Dynamic microstructure and hydration of peroxidized membrane of rat cardiac mitochondria and effects of adriamycin

Nanosecond time-resolved fluorometry of diphenyl hexatriene, DPH, fluorescence was used to study the effects of lipid peroxidation caused by NADH or adriamycin treatment on the dynamic microstructure of mitochondrial membranes from rat myocardium. Isolated mitochondria were incubated with NADH, FeCl3, and ADP, or with adriamycin. Parameters for microdynamics were calculated from the fluorescence intensity and anisotropy decay curves for DPH fluorescence. Peroxidized lipids were measured as malondialdehyde (MDA) resulting from the thiobarbiturate reaction. As peroxidized lipids accumulated, the membrane viscosity increased and the wobbling angle of the phospholipids decreased. The structural changes induced in unsaturated phospholipids by peroxidation probably increased the friction of neighboring phospholipids and restricted the range of their wobbling motion. The fluorescence intensity and fluorescence lifetimes decreased significantly when MDA was higher than 10 nmol/mg protein. These alterations in the behavior of DPH fluorescence strongly suggest that the hydration of the phospholipid layer of the mitochondria is occurring as a consequence of lipid peroxidation, since the fluorophore, DPH, is hydrophobic and its fluorescence is known to be quenched by increasing the dielectric constant of the surrounding media. The present results provide experimental supports to the hypothesis of membrane hydration induced by lipid peroxidation.     

Evaluation of age-related changes of physicochemical properties and functional activity of rat adipose plasma membranes and their possible relationship

Studies were carried out to evaluate structural state of adipose plasma membranes (PM) from mature adult and aged Wistar rats and its possible relation to functional changes in aging. PM lipids were probed by fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene. The data on DPH anisotropy, pyrene excimerization and induction resonance energy transfer (IRET) from PM proteins to pyrene provided the evidence for age-dependent decrease in PM lipid phase fluidity inclusive of that of annular lipid. Interaction between PM lipids and integral proteins also changed in aging. The observed structural changes were due to age-related lipid compositional modification. Evidence for this conclusion was provided by the following data: (i) DPH anisotropy was increased in aging both in PM and liposomes from the same PM lipids; (ii) both saturation/unsaturation fatty acid ratio and relative content of phosphatidylethanolamine (PE) increased in aged PM, whereas cholesterol/phospholipid and lipid/protein ratios were age-independent; (iii) PM polypeptide composition remained practically unchanged during aging. Age-related changes of the PM structure were accompanied by a twofold decrease of high affinity insulin binding sites and elevation of their affinity to insulin. It was suggested that age-related changes of physicochemical properties of PM lipid phase might affect protein molecular conformation and function through either changes in bulk lipid fluidity or through specific lipid-protein interactions.     

Serotonin transport in reconstituted endothelial cell plasma membrane proteoliposomes: effect of hypoxia.

To determine whether changes in the lipid dynamics of the plasma membrane bilayer are responsible for hypoxic stimulation of serotonin (5-hydroxytryptamine [5-HT]) transport in pulmonary artery endothelial cells, we solubilized and isolated phospholipid and protein fractions from plasma membrane vesicles derived from endothelial cells exposed to 20% O2 (normoxia) or 0% O2 (hypoxia) for 24 h. Four different combinations of proteoliposomes were prepared by reconstituting (1) normoxic protein and normoxic phospholipid, (2) normoxic protein and hypoxic phospholipid, (3) hypoxic protein and normoxic phospholipid, and (4) hypoxic protein and hypoxic phospholipid. Fluorescence anisotropy of diphenylhexatriene (DPH), a measure of fluidity, and 5-HT transport were evaluated in each of the four groups of reconstituted proteoliposomes. 5-HT transport by the reconstituted proteoliposomes was saturable, linear with protein (5 to 25 micrograms) and time (15 to 60 s), and optimal with a phospholipid-to-protein ratio of 3:1. There were no significant differences in intravesicular volume, phospholipid-to-protein ratio, and size distribution among the four different groups of proteoliposomes. 5-HT transport was significantly higher and fluorescence anisotropy of DPH was significantly lower in proteoliposomes made from hypoxic phospholipids irrespective of the source of protein. Hypoxia also had a direct effect on the 5-HT transporter since uptake was increased slightly in proteoliposomes from group 3. These results indicate that changes in the plasma membrane phospholipids, and to a much lesser extent changes in the 5-HT transporter, are responsible for increases in the transmembrane transport of 5-HT by hypoxic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical characterization of myelin subfractions obtained from bovine brain stem by zonal centrifugation

