Biology ,Plants ,Sphingolipid ,Yeast ,Cell biology ,030104 developmental biology ,Membrane ,medicine.anatomical_structure ,Sphingolipid metabolism ,lipids (amino acids, peptides, and proteins) ,Metabolic Networks and Pathways ,Function (biology) - Abstract
This review is focused on sphingolipid backbone hydroxylation, a small but widespread structural feature with profound impact on membrane biophysical properties. We start by summarizing sphingolipid metabolism in mammalian cells, yeast and plants, focusing on how distinct hydroxylation patterns emerge in different eukaryotic kingdoms. Then, a comparison of the biophysical properties in membrane model systems and cellular membranes from diverse organisms is made. From an integrative perspective, these results can be rationalized considering that superficial hydroxyl groups in the backbone of sphingolipids (by intervening in the H-bond network) alter the balance of favorable interactions between membrane lipids. They may strengthen the bonding or compete with other hydroxyl groups, in particular the one of membrane sterols. Different sphingolipid hydroxylation patterns can stabilize/disrupt specific membrane domains or change whole plasma membrane properties, and therefore be important in the control of protein distribution, function and lateral diffusion and in the formation and overtime stability of signaling platforms. The recent examples explored throughout this review unveil a potentially key role for sphingolipid backbone hydroxylation in both physiological and pathological situations, as it can be of extreme importance for the proper organization of cell membranes in mammalian cells, yeast and, most likely, also in plants.
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- 2018
15. Differential targeting of membrane lipid domains by caffeic acid and its ester derivatives
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Joaquim T. Marquês, Hugo A. L. Filipe, Ana S. Viana, Rodrigo F.M. de Almeida, Diogo Vila-Viçosa, Carla Sousa, M. Soledade C.S. Santos, António de Granada-Flor, and Miguel Machuqueiro
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0301 basic medicine ,Lipid Bilayers ,Phospholipid ,Molecular Dynamics Simulation ,Depsides ,01 natural sciences ,Biochemistry ,Membrane Potentials ,Cell membrane ,Membrane Lipids ,03 medical and health sciences ,chemistry.chemical_compound ,Caffeic Acids ,Membrane Microdomains ,Physiology (medical) ,0103 physical sciences ,Hydroxybenzoates ,medicine ,Humans ,Lipid bilayer ,Lipid raft ,Cells, Cultured ,Phospholipids ,010304 chemical physics ,Chemistry ,Bilayer ,Cell Membrane ,Esters ,Phenolic acid ,Sphingomyelins ,Cholesterol ,030104 developmental biology ,medicine.anatomical_structure ,Membrane ,Cinnamates ,Biophysics ,Chlorogenic Acid ,Sphingomyelin - Abstract
Phenolic acids have been associated to a wide range of important health benefits underlain by a common molecular mechanism of action. Considering that significant membrane permeation is prevented by their hydrophilic character, we hypothesize that their main effects result from the interplay with cell membrane surface. This hypothesis was tested using the paradigmatic caffeic acid (CA) and two of its ester derivatives, rosmarinic (RA) and chlorogenic (CGA) acids, for which we predict, based on molecular dynamics simulations, a shallow location in phospholipid bilayers dependent on the protonation-state. Using complementary experimental approaches, an interaction with the membrane was definitely revealed for the three compounds, with RA exhibiting the highest lipid bilayer partition, and the redox signals of membrane-bound RA and CA being clearly detected. Cholesterol decreased the compounds bilayer partition, but not their ability to lower membrane dipole potential. In more complex membrane models containing also sphingomyelin, with liquid disordered (ld)/ liquid ordered (lo) phases coexistence, mimicking domains in the external leaflet of human plasma membrane, all compounds were able to affect nanodomains lateral organization. RA, and to a lesser extent CGA, decreased the size of lo domains. The most significant effect of CA was the possible formation of a rigid gel-like phase, enriched in sphingomyelin. In addition, all phenolic acids decreased the order of lo domains. In sum, phenolic acid effects on the membrane are enhanced in cholesterol-rich lo phases, which predominate in the outer leaflet of human cell membranes and are involved in many key cellular processes.
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- 2018
16. Biophysical impact of sphingosine and other abnormal lipid accumulation in Niemann-Pick disease type C cell models
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Ana C. Carreira, Šárka Pokorná, Rodrigo F.M. de Almeida, Ana E. Ventura, Emyr Lloyd-Evans, Anthony H. Futerman, Mathew W. Walker, and Liana C. Silva
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Cell ,CHO Cells ,Endosomes ,03 medical and health sciences ,chemistry.chemical_compound ,Cricetulus ,0302 clinical medicine ,Sphingosine ,Lysosome ,otorhinolaryngologic diseases ,medicine ,Membrane fluidity ,Animals ,Molecular Biology ,030304 developmental biology ,Sphingolipids ,0303 health sciences ,Chemistry ,Cholesterol ,Chinese hamster ovary cell ,Cell Membrane ,Niemann-Pick Disease, Type C ,Biological membrane ,Cell Biology ,Cell biology ,stomatognathic diseases ,Phenotype ,medicine.anatomical_structure ,NPC1 ,Lysosomes ,030217 neurology & neurosurgery - Abstract
Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC.
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- 2021
17. Studies on the mechanism of action of antitumor bis(aminophenolate) ruthenium(III) complexes
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Tamás Kiss, Fernanda Marques, Cristina P. Matos, A. P. Alves de Matos, Orsolya Dömötör, Rodrigo F.M. de Almeida, Ana Isabel Tomaz, Éva A. Enyedy, Leonor Côrte-Real, Carla Real, and M. Helena Garcia
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Models, Molecular ,Stereochemistry ,chemistry.chemical_element ,Antineoplastic Agents ,Apoptosis ,Aminophenols ,010402 general chemistry ,Cell morphology ,01 natural sciences ,Biochemistry ,Ruthenium ,Fluorescence spectroscopy ,Inorganic Chemistry ,chemistry.chemical_compound ,Microscopy, Electron, Transmission ,Coordination Complexes ,Cell Line, Tumor ,medicine ,Humans ,DAPI ,Methylene ,010405 organic chemistry ,Cell Cycle ,DNA ,Fluorescence ,0104 chemical sciences ,Mechanism of action ,chemistry ,Drug Screening Assays, Antitumor ,medicine.symptom ,Ethidium bromide - Abstract
Two recently published Ru(III) complexes bearing (N2O2) tetradentate bis(aminophenolate) ligands, formulated as [Ru(III)(salan)(PPh3)Cl] (salan is the tetradentate ligand 6,6'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol) in complex 1, or 2,2'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol) in complex 2; PPh3 is triphenylphosphane) and found very active against ovarian and breast adenocarcinoma human cells were studied to outline their antitumor mode of action. The human cisplatin-sensitive ovarian adenocarcinoma line A2780 was used herein as the cell model. At a 24h challenge (similarly as found before for 72h) both complexes are active, their cytotoxicity being comparable to that of cisplatin in the same conditions. As a possible target in the cell for their action, the interaction of 1 and 2 with DNA was assessed through displacement of well-established DNA fluorescent probes (ethidium bromide, EB, and 4',6-diamidino-2-phenylindole, DAPI) through steady-state and time-resolved fluorescence spectroscopy. The whole emission spectra were analyzed globally for the binary DNA-probe and ternary DNA-probe-Ru(III) complex systems. Both Ru(III) complexes can displace EB and bind to DNA with similar and moderate strong affinity with conditional stability constants of logK'=(5.05±0.01) for 1 and logK'=(4.79±0.01) for 2. The analysis of time-domain fluorescence intensity decays confirmed both qualitatively and quantitatively the model used to describe the binding and competition processes. Cell studies indicated that apoptosis is the major mechanism of cell death for both complexes, with 2 (the more active complex) promoting that process more efficiently than 1. Transmission electron micrographs revealed clear alterations on intracellular organization consistent with the induction of programmed cell death processes.
