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Find video protocols related to scientific articles indexed in Pubmed.
Antioxidant capacity of Ugni molinae fruit extract on human erythrocytes: an in vitro study.
J. Membr. Biol.
PUBLISHED: 03-27-2014
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Ugni molinae is an important source of molecules with strong antioxidant activity widely used as a medicinal plant in Southern Chile-Argentina. Total phenol concentration from its fruit extract was 10.64 ± 0.04 mM gallic acid equivalents. Analysis by means of HPLC/MS indicated the presence of the anthocyanins cyanidin and peonidin, and the flavonol quercitin, all in glycosylated forms. Its antioxidant properties were assessed in human erythrocytes in vitro exposed to HClO oxidative stress. Scanning electron microscopy showed that HClO induced an alteration in erythrocytes from a normal shape to echinocytes; however, this change was highly attenuated in samples containing U. molinae extracts. It also had a tendency in order to reduce the hemolytic effect of HClO. In addition, X-ray diffraction experiments were performed in dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine bilayers, classes of lipids preferentially located in the outer and inner monolayers, respectively, of the human erythrocyte membrane. It was observed that U. molinae only interacted with DMPC. Results by fluorescence spectroscopy on DMPC large unilamellar vesicles and isolated unsealed human erythrocyte membranes also showed that it interacted with the erythrocyte membrane and DMPC. It is possible that the location of U. molinae components into the membrane outer monolayer might hinder the diffusion of HClO and of free radicals into cell membranes and the consequent decrease of the kinetics of free radical reactions.
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Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes.
Arch. Biochem. Biophys.
PUBLISHED: 08-07-2013
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Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures.
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Structural effects of the Solanum steroids solasodine, diosgenin and solanine on human erythrocytes and molecular models of eukaryotic membranes.
Biochim. Biophys. Acta
PUBLISHED: 03-19-2013
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This report presents evidence that the following Solanum steroids: solasodine, diosgenin and solanine interact with human erythrocytes and molecular models of their membranes as follows: a) X-ray diffraction studies showed that the compounds at low molar ratios (0.1-10.0mol%) induced increasing structural perturbation to dimyristoylphosphatidylcholine bilayers and to a considerable lower extent to those of dimyristoylphosphatidylethanolamine; b) differential scanning calorimetry data showed that the compounds were able to alter the cooperativity of dimyristoylphosphatidylcholine, dimyristoylphosphatidylethanolamine and dimyristoylphosphatidylserine phase transitions in a concentration-dependent manner; c) in the presence of steroids, the fluorescence of Merocyanine 540 incorporated to the membranes decreased suggesting a fluidization of the lipid system; d) scanning electron microscopy observations showed that all steroids altered the normal shape of human erythrocytes inducing mainly echinocytosis, characterized by the formation of blebs in their surfaces, an indication that their molecules are located into the outer monolayer of the erythrocyte membrane.
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Effects of sodium metavanadate on in vitro neuroblastoma and red blood cells.
Arch. Biochem. Biophys.
PUBLISHED: 01-22-2013
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Toxicity of vanadium on cells is one of the less studied effects. This prompted us to study the structural effects induced on neuroblastoma and erythrocytes by vanadium (V) sodium metavanadate. This salt was incubated with mice cholinergic neuroblastoma cells and intact human erythrocytes. To learn whether metavanadate interacts with membrane lipid bilayers it was incubated with bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). These are phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Exposure of neuroblastoma cells to metavanadate showed significant decreases in cell viability as well as in cell number correlating with inhibition of aconitase activity. In scanning electron microscopy (SEM) and defocusing microscopy (DM) it was observed that induced on erythrocytes the formation of echinocytes. However, no effects were obtained when metavanadate was made to interact with DMPC and DMPE multibilayers and liposomes, assays performed by X-ray diffraction and differential scanning calorimetry (DSC), respectively. These results imply that the effects of metavanadate on erythrocytes are through interactions with proteins located in the membrane outer moiety, and could still involve other minor lipid components as well. Also, partly unsaturated lipids could interact differently the fully saturated chains in the model systems.
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Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models.
Biochim. Biophys. Acta
PUBLISHED: 09-15-2011
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Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N(6)-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HP?CD) with 2-hydroxypropyl-?-cyclodextrin (HP?CD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HP?CD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10?M concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1?M induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200?M induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HP?CD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent.
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Human cells and cell membrane molecular models are affected in vitro by the nonsteroidal anti-inflammatory drug ibuprofen.
Biochim. Biophys. Acta
PUBLISHED: 04-06-2011
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This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10?M; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic-hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300?M ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations?300?M induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied.
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Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models.
Biochem. Biophys. Res. Commun.
PUBLISHED: 01-26-2011
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Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 ?M; (c) X-ray diffraction studies showed that PPA in the 0.1-0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.
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Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions.
Biochem. Biophys. Res. Commun.
PUBLISHED: 05-14-2010
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Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl(3) was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 microM; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 microM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 microm-1mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 microM to 100 microM.
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Effects of the nonsteroidal anti-inflammatory drug naproxen on human erythrocytes and on cell membrane molecular models.