Bovine brain stem myelin was found to be distributed in a symmetrical mode on a continuous sucrose gradient with a maximum at 0.59 M sucrose. Increase in absolute quatities from the light to the heavy side of the gradient were observed for proteolipid protein, DM-20 protein and Wolfgram protein, with concomitant decreases in all lipid classes, in particular cerebrosides, while the level of basic protein remained constant. These results demonstrate substantial heterogeneity in myelin composition and preclude the possibility of a 1/1 ratio for basic protein and proteolipid protein.     

Microdynamics of outer and inner membranes of mitochondria from bullfrog myocardium

Outer and inner mitochondrial membranes were separated from bullfrog myocardium. Membrane viscosity and wobbling angle of phospholipids were measured with a nanosecond time-resolved fluorometer using a fluorophore, DPH, in each membrane. Measurements were also made on liposomes prepared from lipids extracted from each membrane. The anisotropy decay curve for DPH fluorescence was assumed to represent a mean value of decays in several microenvironments in membranes. Phospholipid constituents in membranes were analyzed by HPLC. A high proportion of PE and CL, both of which contain large amounts of unsaturated acyl chain, were found in the inner membrane. The low viscosity and large wobbling angle of phospholipids in the liposome from the inner membrane were consistent with the probable high content of unsaturated acyl chains and the low content of cholesterol in the inner membrane. Measurements of the dynamic microstructure of mitochondrial membranes suggested multifactorial characteristics probably resulting from the lipid-protein interactions. The average viscosity was found to be 0.39 +/- 0.08 P in the outer membrane and 0.58 +/- 0.01 P in the inner membrane. The wobbling angle of phospholipids in the outer and the inner membrane was, respectively, 47 and 49 degrees (non-significant difference). The liposomes prepared from lipid extracts of the membranes showed a lower viscosity and/or higher wobbling angle of phospholipids compared with the membranes themselves. The difference in the viscosity and wobbling angle between the mitochondrial membrane and its respective liposome was large in the inner membrane. These results suggest that the motion of phospholipids is limited by membrane proteins and the limitation of molecular motion of phospholipids results in an increase in the average viscosity. The results also suggest that the microdynamic values obtained in the inner membrane fraction reflect the interaction between phospholipid and protein which is abundant in the inner membrane.     

低密度脂蛋白,高脂血清对内皮细胞膜脂流动性的影响

实验用DPH荧光偏振技术测定经人低密度脂蛋白(LDL)及兔高脂血清(HRS)孵育的牛主动脉内皮细胞(EC)的膜脂微粘度。结果表明LDL孵育EC12小时导致细胞膜脂流动性下降,过氧化脂质(Lpo)含量增高;EC经HRS作用36小时后也发生相似的变化。形态观察证明EC胞浆中有大量脂滴样结构。结果提示高胆固醇含量的LDL和HRS可能通过增加膜的胆固醇含量而降低膜的流动性,高浓度Lpo可能也起重要作用。     

A New Approach for the Immunogenic Presentation of Membrane-Bound Human Colon Tumor Antigens

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.     

A new approach for the immunogenic presentation of membrane-bound human colon tumor antigens

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.     