- Published
- 2017
18. A route to understanding yeast cellular envelope – plasma membrane lipids interplaying in cell wall integrity
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Rodrigo F.M. de Almeida
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0301 basic medicine ,Membrane lipids ,Saccharomyces cerevisiae ,Biochemistry ,Cell wall ,Cell membrane ,Membrane Lipids ,03 medical and health sciences ,chemistry.chemical_compound ,Cell Wall ,Ergosterol ,medicine ,Molecular Biology ,Sphingolipids ,biology ,Cell Membrane ,Cell Biology ,biology.organism_classification ,Sphingolipid ,Yeast ,Cell biology ,030104 developmental biology ,Membrane ,medicine.anatomical_structure ,chemistry ,lipids (amino acids, peptides, and proteins) - Abstract
Sphingolipids and sterols are major components of the plasma membrane in eukaryotic cells. A recent study provides new insight into the intricate but fundamental relationship between Saccharomyces cerevisiae membrane lipid composition and membrane biophysical properties, by showing that sphingolipids and sterols co-ordinately regulate cell wall integrity.
- Published
- 2018
19. Interaction with Blood Proteins of a Ruthenium(II) Nitrofuryl Semicarbazone Complex: Effect on the Antitumoral Activity
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Fernanda Marques, Rodrigo F.M. de Almeida, Dinorah Gambino, Andreia Bento-Oliveira, Bruno Demoro, Ana Isabel Tomaz, Lucía Otero, and João Costa Pessoa
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Models, Molecular ,Circular dichroism ,Pharmaceutical Science ,Antineoplastic Agents ,Serum Albumin, Human ,Plasma protein binding ,Article ,Ruthenium ,Analytical Chemistry ,lcsh:QD241-441 ,chemistry.chemical_compound ,lcsh:Organic chemistry ,Coordination Complexes ,Drug Discovery ,medicine ,Humans ,cancer ,Drug Interactions ,Physical and Theoretical Chemistry ,Cytotoxicity ,Semicarbazone ,Semicarbazones ,Molecular Structure ,Chemistry ,Dimethyl sulfoxide ,plasma protein binding ,Organic Chemistry ,Blood Proteins ,Models, Theoretical ,Human serum albumin ,Ligand (biochemistry) ,5-nitrofurylsemicarbazone ,Blood proteins ,Molecular biology ,circular dichroism ,ruthenium(II) ,Chemistry (miscellaneous) ,conditional binding constants ,human serum albumin ,Molecular Medicine ,cytotoxicity ,fluorescence ,human serum apo-transferrin ,Algorithms ,Protein Binding ,medicine.drug - Abstract
The steady rise in the cancer burden and grim statistics set a vital need for new therapeutic solutions. Given their high efficiency, metallodrugs are quite appealing in cancer chemotherapy. This work examined the anticancer activity of an anti-trypanosomal ruthenium-based compound bearing the 5-nitrofuryl pharmacophore, [RuII(dmso)2(5-nitro-2-furaldehyde semicarbazone)] (abbreviated as RuNTF, dmso is the dimethyl sulfoxide ligand). The cytotoxicity of RuNTF was evaluated in vitro against ovarian adenocarcinoma, hormone-dependent breast adenocarcinoma, prostate carcinoma (grade IV) and V79 lung fibroblasts human cells. The activity of RuNTF was similar to the benchmark metallodrug cisplatin for the breast line and inactive against the prostate line and lung fibroblasts. Given the known role of serum protein binding in drug bioavailability and the distribution via blood plasma, this study assessed the interaction of RuNTF with human serum albumin (HSA) by circular dichroism (CD) and fluorescence spectroscopy. The fluorescence emission quenching from the HSA-Trp214 residue and the lifetime data upon RuNTF binding evidenced the formation of a 1:1 {RuNTF-albumin} adduct with log Ksv = (4.58 ±, 0.01) and log KB = (4.55 ±, 0.01). This is supported by CD data with an induced CD broad band observed at ~450 nm even after short incubation times. Importantly, the binding to either HSA or human apo-transferrin is beneficial to the cytotoxicity of the complex towards human cancer cells by enhancing the cytotoxic activity of RuNTF.
- Published
- 2019
20. Evaluation of EGCG Loading Capacity in DMPC Membranes
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Bruno L. Victor, M. Manuela M. Raposo, Filipa Pires, Vananélia P.N. Geraldo, Miguel Machuqueiro, Rodrigo F.M. de Almeida, António de Granada-Flor, Osvaldo N. Oliveira, and Bárbara Rodrigues
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Lipid Bilayers ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Catechin ,Molecular dynamics ,Monolayer ,Electrochemistry ,General Materials Science ,Lipid bilayer ,Spectroscopy ,MEMBRANAS CELULARES ,Liposome ,Chemistry ,Bilayer ,Vesicle ,Surfaces and Interfaces ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,0104 chemical sciences ,Membrane ,Liposomes ,Biophysics ,lipids (amino acids, peptides, and proteins) ,Nanocarriers ,0210 nano-technology ,Dimyristoylphosphatidylcholine - Abstract
Catechins are molecules with potential use in different pathologies such as diabetes and cancer, but their pharmaceutical applications are often hindered by their instability in the bloodstream. This issue can be circumvented using liposomes as their nanocarriers for in vivo delivery. In this work, we studied the molecular details of (-)-epigallocatechin-3-gallate (EGCG) interacting with 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) monolayer/bilayer systems to understand the catechin loading ability and liposome stability, using experimental and computational techniques. The molecular dynamics simulations show the EGCG molecules deep inside the lipid bilayer, positioned below the lipid ester groups, generating a concentration-dependent lipid condensation. This effect was also inferred from the surface pressure isotherms of DMPC monolayers. In the polarization-modulated infrared reflection absorption spectra assays, the predominant effect at higher concentrations of EGCG (e.g., 20 mol %) was an increase in lipid tail disorder. The steady-state fluorescence data confirmed this disordered state, indicating that the catechin-induced liposome aggregation outweighs the condensation effects. Therefore, by adding more than 10 mol % EGCG to the liposomes, a destabilization of the vesicles occurs with the ensuing release of entrapped catechins. The loading capacity for DMPC seems to be limited by its disordered lipid arrangements, typical of a fluid phase. To further increase the clinical usefulness of liposomes, lipid bilayers with more stable and organized assemblies should be employed to avoid aggregation at large concentrations of catechin.
- Published
- 2019
21. Quercetin dual interaction at the membrane level
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Carla Sousa, António de Granada-Flor, Hugo A. L. Filipe, Rodrigo F.M. de Almeida, and M. Soledade C.S. Santos
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Cell ,Lipid Bilayers ,Fluorescence Polarization ,Oxidative phosphorylation ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Catalysis ,chemistry.chemical_compound ,Materials Chemistry ,medicine ,Lipid bilayer ,Sphingolipids ,Binding Sites ,010405 organic chemistry ,Cholesterol ,Metals and Alloys ,General Chemistry ,Sphingolipid ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,medicine.anatomical_structure ,Membrane ,chemistry ,Ceramics and Composites ,Biophysics ,lipids (amino acids, peptides, and proteins) ,Quercetin ,Fluorescence anisotropy - Abstract
Quercetin impact on lipid bilayers suggests a dual action mechanism at cell membranes. This widespread polyphenol displays high partition with low interference in the more fluid membrane domains, more vulnerable to oxidative attack, but strong perturbation of cholesterol/sphingolipid enriched domains, where signalling platforms are expected to assemble.
