Red blood cell ghosts and intact red blood cells as complementary models in photodynamic cell research

Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.     

The sialoglycoprotein subunits of human placental brush border membranes characterized by two-two-dimensional electrophoresis

A brush border membrane enriched fraction was isolated from human full-term placenta. This membrane fraction exhibited large membrane fragments with microvilli projecting from the basal membrane in electron micrographs and was enriched tenfold in alkaline phosphatase, a brush border enzyme marker. The sialoglycoproteins associated with this membrane fraction were tritiated by mild periodate oxidation of sialic acid and reduction with tritiated NaBH4. The membranes were solubilized in 8 M urea, 2 percent Triton X-100, and the tritiated glycoprotein subunits were reduced with beta-mercaptoethanol and characterized by 2-dimensional poly-acrylamide gel electrophoresis using a method similar to that described by O'Farrell and Bhakdi, Knüferman, and Wallach. The tritiated subunits were detected in the gels by autofluorography. The 2-dimensional subunit "maps" resolved at least 17 major sialoglycoprotein subunits whereas only 10 major periodate-Schiff reagent staining components were resolved by 1-dimensional SDS polyacrylamide gel electrophoresis. Placental alkaline phosphatase (PAP) was identified on the subunit maps by inclusion of 32P-labeled PAP in the tritiated membrane sample. The 32P-labeled PAP corresponded to a major tritiated sialoglycoprotein subunit, which was heterogeneous with respect to charge as demonstrated by 3 closely running spots of the same molecular weight.     

Phospholipids Bilayers as Molecular Models for Drug- Membrane Interactions. The Case of the Antiepileptics Phenytoin and Carbamazepine

With the aim to better understand the molecular mechanisms of the interaction of phenytoin and carbamazepine with cell membranes we utilized a well-established model consisting in intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of its membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidyl-ethanolamine (DMPE), representative of phospholipid classes respectively located in the outer and inner monolayers of erythrocytes and other cell membranes. This report presents the following evidence that phenytoin and carbamazepine interact with membrane phospholipids: a) X-ray diffraction and fluorescence spectroscopy showed that both drugs preferentially interacted with DMPC; b) in IUM, the drugs induced a disordering effect on the polar head groups and acyl chains of the eryhrocyte membrane lipid bilayers; c) electron microscopy observations of human erythrocytes showed the echinocyte formation, an effect due to phenytoin and carbamazepine insertion in the outer monolayer of the red cell membrane.     

Heterogeneity in the conformation of different protein fractions from the human erythrocyte membrane.

We have isolated 5 families of proteins from human red blood cell membranes and characterized their secondary structure by ultraviolet circular dichroism measurements. The protein families were prepared by selective solubilization from ghosts under nondenaturing conditions. We find that the intact ghost has a mean alpha-helix fraction of 0.37, whereas a low-ionic-strength extract (bands 1, 2, 5, "spectrin") has a substantially higher helix fraction, 0.55. Further extraction of the ghosts with para-chloromercuribenzoate yields bands 2.1, 4.1, 4.2, and 6; their helix content is only 0.17. Finally, the major intrinsic protein, band 3, was solubilized by a non-ionic detergent. Its helix fraction is 0.38.     

Interaction of pentoxifylline with human erythrocytes. I. Interaction of xanthine derivatives with human erythrocyte ghosts

The interaction of pentoxifylline and other xanthine derivatives with human erythrocyte ghosts was studied. By fluorescence spectroscopy it was found that xanthine derivatives have two modes of binding to erythrocyte ghosts. One is a high-capacity binding to erythrocyte membranes. It seems that the 5-oxohexyl side chain of pentoxifylline is important for this. The second type may be a binding to proteins on the membranes and is specific for pentoxifylline and caffeine. From the circular dichroism spectra, it was presumed that the second binding mode of pentoxifylline occurs at hydrophobic regions of beta-structure of the membrane proteins. The relative high specificity in the interaction of pentoxifylline with erythrocytes should be related to its unique physiological activity on erythrocytes.     