Biophys. Chem.
PUBLISHED: 01-04-2010
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Naproxen, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of lipids found in the outer and inner moieties of the erythrocyte and most cell membranes, respectively. This report presents evidence that naproxen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes it has been observed that the drug induced shape changes, forming echinocytes at a concentration as low as 10microM; b) X-ray diffraction showed that naproxen strongly interacted with DMPC multilayers; in contrast, no perturbing effects on DMPE multilayers were detected; c) differential scanning calorimetry (DSC) data showed a decrease in the melting temperature (T(m)) of DMPC liposomes, which was attributed to a destabilization of the gel phase, effect that was less pronounced for DMPE. These experimental results were observed at concentrations lower than those reported for plasma after therapeutic administration. This is the first time in which the structural effects of naproxen on the human erythrocyte membrane have been described.
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Structural effects in vitro of the anti-inflammatory drug diclofenac on human erythrocytes and molecular models of cell membranes.
Biophys. Chem.
PUBLISHED: 01-23-2009
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Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly on those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated and reported. This report presents the following evidence that diclofenac interacts with red cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that diclofenac interacted with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes it was observed that the drug induced changes different from the normal biconcave morphology of most red blood cells. This is the first time in which structural effects of diclofenac on the human erythrocyte membrane have been described.
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The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: a physico-chemical study with lecithin phospholipids.
Biochim. Biophys. Acta
PUBLISHED: 01-21-2009
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Nonsteroidal anti-inflammatory drugs (NSAIDs) represent non-specific inhibitors of the cycloxygenase pathway of inflammation, and therefore an understanding of the interaction process of the drugs with membrane phospholipids is of high relevance. We have studied the interaction of the NSAIDs with phospholipid membranes made from dimyristoylphosphatidylcholine (DMPC) by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). FTIR data obtained via attenuated total reflectance (ATR) show that the interaction between DMPC and NSAIDs is limited to a strong interaction of the drugs with the phosphate region of the lipid head group. The FTIR transmission data furthermore are indicative of a strong effect of the drugs on the hydrocarbon chains inducing a reduction of the chain-chain interactions, i.e., a fluidization effect. Parallel to this, from the DSC data beside the decrease of T(m) a reduction of the peak height of the melting endotherm connected with its broadening is observed, but leaving the overall phase transition enthalpy constant. Additionally, phase separation is observed, inducing the formation of a NSAID-rich and a NSAID-poor phase. This is especially pronounced for Diclofenac. Despite the strong influence of the drugs on the acyl chain moiety, FRET data do not reveal any evidence for drug incorporation into the lipid matrix, and ITC measurements performed do not exhibit any heat production due to drug binding. This implies that the interaction process is governed by only entropic reactions at the lipid/water interface.
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Human erythrocytes and molecular models of cell membranes are affected in vitro by Balbisia peduncularis (Amancay) extracts.
Chem. Biol. Interact.
PUBLISHED: 01-17-2009
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Balbisia peduncularis, also known as "Amancay", is a plant of the Ledocarpaceae family that can be found in the Atacama Desert in northern Chile. Infusions of the plant have long being used in traditional herbal medicine. Its chemical composition indicates the presence of flavonoids, which have antioxidant properties. Aqueous extracts from its stems were prepared to induce their interaction with human erythrocytes and their membrane models in order to elucidate whether this rare and unstudied plant produced perturbations to cell membranes. Scanning electron microscopy (SEM) of intact human red blood cells showed that the extract changed the normal erythrocytes morphology as a function of its concentration, first inducing echinocytes, and then stomatocytes and spherocytes. According to the bilayer couple hypothesis, the shape changes indicated that the flavonoids were first located in the outer monolayer of the erythrocyte membrane, and at the highest assayed concentration in both monolayers. The results obtained by fluorescence spectroscopy measurements of isolated unsealed human erythrocytes (IUM), of unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC), and by X-ray diffraction of DMPC and dimyristoylphosphatidylethanolamine (DMPE) multilayers, confirmed this conclusion. In fact, they showed that the plant aqueous extract molecules were located in both the hydrophilic polar head and in the hydrophobic acyl chain regions of the lipid bilayers. As a consequence, perturbations of the phospholipid bilayer packing arrangement were produced.
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Real-time study of shape and thermal fluctuations in the echinocyte transformation of human erythrocytes using defocusing microscopy.
J Biomed Opt
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We present a real-time method to measure the amplitude of thermal fluctuations in biological membranes by means of a new treatment of the defocusing microscopy (DM) optical technique. This approach was also applied to study the deformation of human erythrocytes to its echinocyte structure. This was carried out by making three-dimensional shape reconstructions of the cell and measuring the thermal fluctuations of its membrane, as the cell is exposed to the anti-inflammatory drug naproxen and as it recovers its original shape, when it is subsequently cleansed of the drug. The results showed biomechanical changes in the membrane even at low naproxen concentration (0.2 mM). Also, we found that when the cell recovered its original shape, the membrane properties were different compared to the nondrugged initial erythrocyte, indicating that the drug administration-recovery process is not completely reversible.
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