排序方式: 共有10条查询结果,搜索用时 406 毫秒
1
1.
Bruna?R. Casadei Cleyton?C. Domingues Eneida de?Paula Karin?A. Riske 《Biophysical journal》2014,106(11):2417-2425
The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition. 相似文献
2.
Daniel Martins Bruno Menezes de Oliveira Alessandro dos Santos Farias Ricardo Shiniti Oka Horiuchi Cleyton Crepaldi Domingues Eneida de Paula Sérgio Marangoni Wagner Farid Gattaz Emmanuel Dias-Neto José Camillo Novello 《Briefings in Functional Genomics and Prot》2007,6(1):70-75
Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB-16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14. 相似文献
3.
Holetz FB Ueda-Nakamura T Dias Filho BP Mello JC Morgado-Díaz JA Toledo CE Nakamura CV 《Memórias do Instituto Oswaldo Cruz》2005,100(4):397-401
We report the effect of Stryphnodendron adstringens on the trypanosomatid Herpetomonas samuelpessoai. The parasites were grown at 28 degrees C in a chemically defined medium containing crude extract and fractions at concentrations from 100 to 5000 microg/ml obtained from S. adstringens. Concentrations of 500, 1000, 2500, and 5000 microg/ml both crude extract and semi-purified fraction progressively inhibited the protozoans' growth. At a concentration of 100 microg/ml, crude extract or a semi-purified (F3) fraction did not affect the growth of the protozoans. The F3-9 - F3-12 sub-fractions, at a concentration of 1000 microg/ml, also showed increased inhibitory activity on H. samuelpessoai. The IC50 of the crude extract and the F3 fraction were 538 and 634 microg/ml, respectively. Ultrastructural and enzymatic alterations in the trypanosomatids were also evaluated. H. samuelpessoai cultivated in the presence of IC50 crude extract showed considerable ultrastructural alterations, such as marked mitochondrial swelling with a large number of cristae and evident Golgi complex vesiculation, as observed by transmission electron microscopy. Cells exposed to 538 microg/ml of crude extract at 28 degrees C for 72 h, showed decreased activity of the enzyme succinate cytochrome c reductase, a typical mitochondrion marker, as compared to untreated cells. 相似文献
4.
Karin A. Riske Cleyton C. Domingues Bruna R. Casadei Bruno Mattei Amanda C. Caritá Rafael B. Lira Paulo S. C Preté Eneida de Paula 《Biophysical reviews》2017,9(5):649-667
Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose Re sat—the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C12E8 and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only. 相似文献
5.
6.
Juliana F. Santos-Gandelman Olinda C. S. Santos Paula V. M. Pontes Cleyton Lage Andrade Elisa Korenblum Guilherme Muricy Marcia Giambiagi-deMarval Marinella S. Laport 《Marine biotechnology (New York, N.Y.)》2013,15(6):668-676
Among 1,236 colony-forming units (CFU) associated with 11 species of marine sponges collected from a Brazilian coast, a total of 100 morphologically different bacterial strains were analyzed. The phylogenetic diversity of the bacterial isolates was assessed by 16S rRNA gene amplification—restriction fragment length polymorphism (RFLP) analysis, using AluI restriction endonuclease. The RFLP fingerprinting resulted in 21 different patterns with good resolution for the identification of the bacterial isolates at the genus level. The genus Bacillus was the most commonly encountered genus, followed by Kocuria. Regarding the relationship between the morphotypes and species of marine sponges, Mycale microsigmatosa presented major diversity, followed by Dragmacidon reticulatum and Polymastia janeirensis. An antibiotic susceptibility profile of the 100 sponge-associated bacterial strains was determined by the disk diffusion method, and we observed a variable resistance profile, with 15 % of the bacteria being multiresistant. In addition, 71 of 100 strains were able to produce biofilm. These 71 strains were divided into 20 strong biofilm producers, 10 moderate biofilm producers, and 41 weak biofilm producers. The plasmid profile of the 100 bacterial strains was analyzed and 38 (38 %) of these samples possessed one or more plasmids. Studies like this are important to increase the information on these associated bacteria found off the coastline of Brazil, a place which has rich biodiversity that is still unknown. 相似文献
7.
Preté PS Domingues CC Meirelles NC Malheiros SV Goñi FM de Paula E Schreier S 《Biochimica et biophysica acta》2011,1808(1):164-170
The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate-and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111). 相似文献
8.
Cleyton C. Domingues Annarita Ciana Armando Buttafava Bruna Renata Casadei Cesare Balduini Eneida de Paula Giampaolo Minetti 《The Journal of membrane biology》2010,234(3):195-205
Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C12E8) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C12E8 DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C12E8 DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C12E8 treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C12E8 to the study of DRMs. 相似文献
9.
Cleyton?Crepaldi Domingues Annarita?Ciana Armando?Buttafava Cesare?Balduini Eneida?de?Paula Giampaolo?MinettiEmail author 《The Journal of membrane biology》2009,227(1):39-48
Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance
of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are
operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining
membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100
and C12E8. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2
was revealed in trace amounts in DRMs obtained with C12E8, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed
in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin
label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C12E8 in comparison to intact cells, while the difference in the S values between Triton X-100 and C12E8 DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared
with either Triton X-100 or C12E8 detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs. 相似文献
10.
1