Preliminary characterization of the acetylcholine receptor in human erythrocytes.

The response of human erythrocytes to cholinergic ligands was studied with an electron spin resonance assay. The membrane response to carbamyl choline was found to be antagonized by atropine and, in the absence of calcium, by tetrodotoxin. Experiments with resealed ghosts showed that the membrane response to carbamyl choline required ATP and calcium. Reductive alkylation of intact cells eliminated the cholinergic response, but the presence of saturating amounts of carbamyl choline protected the putative receptor against inactivation. Affinity labeling was used to demonstrate an apparent molecular weight of 41,000 for the carbamyl choline-binding species. A lipid vesicle extraction technique was used to induce a specific cation permeability defect in intact cells. Preliminary investigation of this phenomenon is described.     

Insulin binding and degradation by human erythrocytes and erythrocyte membranes

Conclusions By the current results there is evidence that the insulin degrading enzyme activity of the erythrocytes is not located on the inner or outer surface of the plasma membrane but is exclusively cytosolic. On the other hand, specific insulin binding to erythrocytes and erythrocyte membranes has been demonstrated and it could be supposed that the insulin binding region of the plasma membrane is not associated with an insulin degrading activity.In comparison to erythrocytes unsealed and sealed ghosts bound an equal amount of ¹²⁵I-insulin. Since there is binding but not degradation of insulin the membranes of erythrocytes might be a useful tool for the investigation of insulin internalization.

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Insulin-Bindung und -Abbau durch menschliche Erythrocyten und Erythrocyten-Membranen

     

顺二氨二水合铂(Ⅱ)与膜磷脂及膜蛋白相互作用的荧光光谱研究

用Tb~(3+)分别对用和未用顺二氨二水合铂(Ⅱ)(AAP)处理的红细胞膜和磷酰胆碱(PC)脂质体进行荧光滴定,测Tb~(3+)荧光。用AAP滴定红细胞膜,测蛋白质自身荧光变化。用间接Scatchard法求结合参数。表明蛋白质及磷脂均与AAP结合,蛋白质结合能力较强。     

Isolation of Hemoglobin from Bovine Erythrocytes by Controlled Hemolysis in the Membrane Bioreactor

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2–7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.     

USE OF OCTADECYLRHODAMINE FLUORESCENCE DEQUENCHING TO STUDY VESICULAR STOMATITIS VIRUS FUSION WITH HUMAN AGED RED BLOOD CELLS*

Human erythrocytes were separated into five fractions representing different age groups. In each group phospholipid inside-outside translocation was determined by quantitation of the amino phospholipids phosphatidylserine and phosphatidylethanolamine and their lyso-derivatives by thin layer chromatography. To assess the role of transbilayer phospholipid distribution in the recognition and fusion of vesicular stomatitis virus (VSV) and human aged erythrocytes, we monitored the fusion kinetics using the octadecylrhodamine dequenching assay. Fusion of VSV with each single group of red blood cells (RBC) was not detectable with the youngest cells (Fl group) but increased with RBC aging (F2-F5 groups). The same increase in fusion was observed with microvesicles generated from RBC in which aging was mimicked by incubating the cells with Ca²⁺ in the presence of the Ca²⁺ ionophore A23187. Conversion of the aminophospholipids to the trinitrophenyl derivative by reaction with trinitrobenzensulfonate completely inhibits fusion on ghosts in which aging was artificially induced by translocation of aminophospholipids in the outer leaflet (symmetric ghosts). These results indicate that RBC become susceptible to VSV fusion during aging and in all pathology related to the aging process.     