- Published
- 2019
22. Paving the Way to Fight Multi-Drug Resistant Tuberculosis
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Susana G. Santos, Filomena Martins, Catarina Frazão de Faria, Maria da Soledade Santos, Rodrigo F.M. de Almeida, and Joaquim T. Marquês
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business.industry ,Multi-drug-resistant tuberculosis ,Biophysics ,Medicine ,business ,medicine.disease ,Virology - Published
- 2021
23. Impact of Sphingolipid Profile in Yeast Gel Domains and Membrane Compartments
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Andreia Bento-Oliveira, Susana Marinho, Joaquim T. Marquês, Rodrigo F.M. de Almeida, Pedro M. R. Paulo, and Filipa C. Santos
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Membrane ,Chemistry ,Biophysics ,Sphingolipid ,Yeast ,Cell biology - Published
- 2021
24. Fungal Plasma Membrane Lipid Domains Alterations Upon Drug Exposure
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Filipa C. Santos, Arnaldo Videira, Andreia S. Fernandes, and Rodrigo F.M. de Almeida
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Drug ,Membrane ,Chemistry ,media_common.quotation_subject ,Biophysics ,media_common - Published
- 2021
25. NBD derived diphenyl(aminomethyl)phosphane – A new fluorescent dye for imaging of low pH regions and lipid membranes in living cells
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M. Puchalska, Kamila Stokowa-Sołtys, Radosław Starosta, Rodrigo F.M. de Almeida, Tânia C B Santos, Anna Krasowska, Liana C. Silva, and Jakub Suchodolski
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biology ,Endosome ,Chemistry ,Process Chemistry and Technology ,General Chemical Engineering ,Solvatochromism ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,biology.organism_classification ,01 natural sciences ,Fluorescence ,0104 chemical sciences ,Membrane ,Organelle ,Biophysics ,0210 nano-technology ,Candida albicans ,Molecular probe ,Intracellular - Abstract
This work presents the comparative characterization (X-ray crystallography, NMR, solvatochromism and pH dependence of electronic absorption, steady-state and time resolved fluorescence properties) of a novel NBD based phosphane: 7-nitro-4-(4-(diphenylphosphinomethyl-piperazin-1-yl)-2,1,3-benzoxadiazole (1) and an analogous compound: 7-nitro-4-(4-methyl-piperazin-1-yl)-2,1,3-benzoxadiazole (2). Protonated forms of the compounds are highly luminescent, while the neutral ones are not. The pKa values in water solutions are in the range 6.8–7.0 for 1 and 6.4–6.6 for 2. However, we showed that these values do not reflect the luminescence properties in more complex environments, where other equilibria may occur. Performance of 1 and 2 as potential molecular probes for biological imaging of living cells was evaluated towards mammalian (HEK 293T) and fungal cells (two Candida albicans strains: CAF2-1 - parental strain, and DSY1050 - mutant with no functional CDR1, CDR2 or MDR1 genes). For the HEK 293T cells, both compounds showed an intracellular punctuated bright staining typical of acidic compartments (late endosomes/lysosomes), as confirmed by co-localization with LysoView 633. In C. albicans the fungal cells, 1 labeled both organelle and plasma membranes, with similar behavior in both strains. For 2 the results indicated a different intracellular localization and an export of 2 by the cells through MDR transporters. In conclusion, our studies show that 1 and 2 can be the efficient cellular stains. Moreover, we expect that 1 can be also successfully used as a transition metal complexes fluorescent label, being a substitute for PPh3 or related phosphane ligands.
- Published
- 2021
26. Human and bovine serum albumin time-resolved fluorescence: Tryptophan and tyrosine contributions, effect of DMSO and rotational diffusion
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Radosław Starosta, Rodrigo F.M. de Almeida, and Filipa C. Santos
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biology ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Serum albumin ,Tryptophan ,Rotational diffusion ,010402 general chemistry ,01 natural sciences ,Fluorescence ,Small molecule ,0104 chemical sciences ,Analytical Chemistry ,Inorganic Chemistry ,biology.protein ,Biophysics ,Bovine serum albumin ,Spectroscopy ,Fluorescence anisotropy ,Rotational correlation time - Abstract
Albumin is the most abundant protein in mammalian serum and one of the major natural drug carriers in human body, therefore the study of small molecule interactions and other physicochemical processes such as oligomerization or supramolecular assembly have been intensely studied using both human (HSA) and bovine (BSA) serum albumin. The importance of these proteins also supports the need for fundamental and systematic studies of their properties, particularly, their photophysical properties, since a large number of studies relies on their intrinsic fluorescence. Here, multi-wavelength fluorescence intensity decays upon excitation at 280 nm were measured for HSA and BSA, the maximum absorption wavelength of tryptophan. Global analysis of the decay curves revealed the presence of four exponential components and the decay-associated spectra showed small contributions of tyrosine residues to the overall protein luminescence for emission below 340 nm, especially for HSA, in agreement with the fluorescence anisotropy decays obtained for each protein. We also showed that the addition of 2% DMSO, a commonly used drug co-solvent, diminishes the contribution of tyrosine residues. The fluorescence anisotropy decays at 280 nm excitation allowed to determine with high precision a short (0.28 ± 0.10 ns (HSA) and 0.29 ± 0.02 ns (BSA)) and a long (27.1 ± 0.3 ns (HSA) and 27.6 ± 0.1 ns (BSA)) rotational correlation time. Using our experimentally determined viscosity values, they could be assigned to the wobbling of the fluorescent residues side-chains (short time) and to the rotation of the protein molecule (long time). While the former were surprisingly similar despite the different contributions of tyrosine and tryptophan residues to the emission in HSA and BSA, the latter were also identical for both proteins and consistent with hard sphere and oblate ellipsoid models.
- Published
- 2020
27. The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells
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Filipe Vilas-Boas, Andreia Pena, Ana Bagulho, Filipa C. Santos, Ana Jerónimo, Catarina Peneda, Rodrigo F.M. de Almeida, and Carla Real
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PTEN ,Redox signaling ,Membrane permeability ,Clinical Biochemistry ,Protein oxidation ,Biochemistry ,Extracellular matrix ,Endothelial cell ,Cell Adhesion ,Human Umbilical Vein Endothelial Cells ,Humans ,Cell adhesion ,Cells, Cultured ,chemistry.chemical_classification ,Glutathione Peroxidase ,Reactive oxygen species ,biology ,Organic Chemistry ,PTEN Phosphohydrolase ,Hydrogen Peroxide ,Catalase ,Cell biology ,Endothelial stem cell ,chemistry ,biology.protein ,Oxidation-Reduction ,Function (biology) ,Research Paper - Abstract
The molecular processes that are crucial for cell function, such as proliferation, migration and survival, are regulated by hydrogen peroxide (H2O2). Although environmental cues, such as growth factors, regulate redox signaling, it was still unknown whether the ECM, a component of the cell microenvironment, had a function in this process. Here, we showed that the extracellular matrix (ECM) differently regulated H2O2 consumption by endothelial cells and that this effect was not general for all types of cells. The analysis of biophysical properties of the endothelial cell membrane suggested that this modification in H2O2 consumption rates was not due to altered membrane permeability. Instead, we found that the ECM regulated GPx activity, a known H2O2 scavenger. Finally, we showed that the extent of PTEN oxidation was dependent on the ECM, indicating that the ECM was able to modulate H2O2-dependent protein oxidation. Thus, our results unraveled a new mechanism by which the ECM regulates endothelial cell function by altering redox balance. These results pinpoint the ECM as an important component of redox-signaling., Graphical abstract fx1, Highlights • The extracellular matrix (ECM) regulates H2O2 consumption by endothelial cells. • Membrane biophysical properties are not affected by the ECM. • The ECM regulates GPx activity and the extent of PTEN oxidation. • We propose that the ECM modulates redox-signaling by controlling H2O2 degradation.
- Published
- 2015
28. Yeast Sphingolipid-Enriched Domains and Membrane Compartments in the Absence of Mannosyldiinositolphosphorylceramide
- Author
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Joaquim T. Marquês, H. Susana Marinho, Thomas Korte, Andreia Bento-Oliveira, Andreas Herrmann, Pedro M. R. Paulo, Rodrigo F.M. de Almeida, and Filipa C. Santos
- Subjects
Proton ATPase ,Saccharomyces cerevisiae Proteins ,Membrane lipids ,membrane compartments ,Saccharomyces cerevisiae ,lcsh:QR1-502 ,Biochemistry ,Article ,Glycosphingolipids ,lcsh:Microbiology ,Green fluorescent protein ,03 medical and health sciences ,Pma1p ,0302 clinical medicine ,fungal plasma membrane ,Protein Domains ,Molecular Biology ,sphingolipids ,Can1p ,fluorescence lifetime imaging microscopy (FLIM) ,inositolphosphorylceramides ,fluorescence spectroscopy ,giant unilamellar vesicles (GUVs) ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,Vesicle ,Cell Membrane ,biology.organism_classification ,Sphingolipid ,Membrane ,Symporter ,Biophysics ,lipids (amino acids, peptides, and proteins) ,030217 neurology & neurosurgery - Abstract
The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.
- Published
- 2020
29. Biophysical Implications of Sphingosine Accumulation in Membrane Properties at Neutral and Acidic pH
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Ana C. Carreira, Rodrigo F.M. de Almeida, Liana C. Silva, and Eva Zupancic
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Membrane Fluidity ,Endosome ,Static Electricity ,Fluorescence Polarization ,Phase Transition ,chemistry.chemical_compound ,Membrane Microdomains ,Sphingosine ,Static electricity ,Materials Chemistry ,Membrane fluidity ,Physical and Theoretical Chemistry ,POPC ,technology, industry, and agriculture ,Biological membrane ,Hydrogen-Ion Concentration ,Sphingomyelins ,Surfaces, Coatings and Films ,Cholesterol ,Membrane ,chemistry ,Liposomes ,Phosphatidylcholines ,Biophysics ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin - Abstract
Sphingosine (Sph) is a simple lipid involved in the regulation of several biological processes. When accumulated in the late endosomal/lysosomal compartments, Sph causes changes in ion signaling and membrane trafficking, leading to the development of Niemann-Pick disease type C. Little is known about Sph interaction with other lipids in biological membranes; however, understanding the effect of Sph in the physical state of membranes might provide insights into its mode of action. Using complementary established fluorescence approaches, we show that Sph accumulation leads to the formation of Sph-enriched gel domains in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (Chol) model membranes. These domains are more easily formed in membrane models mimicking the neutral pH plasma membrane environment (PM) as compared to the acidic lysosomal membrane environment (LM), where higher Sph concentrations (or lower temperatures) are required. Electrophoretic light scattering measurements further revealed that in PM-raft models (POPC/SM/Chol), Sph is mainly neutral, whereas in LM models, the positive charge of Sph leads to electrostatic repulsion, reducing the Sph ability to form gel domains. Thus, formation of Sph-enriched domains in cellular membranes might be strongly regulated by Sph charge.
- Published
- 2014
30. Changes in Membrane Organization upon Spontaneous Insertion of 2-Hydroxylated Unsaturated Fatty Acids in the Lipid Bilayer
- Author
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Maitane Ibarguren, Pablo V. Escribá, Alena Khmelinskaia, David J. López, Vanda A. Paixão, Hasna Ahyayauch, Rodrigo F.M. de Almeida, and Félix M. Goñi
- Subjects
Phosphatidylethanolamine ,Chemistry ,Lipid Bilayers ,Isothermal titration calorimetry ,Surfaces and Interfaces ,Condensed Matter Physics ,chemistry.chemical_compound ,Membrane ,Models, Chemical ,Biochemistry ,Phosphatidylcholine ,Fatty Acids, Unsaturated ,Electrochemistry ,Membrane fluidity ,lipids (amino acids, peptides, and proteins) ,General Materials Science ,Lipid bilayer phase behavior ,Lipid bilayer ,Sphingomyelin ,Spectroscopy - Abstract
Recent research regarding 2-hydroxylated fatty acids (2OHFAs) showed clear evidence of their benefits in the treatment of cancer, inflammation, and neurodegenerative disorders such as Alzheimer's disease. Monolayer compressibility isotherms and isothermal titration calorimetry of 2OHFA (C18-C22) in phosphatidylcholine/phosphatidylethanolamine/sphingomyelin/cholesterol (1:1:1:1 mole ratio), a mixture that mimics the composition of mammalian plasma membrane, were performed to assess the membrane binding capacity of 2OHFAs and their natural, nonhydroxylated counterparts. The results show that 2OHFAs are surface-active substances that bind membranes through exothermic, spontaneous processes. The main effects of 2OHFAs are a decrease in lipid order, with a looser packing of the acyl chains, and a decreased dipole potential, regardless of the 2OHFAs' relative affinity for the lipid bilayer. The strongest effects are usually observed for 2-hydroxyarachidonic (C20:4) acid, and the weakest one, for 2-hydroxydocosahexaenoic acid (C22:6). In addition, 2OHFAs cause increased hydration, except in gel-phase membranes, which can be explained by the 2OHFA preference for membrane defects. Concerning the membrane dipole potential, the magnitude of the reduction induced by 2OHFAs was particularly marked in the liquid-ordered (lo) phase (cholesterol/sphingomyelin-rich) membranes, those where order reduction was the smallest, suggesting a disruption of cholesterol-sphingolipid interactions that are responsible for the large dipole potential in those membranes. Moreover, 2OHFA effects were larger than for both lo and ld phases separately in model membranes with liquid disordered (ld)/lo coexistence when both phases were present in significant amounts, possibly because of the facilitating effect of ld/lo domain interfaces. The specific and marked changes induced by 2OHFAs in several membrane properties suggest that the initial interaction with the membrane and subsequent reorganization might constitute an important step in their mechanisms of action.
- Published
- 2014
31. Application of Ratiometric Measurements and Microplate Fluorimetry to Protein Denaturation: An Experiment for Analytical and Biochemistry Students
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Joaquim T. Marquês and Rodrigo F.M. de Almeida
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Chemistry ,Analytical chemistry ,Biophysics ,Denaturation (biochemistry) ,General Chemistry ,Fluorescence ,Fluorescence spectroscopy ,Education ,Biophysical chemistry ,Microplate Reader - Abstract
The number of applications of fluorescence spectroscopy in different areas of chemistry has increased dramatically, in part because a variety of instruments are used to measure fluorescence, including high-throughput microplate readers. Therefore, it is important to introduce students to different instruments. With many instruments, several experimental limitations hamper quantitative treatment of data, unless ratiometric measurements, that is, the ratio of intensity at two different excitation or emission wavelengths, are made. However, such methods are not always applicable. The denaturation of proteins often induces a red-shift of the tryptophan residues emission. Such a shift permits the use of ratiometric measures to obtain the fraction of native and denatured protein. To our knowledge, the use of ratiometric analysis with fluorescence measurements obtained from a microplate reader for the study of protein (biomolecular) denaturation has not been applied as a teaching exercise. In this experiment, th...
- Published
- 2013
32. New polydentate Ru(III)-Salan complexes: Synthesis, characterization, anti-tumour activity and interaction with human serum proteins
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Rodrigo F.M. de Almeida, Pedro Adão, João Costa Pessoa, Isabel Santos, M. Paula Robalo, Andreia Valente, M. Helena Garcia, Ana Isabel Tomaz, Cristina P. Matos, and Fernanda Marques
- Subjects
Denticity ,biology ,Ligand ,Stereochemistry ,Chemistry ,Serum albumin ,Human serum albumin ,Blood proteins ,Inorganic Chemistry ,Blood serum ,Octahedral molecular geometry ,Materials Chemistry ,biology.protein ,medicine ,Physical and Theoretical Chemistry ,Cytotoxicity ,medicine.drug - Abstract
Two new Ru(III) complexes bearing tetradentate N 2 O 2 bis(aminophenolate) ligands ( i.e. Salan-type ligands), were synthesized and characterized. The paramagnetism of the new [Ru III (Salan)Cl(PPh 3 )] (Salan = 4-methoxy/5-methoxy derivatives of 1,4-bis(salycilidene)cyclohexanediamine, PPh 3 = triphenylphosphane) was proved by spectroscopic studies. These complexes exhibit a 4d 5 low-spin distorted octahedral geometry. The anti-tumour activity of ligands and complexes was screened in vitro against A2780, MCF7 and MDAMB231 human cancer cell lines. Both ligands and complexes exhibit moderate to high cytotoxicity against all investigated cell lines, in some cases surpassing that of Cisplatin . Coordination to the Ru(III) center enhanced the cytotoxicity of each bis(aminophenolate) ligand by at least twofold. Binding of both Ru(III)-Salan complexes to human serum albumin is strong, as evaluated by steady-state and time-resolved fluorescence spectroscopy, suggesting that this protein might be a transport vehicle in the blood serum for these agents. The cytotoxicity of the protein-bound Ru(III)-Salan complex was assessed, as well as the effect of serum albumin binding on the activity of these complexes. These new Ru(III)-Salan are the first compounds of this class studied for anti-tumour proposes reported in the literature.
- Published
- 2013
33. The role of membrane fatty acid remodeling in the antitumor mechanism of action of 2-hydroxyoleic acid
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Rodrigo F.M. de Almeida, Gwendolyn Barceló-Coblijn, Pablo V. Escribá, Maria Laura Martin, Gerd Schmitz, Xavier Busquets, Gerhard Liebisch, Maria Antònia Noguera-Salvà, Mónica Higuera, and Silvia Terés
- Subjects
Time Factors ,Membrane lipid therapy ,Lipid Bilayers ,Biophysics ,Antineoplastic Agents ,Oleic Acids ,Biology ,Biochemistry ,Sphingolipid ,Biophysical Phenomena ,Mass Spectrometry ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Membrane Lipids ,0302 clinical medicine ,2-Hydroxyoleic Acid ,Lipid droplet ,Cell Line, Tumor ,Neoplasms ,Humans ,Lipid bilayer ,Phospholipids ,Triglycerides ,Cancer ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Liposome ,Phosphatidylethanolamines ,Cell Membrane ,Fatty Acids ,Fatty acid ,Cell Biology ,Cell biology ,Oleic acid ,chemistry ,Phosphatidylcholines ,lipids (amino acids, peptides, and proteins) ,Chromatography, Thin Layer ,Sphingomyelin ,030217 neurology & neurosurgery ,Stearoyl-CoA Desaturase - Abstract
The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is a potent antitumor drug that we rationally designed to regulate the membrane lipid composition and structure. The lipid modifications caused by 2OHOA treatments induce important signaling changes that end up with cell death (Terés et al., 2012 [1]). One of these regulatory effects is restoration of sphingomyelin levels, which are markedly lower in cancer cells compared to normal cells (Barceló‐Coblijn et al., 2011 [2]). In this study, we report another important regulatory effect of 2OHOA on cancer cell membrane composition: a large increase in 2OHOA levels, accounting for ~15% of the fatty acids present in membrane phospholipids, in human glioma (SF767 and U118) and lung cancer (A549) cells. Concomitantly, we observed marked reductions in oleic acid levels and inhibition of stearoyl-CoA desaturase. The impact of these changes on the biophysical properties of the lipid bilayer was evaluated in liposomes reconstituted from cancer cell membrane lipid extracts. Thus, 2OHOA increased the packing of ordered domains and decreased the global order of the membrane. The present results further support and extend the knowledge about the mechanism of action for 2OHOA, based on the regulation of the membrane lipid composition and structure and subsequent modulation of membrane protein-associated signaling.
- Published
- 2013
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34. [RuII(η5-C5H5)(bipy)(PPh3)]+, a promising large spectrum antitumor agent: Cytotoxic activity and interaction with human serum albumin
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M. Helena Garcia, Filipa Mendes, Fernanda Marques, João Costa Pessoa, Éva A. Enyedy, Isabel Santos, Tânia S. Morais, Tamás Kiss, Ana Isabel Tomaz, Rodrigo F.M. de Almeida, and Tamás Jakusch
- Subjects
Cisplatin ,Chemistry ,Stereochemistry ,chemistry.chemical_element ,Plasma protein binding ,Human serum albumin ,Biochemistry ,In vitro ,Ruthenium ,Inorganic Chemistry ,Mechanism of action ,Apoptosis ,medicine ,medicine.symptom ,Cytotoxicity ,medicine.drug - Abstract
Ruthenium complexes hold great potential as alternatives to cisplatin in cancer chemotherapy. We present results on the in vitro antitumor activity of an organometallic 'Ru(II)Cp' complex, [Ru(II)Cp(bipy)(PPh(3))][CF(3)SO(3)], designated as TM34 (PPh(3) = triphenylphosphine; bipy = 2,2'-bipyridine), against a panel of human tumor cell lines with different responses to cisplatin treatment, namely ovarian (A2780/A2780cisR, cisplatin sensitive and resistant, respectively), breast (MCF7) and prostate (PC3) adenocarcinomas. TM34 is very active against all tumorigenic cell lines, its efficacy largely surpassing that of cisplatin (CisPt). The high activity of TM34 towards CisPt resistant cell lines possibly suggests a mechanism of action distinct from that of CisPt. The effect of TM34 on the activity of the enzyme poly(ADP-ribose) polymerase 1 (PARP-1) involved in DNA repair mechanisms and apoptotic pathways was also evaluated, and it was found to be a strong PARP-1 ruthenium inhibitor in the low micromolar range (IC(50)=1.0 ± 0.3 μM). TM34 quickly binds to human serum albumin forming a 1:1 complex with a conditional stability constant (log K'~4.0), comparable to that of the Ru(III) complex in clinical trial KP1019. This indicates that TM34 can be efficiently transported by this protein, possibly being involved in its distribution and delivery if the complex is introduced in the blood stream. Albumin binding does not affect TM34 activity, yielding an adduct that maintains cytotoxic properties (against A2780 and A2780cisR cells). Altogether, the properties herein evaluated suggest that TM34 could be an anticancer agent of highly relevant therapeutic value.
- Published
- 2012
35. Development of lysosome-mimicking vesicles to study the effect of abnormal accumulation of sphingosine on membrane properties
- Author
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Rodrigo F.M. de Almeida, Ana C. Carreira, and Liana C. Silva
- Subjects
0301 basic medicine ,Cell Membrane Permeability ,Membrane permeability ,Science ,Lipid Bilayers ,Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Sphingosine ,Lysosome ,medicine ,Lipid bilayer ,POPC ,Multidisciplinary ,030102 biochemistry & molecular biology ,Vesicle ,Cell Membrane ,technology, industry, and agriculture ,Membranes, Artificial ,030104 developmental biology ,medicine.anatomical_structure ,Membrane ,chemistry ,Biochemistry ,Biophysics ,Medicine ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin ,Lysosomes - Abstract
Synthetic systems are widely used to unveil the molecular mechanisms of complex cellular events. Artificial membranes are key examples of models employed to address lipid-lipid and lipid-protein interactions. In this work, we developed a new synthetic system that more closely resembles the lysosome – the lysosome-mimicking vesicles (LMVs) – displaying stable acid-to-neutral pH gradient across the membrane. To evaluate the advantages of this synthetic system, we assessed the distinct effects of sphingosine (Sph) accumulation in membrane structure and biophysical properties of standard liposomes (no pH gradient) and in LMVs with lipid composition tuned to mimic physiological- or NPC1-like lysosomes. Ternary 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/Sphingomyelin (SM)/Cholesterol (Chol) mixtures with, respectively, low and high Chol/SM levels were prepared. The effect of Sph on membrane permeability and biophysical properties was evaluated by fluorescence spectroscopy, electrophoretic and dynamic light scattering. The results showed that overall Sph has the ability to cause a shift in vesicle surface charge, increase membrane order and promote a rapid increase in membrane permeability. These effects are enhanced in NPC1- LMVs. The results suggest that lysosomal accumulation of these lipids, as observed under pathological conditions, might significantly affect lysosomal membrane structure and integrity, and therefore contribute to the impairment of cell function.
- Published
- 2016
36. Reorganization of plasma membrane lipid domains during conidial germination
- Author
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Rodrigo F.M. de Almeida, Arnaldo Videira, Andreia S. Fernandes, Catarina A.C. Antunes, H. Susana Marinho, Filipa C. Santos, and Filipe P. Moreira
- Subjects
0301 basic medicine ,Spores ,Membrane Fluidity ,Saccharomyces cerevisiae ,Neurospora crassa ,Cell wall ,Fungal Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Membrane Lipids ,Cell Wall ,Membrane fluidity ,Spore germination ,Molecular Biology ,Ergosterol ,Sphingolipids ,Plasma membrane organization ,Membranes ,030102 biochemistry & molecular biology ,biology ,fungi ,Cell Membrane ,Crassa ,Cell Biology ,Spores, Fungal ,biology.organism_classification ,030104 developmental biology ,chemistry ,Biochemistry - Abstract
Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30 °C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.
- Published
- 2016
37. Biomimetic membrane rafts stably supported on unmodified gold
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Rodrigo F.M. de Almeida, Joaquim T. Marquês, and Ana S. Viana
- Subjects
Vesicle fusion ,Materials science ,Bilayer ,Biological membrane ,Nanotechnology ,General Chemistry ,Model lipid bilayer ,Condensed Matter Physics ,Chemical engineering ,lipids (amino acids, peptides, and proteins) ,Lipid bilayer phase behavior ,Cyclic voltammetry ,Lipid bilayer ,Lipid raft - Abstract
The formation of lipid bilayers on bare gold containing gel/fluid and liquid disordered/liquid ordered domains (lipid rafts), essential for the functioning of biological membranes, is reported here for the first time. Such binary and ternary lipid mixtures deposited on gold are improved biomimetic platforms. However, gold's hydrophobic nature has been an obstacle for direct deposition, and most studies rely on previous modification of its surface. In this work, lipid mixtures were deposited under different experimental conditions, including those commonly used for other solid supports such as mica, which are known to yield planar and organized bilayers. Atomic force microscopy imaging was used to study the topography of the lipid films at the nanoscale. The coverage, continuity and packing were addressed by ellipsometry and cyclic voltammetry, taking advantage of gold optical/electrical properties. A high quality bilayer displaying well organized lipid rafts is obtained by small or large unilamellar vesicle fusion in 10 mM Hepes buffer without added salt, while the presence of NaCl inhibits the formation of a lipid bilayer and leads to tubular structures. The raft-containing bilayer is stable over a wide range of potential sweep, enabling the development of new lipid raft based biosensing interfaces.
- Published
- 2012
38. Organization and Dynamics of Fas Transmembrane Domain in Raft Membranes and Modulation by Ceramide
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Manuel Prieto, Aleksander Fedorov, Bruno M. Castro, Erik Goormaghtigh, and Rodrigo F.M. de Almeida
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Ceramide ,Molecular Sequence Data ,Biophysics ,Apoptosis ,Fluorescence Polarization ,Ceramides ,03 medical and health sciences ,chemistry.chemical_compound ,Hydrophobic mismatch ,Membrane Microdomains ,0302 clinical medicine ,Spectroscopy, Fourier Transform Infrared ,Amino Acid Sequence ,fas Receptor ,Lipid raft ,030304 developmental biology ,0303 health sciences ,Chemistry ,Membrane ,Raft ,Protein Structure, Tertiary ,Protein Transport ,Transmembrane domain ,Biochemistry ,030220 oncology & carcinogenesis ,lipids (amino acids, peptides, and proteins) ,Receptor clustering ,Sphingomyelin - Abstract
To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events.
- Published
- 2011
39. Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae
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Fernando Antunes, H. Susana Marinho, Rodrigo F.M. de Almeida, André M. Cordeiro, Luísa Cyrne, and Francisco Aresta-Branco
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Diphenylhexatriene ,Ergosterol ,Peripheral membrane protein ,Cell Biology ,Biology ,Biochemistry ,Membrane contact site ,Sphingolipid ,chemistry.chemical_compound ,Membrane ,chemistry ,Membrane fluidity ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Lipid raft - Abstract
The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.
- Published
- 2011
40. Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts
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Joaquim T. Marquês, Rodrigo F.M. de Almeida, and Ana S. Viana
- Subjects
Sphingomyelin ,Silicon ,1,2-Dipalmitoylphosphatidylcholine ,Bilayer expansion ,Membrane Fluidity ,Liquid ordered phase ,Lipid Bilayers ,Biophysics ,02 engineering and technology ,Model lipid bilayer ,Microscopy, Atomic Force ,Biochemistry ,03 medical and health sciences ,Membrane Microdomains ,Ethanol–membrane interactions ,Membrane fluidity ,Lipid bilayer phase behavior ,Lipid bilayer ,Phospholipids ,030304 developmental biology ,0303 health sciences ,Chromatography ,Ethanol ,Chemistry ,Bilayer ,technology, industry, and agriculture ,Cell Biology ,Lipid bilayer mechanics ,Liquid ordered liquid disordered ,021001 nanoscience & nanotechnology ,Protein Structure, Tertiary ,Alcohols ,Phosphatidylcholines ,lipids (amino acids, peptides, and proteins) ,Liquid AFM ,0210 nano-technology ,Gels ,Membrane biophysics - Abstract
Ethanol–lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.
- Published
- 2011
41. Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains
- Author
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Alexander Fedorov, Manuel Prieto, Bruno M. Castro, Rodrigo F.M. de Almeida, and Liana C. Silva
- Subjects
Ceramide ,Cholesterol oxidase ,Membrane lipids ,Cell Biology ,Biology ,Biochemistry ,chemistry.chemical_compound ,Membrane ,chemistry ,Cholesterol oxidase activity ,Membrane fluidity ,lipids (amino acids, peptides, and proteins) ,Lipid bilayer ,Sphingomyelin ,Molecular Biology - Abstract
A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.
- Published
- 2009
42. FRET analysis of domain formation and properties in complex membrane systems
- Author
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Manuel Prieto, Rodrigo F.M. de Almeida, Liana C. Silva, and Luís M. S. Loura
- Subjects
Analytical chemistry ,Biophysics ,Model lipid bilayer ,01 natural sciences ,7. Clean energy ,Models, Biological ,Biochemistry ,Fluorescence ,Lipid bilayer ,Membrane phase separation ,03 medical and health sciences ,Membrane Microdomains ,Phase (matter) ,0103 physical sciences ,Membrane fluidity ,Fluorescence Resonance Energy Transfer ,Animals ,Humans ,Lipid bilayer phase behavior ,Lipid raft ,Phospholipids ,030304 developmental biology ,0303 health sciences ,010304 chemical physics ,Molecular Structure ,Chemistry ,Cell Membrane ,Cell Biology ,Phase diagram ,Förster resonance energy transfer ,Membrane ,Models, Chemical ,Chemical physics ,Thermodynamics - Abstract
The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.
- Published
- 2009
- Full Text
- View/download PDF
43. Modulation of plasma membrane lipid profile and microdomains by H2O2 in Saccharomyces cerevisiae
- Author
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Enrique Herrero, Carlos Borges, Luísa Cyrne, Rui Malhó, Ana C. Matias, Fernando Antunes, Rodrigo F.M. de Almeida, H. Susana Marinho, and Nuno M. Pedroso
- Subjects
Squalene ,ERG1 Potassium Channel ,Cell Membrane Permeability ,Saccharomyces cerevisiae Proteins ,Very long chain fatty acid ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,chemistry.chemical_compound ,Membrane Microdomains ,Acetyltransferases ,Ergosterol ,Physiology (medical) ,Lipid droplet ,Phosphatidylcholine ,Animals ,Ceramide synthase ,Lipid raft ,Phosphatidylethanolamine ,Plasma membrane permeability ,Phosphatidylethanolamines ,Cell Membrane ,Membrane Proteins ,Lipid metabolism ,Hydrogen Peroxide ,Lipid Metabolism ,Ether-A-Go-Go Potassium Channels ,Very long chain fatty acids ,Sterols ,Gene Expression Regulation ,chemistry ,H2O2 adaptation ,Phosphatidylcholines ,Fatty acid elongation ,lipids (amino acids, peptides, and proteins) ,Fatty Acid Synthases ,Oxidoreductases ,Oleic Acid - Abstract
In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H(2)O(2)) diffusion through the plasma membrane decreases during adaptation to H(2)O(2) by a still unknown mechanism. Here, adaptation to H(2)O(2) was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H(2)O(2) also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80% decrease in 2-hydroxy-C26:0 levels and a 50% decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H(2)O(2) leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H(2)O(2), and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.
- Published
- 2009
44. Membrane Domain Formation, Interdigitation, and Morphological Alterations Induced by the Very Long Chain Asymmetric C24:1 Ceramide
- Author
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Liana C. Silva, Manuel Prieto, Rodrigo F.M. de Almeida, and Sandra N. Pinto
- Subjects
Ceramide ,Biophysics ,Analytical chemistry ,Ceramides ,Cell membrane ,chemistry.chemical_compound ,Differential scanning calorimetry ,X-Ray Diffraction ,Phase (matter) ,medicine ,Fluorescence microscope ,POPC ,Unilamellar Liposomes ,Phase diagram ,Membranes ,Microscopy, Confocal ,Calorimetry, Differential Scanning ,Cell Membrane ,Spectrometry, Fluorescence ,medicine.anatomical_structure ,Membrane ,Solubility ,chemistry ,Phosphatidylcholines ,lipids (amino acids, peptides, and proteins) - Abstract
Ceramide (Cer) is involved in the regulation of several biological processes, such as apoptosis and cell signaling. The alterations induced by Cer in the biophysical properties of membranes are thought to be one of the major routes of Cer action. To gain further knowledge about the alterations induced by Cer, membrane reorganization by the very long chain asymmetric nervonoylceramide (NCer) was studied. The application of an established fluorescence multiprobe approach, together with x-ray diffraction, differential scanning calorimetry, and confocal fluorescence microscopy, allowed the characterization of NCer and the determination of the phase diagram of palmitoyloleoylphosphatidylcholine (POPC)/NCer binary mixtures. Nervonoylceramide undergoes a transition from a mixed interdigitated gel phase to a partially interdigitated gel phase at approximately 20 degrees C, and a broad main transition to the fluid phase at approximately 52 degrees C. The solubility of NCer in the fluid POPC is low, driving gel-fluid phase separation, and the binary-phase diagram is characterized by multiple and large coexistence regions between the interdigitated gel phases and the fluid phase. At 37 degrees C, the relevant phases are the fluid and the partially interdigitated gel. Moreover, the formation of NCer interdigitated gel phases leads to strong morphological alterations in the lipid vesicles, driving the formation of cochleate-type tubular structures.
- Published
- 2008
45. Structural and Dynamic Characterization of the Interaction of the Putative Fusion Peptide of the S2 SARS-CoV Virus Protein with Lipid Membranes
- Author
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Manuel Prieto, José Villalaín, Rodrigo F.M. de Almeida, and Jaime Guillén
- Subjects
Protein Conformation ,Cell ,Phospholipid ,Peptide ,Membrane Fusion ,Virus ,chemistry.chemical_compound ,Viral Envelope Proteins ,Materials Chemistry ,medicine ,Physical and Theoretical Chemistry ,skin and connective tissue diseases ,chemistry.chemical_classification ,Membrane Glycoproteins ,fungi ,Biological membrane ,Lipids ,Fusion protein ,Surfaces, Coatings and Films ,body regions ,Kinetics ,Spectrometry, Fluorescence ,Membrane ,medicine.anatomical_structure ,Biochemistry ,chemistry ,Spike Glycoprotein, Coronavirus ,Biophysics ,lipids (amino acids, peptides, and proteins) ,Glycoprotein - Abstract
The SARS coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a Class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. In the present work, we report a study of the binding and interaction with model membranes of a peptide pertaining to the putative fusion domain of SARS-CoV, SARS FP, as well as the structural changes that take place in both the phospholipid and the peptide molecules upon this interaction. From fluorescence and infrared spectroscopies, the peptide ability to induce membrane leakage, aggregation and fusion, as well as its affinity toward specific phospholipids, was assessed. We demonstrate that SARS FP strongly partitions into phospholipid membranes, more specifically with those containing negatively charged phospholipids, increasing the water penetration depth and displaying membrane-activity modulated by the lipid composition of the membrane. Interestingly, peptide organization is different depending if SARS FP is in water or bound to the membrane. These data suggest that SARS FP could be involved in the merging of the viral and target cell membranes by perturbing the membrane outer leaflet phospholipids and specifically interacting with negatively charged phospholipids located in the inner leaflet.
- Published
- 2008
46. Complexity of Lipid Domains and Rafts in Giant Unilamellar Vesicles Revealed by Combining Imaging and Microscopic and Macroscopic Time-Resolved Fluorescence
- Author
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Jan Willem Borst, Antonie J. W. G. Visser, Alexander Fedorov, Rodrigo F.M. de Almeida, and Manuel Prieto
- Subjects
Fluorescence-lifetime imaging microscopy ,phase-equilibria ,Microscope ,resonance energy-transfer ,1,2-Dipalmitoylphosphatidylcholine ,model membranes ,Biophysics ,Biochemie ,correlation spectroscopy ,Biochemistry ,Biophysical Phenomena ,law.invention ,chemistry.chemical_compound ,cell-membranes ,Membrane Microdomains ,Nuclear magnetic resonance ,law ,Phase (matter) ,phosphatidylcholine ,Fluorescent Dyes ,VLAG ,fluid ,Liposome ,Membranes ,Rhodamines ,Phosphatidylethanolamines ,Vesicle ,cholesterol ,Fluorescence ,Spectrometry, Fluorescence ,mixtures ,Microscopy, Fluorescence ,chemistry ,Dipalmitoylphosphatidylcholine ,Liposomes ,Phosphatidylcholines ,lipids (amino acids, peptides, and proteins) ,Time-resolved spectroscopy ,differential scanning calorimetry - Abstract
The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (l(o))/liquid-disordered (l(d)) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser-beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of l(o) raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the l(o) phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed.
