Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.     

Enzymatic,biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5′-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.     

Aging and the biophysical properties of cell membranes

Fluorescence spectroscopy was used to examine the biophysical characteristics of human platelet membranes as a function of subject age. The structural order of membrane lipid domains was determined with the use of 1,6-diphenyl-1,3,5-hexatriene (DPH), a fluorescent probe that preferentially localizes in the hydrocarbon core of synthetic and biological membranes. Over the age range of subjects examined (17 to 86 years) the structural order of platelet membranes, as reflected by the steady-state fluorescence polarization of DPH, increased substantially. The magnitude of the observed increase in membrane structural order is sufficient to affect membrane-related cell functions including platelet aggregation. A major contributor to the increase in structural order of platelet membranes may have been an increase in the concentration of cholesterol in serum and tissue with age. The changes observed here in platelet membranes may be a general phenomenon of aging, as changes of similar type and magnitude have been observed in lymphocyte membranes and brain with age in other studies.     

Composition and physical properties of lipids from plasma membranes of dog kidney

Lipid composition, physical state of major phospholipid classes and transbilayer migration of phosphatidylcholine have been determined in plasma membranes of the dog kidney. The lipid composition of brush-border membranes markedly differs from that of antiluminal membranes with respect to: (a) the total phospholipid content; (b) the cholesterol to phospholipid ratio (C/P); (c) the distribution of the major phospholipid classes. Sphingomyelin present in large amounts in both luminal and antiluminal membranes extracts exhibits a transition of phase between 20 and 44 degrees C approximately. In the range of temperature studied (5-55 degrees C) no phase transitions were detected for the other phospholipid species. Our data suggest that: (1) at physiological temperature the higher C/P ratio of brush-border membranes is in large part responsible for their lower fluidity; (2) both the relatively low cholesterol and high sphingomyelin contents contribute to the thermotropic transitions observed in intact membranes. Finally transbilayer migration of phosphatidylcholine in brush-border membranes is a very slow process with a half time of 6.5 h at 37 degrees C which compares with that of other biological membranes.     

Composition ofNeurospora crassa vacuolar membranes and comparison to endoplasmic reticulum,plasma membranes,and mitochondrial membranes

We have determined the carbohydrate and lipid contents of vacuolar membranes fromNeurospora crassa, and have compared them to mitochondrial membranes, endoplasmic reticulum, and plasma membranes. These four membrane fractions were clearly distinct from each other in polypeptide composition, as judged by polyacrylamide gel electrophoresis. The vacuolar membranes proved unusual in two respects: the contained very high amounts of carbohydrate and were the only membranes with significant levels of phosphatidylserine. As in other eucaryotic cells, the mitochondrial membranes were unique in having high amounts of cardiolipin but virtually no sterol. Although the endoplasmic reticulum and plasma membranes were qualitatively similar to each other, the plasma membranes could be distinguished by a higher carbohydrate content, whereas the endoplasmic reticulum had a characteristically high ratio of phosphatidylcholine to phosphatidylethanolamine. The fatty acid compositions of all four membranes were similar, except that mitochondrial membranes contained about half as much saturated fatty acids as the other three fractions.     

Endothelial cells as early sensors of pulmonary interstitial edema.

We studied responses of endothelial and epithelial cells in the thin portion of the air-blood barrier to a rise in interstitial pressure caused by an increase in extravascular water (interstitial edema) obtained in anesthetized rabbits receiving saline infusion (0.5 ml.kg(-1).min(-1) for 3 h). We obtained morphometric analyses of the cells and of their microenvironment (electron microscopy); furthermore, we also studied in lung tissue extracts the biochemical alterations of proteins responsible for signal transduction (PKC, caveolin-1) and cell-cell adhesion (CD31) and of proteins involved in membrane-to-cytoskeleton linkage (alpha-tubulin and beta-tubulin). In endothelial cells, we observed a folding of the plasma membrane with an increase in cell surface area, a doubling of plasmalemma vesicular density, and an increase in cell volume. Minor morphological changes were observed in epithelial cells. Edema did not affect the total plasmalemma amount of PKC, beta-tubulin, and caveolin-1, but alpha-tubulin and CD-31 increased. In edema, the distribution of these proteins changed between the detergent-resistant fraction of the plasma membrane (DRF, lipid microdomains) and the rest of the plasma membrane [high-density fractions (HDFs)]. PKC and tubulin isoforms shifted from the DRF to HDFs in edema, whereas caveolin-1 increased in DRF at the expense of a decrease in phosphorylated caveolin-1. The changes in cellular morphology and in plasma membrane composition suggest an early endothelial response to mechanical stimuli arising at the interstitial level subsequently to a modest (approximately 5%) increase in extravascular water.     

Radiographic evidence of interstitial pulmonary edema after exercise at altitude

Pulmonary functionabnormalities after exercise are suggestive of pulmonary edema;however, radiographic evidence is lacking. Well-trained cyclists werestudied to determine whether there is radiographic evidence ofpulmonary edema after endurance exercise (cycling distance5.3-131.5 km) at altitude. Chest radiographs obtained beforeexercise were coded for later interpretation. Films obtained afterexercise were coded with a different number. A total of 74 sets ofposteroanterior and lateral films were analyzed by three radiologistsfor signs of pulmonary edema. Radiographic changes were graded on athree-point scale. An edema score was calculated by summing the scorefor each individual radiographic finding for each radiologist and anoverall edema score representing the mean scores from all threeradiologists. The overall edema score increased from 0.8 ± 1.2 before exercise to 1.8 ± 1.6 after exercise(P < 0.01). These results suggestthat, after prolonged high-intensity exercise at moderate altitude,there is radiographic evidence of early pulmonary edema in some cyclists.     

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals.When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.     

大鼠神经源性肺水肿时交感神经放电活动,血浆儿...

Neurogenic pulmonary edema (NPE) was induced by microinjection of kainic acid into bilateral preoptic area (POA) of the hypothalamus in rats. Sympathetic discharge of the left adrenal branch was recorded, plasma catecholamine (CA) was assayed and the physical properties of pulmonary surfactant (PS) of the lung lavage were measured. The results showed that some physical properties of PS were changed in NPE, i.e. maximal surface tension (gamma Max) decreased, minimum surface tension (gamma Min) increased, recruitment index (RI), stability index and area of hysteresis loop (H-area) decreased. After injection of kainic acid into POA, the sympathetic discharge increased by 22.8 +/- 7.20% and 32.2 +/- 8.0% respectively at 30 and 60 min after injection and paralleled by a marked elevation of plasma catecholamine (CA). The results showed that PS activity had decreased at an early stage of NPE and the change of the plasma CA level was parallel to that of sympathetic discharge. It is suggested that generation of NPE may be related to some disorder of the autonomic nervous system at the level of hypothalamus, increase of sympathetic discharge, elevation of plasma CA level and fall of PS activity.     

Ablation of ceramide synthase 2 strongly affects biophysical properties of membranes

Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a ceramide synthase 2 (CerS2)-null mouse, which is unable to synthesize C22-C24 ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.     

Sulfoquinovosyldiacylglycerol and phosphatidylglycerol bilayers share biophysical properties and are good mutual substitutes in photosynthetic membranes