The Interaction of Antibodies to two Myelin Proteins

Antibody has been prepared to a hydrophobic myelin protein in rabbits using several different adjuvants. This antibody was found to react with the myelin basic protein. This latter activity could be absorbed out with basic protein leaving the reactivity to the hydrophobic protein, suggesting that the two myelin proteins shared a common antigenic determinant. In addition, the hydrophobic protein appears to possess a unique antigenic determinant.     

The interaction of antibodies to two myelin proteins.

Antibody has been prepared to a hydrophobic myelin protein in rabbits using several different adjuvants. This antibody was found to react with the myelin basic protein. This latter activity could be absorbed out with basic protein leaving the reactivity to the hydrophobic protein, suggesting that the two myelin proteins shared a common antigenic determinant. In addition, the hydrophobic protein appears to possess a unique antigenic determinant.     

Buoyant density and lipid composition of purified myelin of aging human brain

Purified myelin of human brain from 15 young adult (below 50 years of age) and old (above 70 years of age) autopsy cases each was examined by isopycnic centrifugation in continuous sucrose gradients, and for lipid composition. The mean buoyant density of myelin was the same in both groups. Apparent features of old age were a wide range of density values, less compact myelin bands, and the dissociation of myelin into two bands in six of 15 old cases. Lipid analyses of randomly selected myelin samples of each group revealed an inverse relationship between the total lipid to protein ratio and density of myelin. In old age total lipids decreased by an average 10 mol lipid per mol protein. This decrease was accounted for by cholesterol, phosphatidylserine and cerebrosides. Changes in fatty acid moieties mainly affected sphingolipids. C20:0 and C24:0 of sphingomyelin increased, as did even more markedly the more hydrophilic OH-fatty acids of cerebrosides. Correlations with buoyant density existed for the ratios of cholesterol to protein in young adult cases, and those of galactolipids to protein in old cases. The results suggest that old age is associated with impaired stability and altered lipid composition of myelin.     

Effect of optic nerve transection upon myelin protein gene expression by oligodendrocytes: evidence for axonal influences on gene expression

Summary The effect of optic nerve transection on myelin protein gene expression was studied in rats following axotomy at two ages: during active myelination (17 days of age) and after peak expression of the genes (35 days of age). mRNA levels for proteolipid protein, myelin basic protein and myelin-associated glycoprotein were assessed by northern and dot blotting and byin situ hybridization using tissue sections and cultured individual oligodendrocytes. Transection at 17 days caused down-regulation of mRNAs for proteolipid protein, myelin basic protein and myelin-associated glycoprotein by 5 days after axotomy with an increase in GFAP mRNA. A more protracted change followed axotomy at 35 days of age. The abundance of mRNAs for proteolipid protein and myelin basic protein was significantly reduced by 28 days after transection in the affected nerve. Quantification of proteolipid protein mRNA expression in individual oligodendrocytes confirmed the down-regulation. However, in contrast to the effects on the major myelin proteins, the abundance of myelin-associated glycoprotein mRNA increased in the affected nerve for at least the initial month after lesioning at 35 days.The results show that optic nerve transection has significant effects on myelin protein mRNA expression in oligodendrocytes of optic nerve. However, the changes in myelin protein gene activity are relatively small and more protracted than those seen in Schwann cells after peripheral nerve section. Because axotomy also causes marked changes in the glial population of the optic nerve it is not possible unequivocally to ascribe the alteration in gene expression to loss of axons. However, the data may provide evidence that axons do influence myelin protein genes in oligodendrocytes and are necessary for them to develop their full expression.     

Brain membrane fluidity and lipid peroxidation in Alzheimer's disease

Membrane fluidity and lipid peroxidation in 4 brain areas from patients with Alzheimer's disease (AD) and matched controls were determined by measuring fluorescence anisotropy of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and by the thiobarbituric acid test respectively. Fluorescence anisotropy of DPH was not changed in any of the 4 areas in AD compared to controls. Basal levels of malondialdehyde (MDA; an intermediate in the lipid peroxidation process) were also not changed in different brain regions of AD and controls. However, stimulated MDA production determined by incubating tissue with FeSO4 plus H2O2 produced significantly higher MDA levels in AD brain than in controls.     