Phospholipid Bilayers as Molecular Models for Drug-Cell Membrana Interactions. The Case of the Antiinflamatory Drug Diclofenac

Phospholipids are amphipatic molecules with long hydrophobic acyl chains and zwitterionic polar heads which assemble into different types of molecular aggregates. The most relevant is the bilayer because of its relation with cell membranes, which are very complex entities. For this reason, simpler molecular models based on phospholipids bilayers are widely used. We have determined the bilayer structure of phospholipids located in the outer and inner monolayers of most cell membranes, and use them as molecular models to study the way different chemicals of biological interest interact with cell membranes. We present the results of our studies on the nonsteroidal anti-inflammatory drug diclofenac, from which little is known about its effects on human erythrocytes. This report presents the following evidence that diclofenac interacts with the human red cell membrane: a) X-ray diffraction and fluorescence spectroscopy of phospholipids bilayers show that diclofenac interacts with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy studies on human erythrocytes it was observed that the drug induced morphological changes different from their normal biconcave shape.     

Analysis of human erythrocyte membrane vesicles produced by shearing

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg++-ATPase, Na+,K+-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogenase, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.     

Investigation of surface and shape changes accompanying the membrane alteration responsible for the heat-induced lysis of human erythrocytes

Heat induces several successive events in erythrocyte membrane; the denaturation of spectrin at about 50°C, thermoporation at 62°C and denaturation of the anion channel at 67°C. The heat denaturation of major membrane proteins, spectrin and the anion channel, is not needed for the thermoporation which is involved in thermohemolysis. This study reports about the surface and shape changes which are specific for thermoporated membranes with spectrin and anion channel preserved intact. Thermoporation was produced exposing human erythrocytes to 39.5°C for 3 min in isotonic medium containing 18% (v/v) ethanol as membrane fluidizer and sucrose as osmotic protectant which prevents hemolysis (Ivanov, J. Therm. Biol. 1996). The control cells were processed similarly except that they were incubated at 23°C, thus avoiding thermoporation. In control and porated membranes the overall structure of spectrin and the anion channel was retained inasmuch as their enthalpies and denaturation temperatures were microcalorimetrically found preserved. Nevertheless, irregular shape, grainy surface and asymmetric spicules were apparent in porated cells through scanning electron microscopy. A decrease in the number of binding sites for Alcian blue and an increased binding of eosine was established in the membranes of porated cells. After poration the hexane/aqueous partition coefficient K_d of cells increased from 5 to about 220 and the electrophoretic mobility of cells decreased by about 25% indicating marked increase in cell surface hydrophobicity and a decrease in surface charge, respectively. In addition, adhesivity to hydrophobic interfaces and aggregability in low ionic media strongly increased after poration. In contrast to intact and control cells, the porated ones (all prefixed with 0.2% glutaraldehyde) made molecular contacts with inclined hydrophobic interfaces at low (5 mM NaCl) but not at high (150 mM NaCl) ionic media. Thus, the microtopological shape changes and exposure of new hydrophobic and charge groups over the outer cell surface, without major thermal unfolding, possibly indicates an irreversible redistribution of membrane material and disturbed lipid–protein complementation during thermoporation.     

Membrane microextension: a possible mechanism for establishing molecular contact in electrofusion.

True cell membrane contact is an essential condition for electro-pulsed cell fusion, but initial morphological perturbation leading to true contact is still not clear. Dielectrophoresis mediated compression and fusogenic pulse induced compaction of cells led to rapid merger of tight membranes, and deprived direct microscopic view of surface membrane perturbation. Freely suspending cells with large and different cell-cell gaps may proceed to electrofusion with perturbed membrane and initiates fusion events at different time. These pulsed exposed cells can be used for capturing changes in the membrane surface and early electrofusion events. Early stage of fusion of freely suspended intact human erythrocytes exposed to single exponential decay pulse was studied by scanning electron microscopy (SEM). Field pulse induces small membrane bumps. Interaction of bumps on adjacent membranes lead to true membrane contact and form bridges between the membranes as microextension, combining both membranes into a topologically single structure. Some fusion products showed expanded fusion zones, which suggest indication of open lumen at contact area.     

Analysis of the phospholipid composition of plasmodium knowlesi and rhesus erythrocyte membranes

Methods for the separation and analysis of the phospholipid classes have been studied. The lipid extracts of normal and Plasmodium knowlesi- infected rhesus erythrocytes and of the parasite itself have been examined for phospholipid composition on an animal-to-animal basis. Several differences were apparent between the phospholipids of parasites and infected host cells. Phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine represented larger percentages in the parasite than in the host; the average phosphatidylinositol content was 1.8 % in infected host cells and 4.3 % in parasites. Sphingomyelin and phosphatidylserine were also strikingly different in the two membranes; in the parasite they averaged less than 20 % and 33 % respectively of their level in the infected red blood cell.     

Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver

We isolated and characterized a subcellular fraction derived from the blood-sinusoidal plasma membrane of hepatocytes enriched in caveolin and containing several of the molecular components described to be present in caveolae isolated from other cell types. A morphological study by electron microscopy revealed that it was composed of caveolae-attached membrane profiles. Immunoelectron microscopy of isolated fraction showed the specific labeling of internal caveolae membranes with anti-caveolin antibody. Finally, one- and two-dimensional electrophoresis and Western blotting were used for the biochemical analysis of this new rat liver plasma membrane fraction. From the biochemical and the morphological characterization, we conclude that the caveolae-enriched plasma membrane fraction is a plasma membrane fraction, which originates from specialized regions of the sinusoidal plasma membrane, enriched in caveolae.     

Metabolic fate of [14C]triglyceride-entrapped lactosylceramide-bearing liposomes after intravenous injection into mouse.

We investigated the distribution and fate of liposomes after their intravenous injection into a mouse. Liposomes were composed of dimyristoylphosphatidylcholine, cholesterol and dicetylphosphate (7:2:1, molar ratio) with or without lactosylceramide. They were characterized as small unilamellar vesicles, approximately 100 nm in diameter, using gel-exclusion chromatography on a Sephacryl S-1000, freeze-fracture electron microscope and dynamic light scattering method. Liposomes were very stable in serum as seen by the results of leakage of the entrapped marker and electrophoresis experiments. We demonstrated that liposomes were internalized by way of an endocytotic process via coated vesicles detected in the electron microscope. The increase in liver uptake of lactosylceramide-bearing liposomes was mostly accounted for by enhanced uptake in the parenchymal cells, while uptake by non-parenchymal cells was only slightly increased. This observation supported the notion that a galactose-specific receptor was involved in liver uptake of lactosylceramide liposomes. The lactosylceramide-bearing liposomes were preferentially recovered in the liver and were found to first be predominantly localized in the mitochondria-lysosomal fraction. They were then decomposed by lysosomal enzymes, and the hydrolyzed components were reincorporated into membrane phospholipids in the microsomal fraction. At the same time, a rapid and reversible exchange of phosphatidylcholine between microsomes and mitochondria was demonstrated.     

Separation and determination of lipids by one-dimensional micro-thin-layer chromatography followed by densitometry

A method is described for the rapid analysis of a mixture of phospholipids and neutral lipids, which was used for the analysis of extracts obtained from a nuclear fraction isolated from rat liver. The lipids are separated by one-dimensional thin-layer chromatography on microchromatoplates (48 X 24 mm), using three solvents for development. After spraying the plates with phosphoric acid and heating, the amount of carbon from the charred compounds is measured densitometrically. Only 2-10 microgram of lipid mixture are needed for the determination of the relative amounts of the separated compounds.     

燃料电池聚合物电解质膜的研究进展

本文根据聚合物电解质膜燃料电池操作温度、使用的电解质和燃料的不同,将其分为高温质子交换膜燃料电池、低温质子换膜燃料电池、直接甲醇燃料电池和阴离子交换膜燃料电池,综述了它们所用电解质膜的最新进展.第一部分简要介绍了这4种燃料电池的优点和不足.第二部分首先介绍了Nafion膜的结构模型,并对平行柱状纳米水通道模型在介观尺度上进行了修正;接着分别对应用于不同燃料电池的改性膜的改性思路作了分析;最后讨论了用于不同燃料电池的新型质子交换膜的研究,同时列举了性能突出的改性膜和新型质子交换膜.第三部分介绍了阴离子交换膜的研究现状.第四部分对未来聚合物电解质膜的研究作了展望.