From cyanobacteria to higher plants, photosynthetic membranes are composed of two galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), and two negatively charged lipids, sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). In many environments, plants and algae grow in a shortage of nutrients, leading to the development of nutrient-saving mechanisms. For example, at the cellular level, in phosphate starvation, these mechanisms include conversion of phospholipids into phosphorus-free lipids. In photosynthetic membranes, PG is supposed to be replaced by SQDG in phosphate starvation whereas the opposite occurs in sulfur deprivation. All biological data confirm a complementary relationship between SQDG and PG and suggest the importance of maintaining the total amount of anionic lipids in photosynthetic membranes. Using neutron diffraction on reconstituted SQDG or PG lipid membranes, we demonstrate that, despite chemically different headgroups, PG and SQDG have similar physicochemical properties. With an equivalent diacylglycerol backbone, PG and SQDG membranes have a similar bilayer thickness and bending rigidity. They also have essentially the same response to hydration in terms of repulsion and interaction forces. The results presented here establish that SQDG and PG are good substitutes to each other in nutrient starvation conditions to maintain the chloroplast functional organization and its photosynthesis activity.     

A product of ozonolysis of cholesterol alters the biophysical properties of phosphatidylethanolamine membranes

There is evidence that some products of the reaction of ozone with cholesterol contribute to atherosclerosis. One of these compounds is 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al. We have synthesized this compound and have demonstrated that it reacts with phosphatidylethanolamine to form a Schiff base. The 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al also affects the physical properties of phosphatidylethanolamines. We show by both DSC and X-ray diffraction that it increases the negative curvature of the membrane. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al causes the lamellar phase to become disorganized, resulting in the loss of lamellar periodicity. The chemical and physical interactions of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al with phosphatidylethanolamines may contribute to damaging effects of this lipid on cell membranes, resulting in pathology.     

Changes in biophysical parameters of plasma membranes influence cisplatin resistance of sensitive and resistant epidermal carcinoma cells

The mechanism of resistance of cancer cells to the anticancer drug cisplatin is not fully understood. Using cisplatin-sensitive KB-3-1 and -resistant KCP-20 cells, we found that the resistant cells have higher membrane potential, as determined by membrane potential sensing oxonol dye. Electron spin resonance and fluorescence polarization studies revealed that the resistant cells have more "fluid" plasma membranes than the sensitive cells. Because of this observed difference in membrane "fluidity," we attempted modification of the plasma membrane fluidity by the incorporation of heptadecanoic acid into KB-3-1 and KCP-20 cell membranes. We found that such treatment resulted in increased heptadecanoic acid content and increased fluidity in the plasma membranes of both cell types, and also resulted in increased cisplatin resistance in the KCP-20 cells. This finding is in accord with our results, which showed that the cisplatin-resistant KCP-20 cells have more fluid membranes than the cisplatin-sensitive KB-3-1 cells. It remains to be determined whether the observed differences in biophysical status and/or fatty acid composition alone, or the secondary effect of these differences on the structure or function of some transmembrane protein(s), is the reason for increased cisplatin resistance.     

Synthesis, biophysical, and biochemical properties of PNA-DNA chimeras

Three PNA-DNA chimeric dimer synthons (tT, upT and uhT, see Sch. 1) have been synthesized in solution and used to make T20-analogue chimeras applying standard solid-phase DNA synthesis protocol. Duplex forming ability of chimeras with dA20 and their hydrolyses by 3'- and 5'-exonucleases (snake venom and bovine spleen phosphodiesterase, respectively) have been investigated.     

Proteomic analysis of pulmonary edema fluid and plasma in patients with acute lung injury

Proteomics is the large-scale analysis of protein profiles. This approach has not yet been reported in the study of acute lung injury (ALI). This study details protein profiles in plasma and pulmonary edema fluid (EF) from 16 ALI patients and plasma and bronchoalveolar lavage fluid (BALF) from 12 normal subjects. More than 300 distinct protein spots were evident in the EF and BALF of both normal subjects and ALI patients. Of these, 158 were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the plasma and EF protein profile of ALI patients, there were multiple qualitative changes. For instance, in all normal subjects, but in only one of the ALI patients, seven distinct surfactant protein A isoforms were evident. Nearly all ALI patients also had protein spots that indicate truncation or other posttranslational modifications. Several of these novel changes could serve as new biomarkers of lung injury.