Augmentation of immune-mediated demyelination by lipid haptens

The encephalitogenic effects of bovine galactocerbroside and total myelin lipids given in the presence or absence of a known encephalitogenic dose of bovine myelin basic protein (MBP) in complete Freund's adjuvant have been examined in Hartley strain guinea pigs. The lipid haptens and MBP were given in the ratio in which they occur in intact myelin and were compared with experimental allergic encephalomyelitis (EAE) induced by whole bovine white matter in complete Freund's adjuvant. Clinically, bovine white matter- and MBP-induced EAE were similar, but in lesions in the central nervous system. Lipid/complete Freund's adjuvant emulsions were ineffective both clinically and histologically. In combination with MBP, galactocerebroside and total myelin lipids induced an EAE as severe as MBP-induced disease except that central nervous system lesions also showed demyelination. When given separately into opposite hindfeet, the lipid haptens and MBP produced EAE, but the lesions were not demyelinative. It appears, therefore, that lipid haptens have an augmenting effect on MBP when given in the same emulsion and produce central nervous system lesions which are both inflammatory and demyelinative.     

Isolation and characterization of defective jimpy oligodendrocytes in culture

Summary This study characterizes jimpy oligodendrocyte-enriched secondary cultures isolated from 10–12 daysin vitro primary glial cell cultures derived from 1–2-day-old jimpy mouse brains. Proliferation of defective oligodendrocytes was carefully investigated with regard to the expression of myelin basic protein and proteolipid protein and their respective mRNAs. Less than 5% of contaminating astrocytes (GFAP⁺ cells) were usually present. The identity of jimpy oligodendrocytes was confirmed using an antibody directed against a peptide from the wild type proteolipid protein C-terminal sequence for immunocytochernistry and an oligonucleotide complementary to mRNA derived from exon 5 of the proteolipid protein gene forin situ hybridization. Both the antibody and the probe recognize only normal oligondendrocytes while jimpy oligodendrocytes always remain unstained. Proteolipid protein in normal and jimpy oligodendrocytes was detected with antibody recognizing normal and mutated forms. Between 80 and 95% of the cells in normal and jimpy cultures at 2 and 4 daysin vitro in secondary cultures express myelin basic protein and proteolipid protein and their respective mRNAs. The percentage of oligodendrocytes (PLP⁺ or MBP⁺) in S phase of the cell cycle was 7–10% for both normal and jimpy oligodendrocytes. This contrasts with thein vivo situation where the proliferation rate of oligodendrocytes in jimpy brains is higher than in normal brains. In addition, jimpy oligodendrocytes remain unresponsive to basic fibroblast growth factor treatment while a similar treatment stimulates the proliferation of normal oligodendrocytes.     

Studies of human myelin proteins during old age

The electrophoretic protein patterns of myelin isolated from frontal and callosal white matter were studied in adult man up to the age of 90 years. The proportions of the four major myelin proteins remained virtually unchanged as did the total protein content of white matter and of purified myelin. The total mass of purified myelin that could be recovered from white matter gradually decreased with age, suggesting an age-related loss of myelin sheath and probably neurons as well, without detectable alterations of the regular protein composition of myelin. In most cases basic protein of myelin was preceded by one or two minor protein components on electrophoresis. One of them is tentatively identified as "prebasic" protein similar to the one previously observed in other species, because of its close electrophoretic apposition to the main basic protein. The second component was found less frequently and was thought to arise from specific types of proteolysis of myelin proteins. Prolonged time intervals between death and autopsy had little, if any, effect on the proportions of basic protein and proteolipid protein. Similar results were obtained when bovine brain was incubated under conditions designed to simulate post-mortem autolysis. It was there fore concluded that meaningful data on proteins of human central myelin may be obtained even though an autopsy was not performed within a few hours of death.     