- Published
- 2007
- Full Text
- View/download PDF
47. Formation and Properties of Membrane-Ordered Domains by Phytoceramide: Role of Sphingoid Base Hydroxylation
- Author
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André M. Cordeiro, Rodrigo F.M. de Almeida, Andreas Herrmann, Joaquim T. Marquês, Ana S. Viana, and H. Susana Marinho
- Subjects
Sphingosine ,Molecular Structure ,Lipid Bilayers ,Context (language use) ,Surfaces and Interfaces ,Condensed Matter Physics ,Ceramides ,Hydroxylation ,Sphingolipid ,law.invention ,chemistry.chemical_compound ,Membrane ,chemistry ,Biochemistry ,Confocal microscopy ,law ,Electrochemistry ,lipids (amino acids, peptides, and proteins) ,General Materials Science ,Lipid bilayer ,POPC ,Spectroscopy - Abstract
Phytoceramide is the backbone of major sphingolipids in fungi and plants and is essential in several tissues of animal organisms, such as human skin. Its sphingoid base, phytosphingosine, differs from that usually found in mammals by the addition of a hydroxyl group to the 4-ene, which may be a crucial factor for the different properties of membrane microdomains among those organisms and tissues. Recently, sphingolipid hydroxylation in animal cells emerged as a key feature in several physiopathological processes. Hence, the study of the biophysical properties of phytosphingolipids is also relevant in that context since it helps us to understand the effects of sphingolipid hydroxylation. In this work, binary mixtures of N-stearoyl-phytoceramide (PhyCer) with palmitoyloleoylphosphatidylcholine (POPC) were studied. Steady-state and time-resolved fluorescence of membrane probes, X-ray diffraction, atomic force microscopy, and confocal microscopy were employed. As for other saturated ceramides, highly rigid gel domains start to form with just ∼5 mol % PhyCer at 24 °C. However, PhyCer gel-enriched domains in coexistence with POPC-enriched fluid present additional complexity since their properties (maximal order, shape, and thickness) change at specific POPC/PhyCer molar ratios, suggesting the formation of highly stable stoichiometric complexes with their own properties, distinct from both POPC and PhyCer. A POPC/PhyCer binary phase diagram, supported by the different experimental approaches employed, is proposed with complexes of 3:1 and 1:2 stoichiometries which are stable at least from ∼15 to ∼55 °C. Thus, it provides mechanisms for the in vivo formation of sphingolipid-enriched gel domains that may account for stable membrane compartments and diffusion barriers in eukaryotic cell membranes.
- Published
- 2015
48. Biomembrane Organization and Function
- Author
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Rodrigo F.M. de Almeida, Joaquim T. Marquês, Catarina A.C. Antunes, and Filipa C. Santos
- Subjects
Membrane ,Membrane protein ,Saccharomyces cerevisiae ,lipids (amino acids, peptides, and proteins) ,Biological membrane ,Biology ,biology.organism_classification ,Lipid bilayer ,Lipid raft ,Sphingolipid ,Function (biology) ,Cell biology - Abstract
There has been a great effort to study lipid lateral organization in biomembranes in the past decades, in order to unravel the structural basis and functional significance of membrane lipid domains. However, in both respects fundamental doubts still persist, and recent results have widened this topic well beyond lipid rafts. In particular, the detection of sphingolipid-enriched gel domains in the yeast Saccharomyces cerevisiae plasma membrane, which are not the prototypical liquid ordered, sterol-enriched lipid rafts, will be described. The critical role of ordered lipid domains will be demonstrated with biophysical studies of membrane lipid organization in living cells and in model systems, concerning mammalian and fungal membranes. Membrane interactions with different types of bioactive molecules will be briefly presented, including endogenous molecules such as the hormone epinephrine or membrane proteins, as well as drugs, including anticancer and antitubercular compounds. Strategies to tackle the complexity of living cell membranes will be discussed, in an attempt to reach a compromise between lipid lamellar phases in artificial or reconstituted systems and observations in living cells. The development of new and improved biomimetic systems might provide answers to some of the open questions in the lipid domains field. Therefore, new lipid bilayer membrane models containing lipid domains stably formed on a conducting support (gold), where powerful surface and electrochemical techniques can be employed, will also be presented. The redox behavior of the catecholamine hormone epinephrine studied in such system showed that the lipid bilayer has a crucial role for the hormone chemical stability.
- Published
- 2015
49. Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane
- Author
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A. P. Alves de Matos, Rodrigo F.M. de Almeida, Liana C. Silva, Alexander Fedorov, and Manuel Prieto
- Subjects
Ceramide ,Light ,Lipid Bilayers ,Biophysics ,Phospholipid ,Apoptosis ,Electrons ,Biology ,Ceramides ,Cell membrane ,chemistry.chemical_compound ,Dynamic light scattering ,Fluorescence Resonance Energy Transfer ,medicine ,Scattering, Radiation ,Lipid bilayer ,Molecular Biology ,POPC ,Phospholipids ,Membrane Proteins ,Cell Biology ,Cell biology ,medicine.anatomical_structure ,Membrane ,Förster resonance energy transfer ,chemistry ,Spectrophotometry ,Phosphatidylcholines ,Anisotropy ,lipids (amino acids, peptides, and proteins) ,Signal Transduction - Abstract
To understand the formation and properties of ceramide-enriched domains in cell membranes, the biophysical properties of the binary system palmitoyloleoylphosphatidylcholine (POPC)/palmitoylceramide were thoroughly characterized. Diverse fluorescent probes and parameters were necessary to unravel the complexity of this apparently simple system. For the first time, a complete phase diagram is reported, characterizing the lamellar phases of these mixtures, and providing a quantitative framework integrating biophysical and biological studies. The diagram suggests that in resting cells no ceramide domains exist, but upon apoptotic stimuli, platforms may form. Moreover, our data show that 2 mol% of Cer strongly affects the POPC fluid matrix, suggesting that a small increase in Cer levels can significantly affect cell membrane properties. In this work, we also show that Cer domains, formed in conditions similar to physiological, are extremely ordered and rigid. The domains composition is estimated from the phase diagram and their large size was concluded from fluorescence resonance energy transfer. Dynamic light scattering and electron microscopy were used to characterize the system morphology, which is highly dependent on ceramide content and includes vesiculation and tubular structure formation.
- Published
- 2006
50. Interaction of peptides with binary phospholipid membranes: application of fluorescence methodologies
- Author
-
Rodrigo F.M. de Almeida, Manuel Prieto, Ana Coutinho, and Luís M. S. Loura
- Subjects
chemistry.chemical_classification ,Chemistry ,Molecular Sequence Data ,Organic Chemistry ,Analytical chemistry ,Phospholipid ,Binary number ,Peptide ,Peptide binding ,Cell Biology ,Fluorescence in the life sciences ,Biochemistry ,Fluorescence ,Membrane Lipids ,chemistry.chemical_compound ,Spectrometry, Fluorescence ,Membrane ,Förster resonance energy transfer ,Molecular Probes ,Biophysics ,Amino Acid Sequence ,Peptides ,Molecular Biology ,Phospholipids - Abstract
The application of fluorescence methodologies to obtain information about the extent, dynamics and topology of peptide interaction with binary phospholipid (mainly zwitterionic/anionic) mixtures is reviewed. First, general approaches based on peptide (tryptophan residues) fluorescence properties that give information about its partition, location and dynamics will be presented. Then, methodologies based on membrane probes fluorescence that report the influence of peptide binding and/or incorporation on the lateral organization (phase separation) of membrane phospholipids will be described. Specific examples taken from the literature that illustrate both situations are presented as well as formalisms for data analysis. It is shown that steady-state and time-resolved fluorescence data (particularly important in the case of fluorescence resonance energy transfer studies) give complementary information, allowing a molecular picture of peptide interaction with biphasic systems to be drawn.
- Published
- 2003
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