Expression of myelin protein genes in Schwann cells

Summary The expression of myelin protein genes in Schwann cells has been studied byin situ hybridization.³⁵S-UTP-labelled, antisense and sense RNA probes to the major protein Po, myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and proteolipid protein (PLP) were employed with paraffin-embedded sections, teased fibres and dissociated Schwann cells from sciatic nerves of rats. Teased fibres were also prepared from cervical sympathetic trunks. Po mRNA was strongly expressed in the mid-internodal perinuclear area of Schwann cell cytoplasm. The degree of signal appeared to be related to fibre size. MBP mRNA showed a diffuse pattern along the Schwann cell internode with a marked increase in grains at the paranodal cytoplasm, particularly in larger fibres. This distribution suggests that the paranodal area is a major site of insertion of MBP into myelin membrane. The expression of MAG and PLP mRNA was markedly lower than Po and MBP. Both mRNAs were localized in the perinuclear cytoplasm and showed a dependence on fibre size. No significant signal was present in Schwann cells associated with unmyelinated axons.In addition to providing data on the cellular expression of myelin protein genes, these studies have shown that teased fibres are invaluable in allowing the localization of low abundance mRNAs.     

Immunohistochemical localization of myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase in flattened membrane expansions produced by cultured oligodendrocytes

Oligodendrocytes, the cells responsible for myelin sheath formation in the central nervous system, were isolated from primary dissociated mixed glial cultures prepared from newborn mouse forebrain, and further cultured in a serum-free defined culture medium. Single and double indirect immunofluorescence using antibodies against the myelin glycolipids, galactocerebroside and sulfatide, and the myelin proteins, myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase, was used to investigate the composition of the flat membrane extensions produced by some oligodendrocytes in culture. Galactocerebroside and sulfatide were both expressed on the external surface of the plasma membrane of oligodendrocyte cell bodies and processes and also the membrane expansions. Neither myelin basic protein nor 2',3'-cyclic nucleotide 3'-phosphohydrolase were expressed on the external surface of oligodendrocytes. Myelin basic protein could be localized to the cell body and the membrane expansions but not the major and fine processes. The localization of these myelin components suggests that the expansions have characteristics of the mature myelin membrane. 2',3'-Cyclic nucleotide 3'-phosphohydrolase was found to be localized in the cell body, and in total contrast to myelin basic protein, in the major processes and the fine interconnecting processes, but not the membrane expansions. In some of the cells 2',3'-cyclic nucleotide 3'-phosphohydrolase was present at the outer extremities of the flat membrane sheets, giving the appearance of an extending growth region. Our results thus clearly show that 2',3'-cyclic nucleotide 3'-phosphohydrolase is localized within oligodendrocytes in discrete regions of plasma membranes and suggest that this protein has a possible role in the early stages of myelin formation.     

Hemolysis of human erythrocytes with saponin affects the membrane structure

Incubation of cells and tissues with saponin makes the lipid bilayer permeable to macromolecules. Ghosts (membrane preparations) of saponin-lysed erythrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes with ghosts of cells lysed in hypotonic buffer. Transmission electron microscopy revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponin-lysed ghosts. Freeze-fracture electron microscopy showed regions with crystalline lipids and an increase in particle-free areas on fracture faces. The number of protein sulfhydryl groups and the binding of hemoglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemoglobin binding revealed the decrease of high affinity binding sites. All these results indicate an aggregation of band 3 protein also demonstrated by laser scanning microscopy after incubation of cells labelled with eosin-5-maleimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleton. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the association of spectrin with spectrin-depleted vesicles was decreased. The increased incorporation of the fluorescent probe Merocyanine 540 into saponin-lysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of the lipid bilayer and the changed interaction between membrane lipids and intrinsic membrane proteins. Our results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton.