Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide γ-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10–40°C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10°C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.     

ARAGONITE CRYSTALS IN SPIROGYRA SP. (CHLOROPHYTA)

Intracellular crystals of aragonite have been identified by selected area electron diffraction (SAED) in a species of the freshwater filamentous alga Spirogyra from the Thames River, Ontario, Canada. The crystals are 2 to 24 μm in diameter, and characterized by a unique cross-shaped morphology, in which needle-like, or prismatic outgrowths develop from a common axis. Crystals may be dispersed throughout filaments, but tend to cluster as aggregates towards the centre .     

Effect of Maternal Alcohol Consumption on the Lipid Composition of CNS in the Offspring

Maternal alcohol consumption at a level that does not affect calorie intake increases cholesterol concentration and content as well as incorporation of labeled glucose into cholesterol in the brain and spinal cord of newborn rat pups. Continued consumption of alcohol during lactation also affects the galactolipid concentration in the brain and spinal cord of pups at 21 days of age, and this increase seems mainly to be due to an increase in content of myelin lipids. Analysis of myelin shows that the concentration of phospholipids also increases in this fraction. The increase in incorporation of labeled glucose into these membrane lipids suggests an increase in the synthesis of these lipids, which prevents fluidization of the membrane by alcohol. That in the brainstem the increase in levels of cholesterol and galactolipids is higher than in other regions and that there is also an increase in content of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine suggest that the brainstem needs better protection against fluidization.     

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions.     

The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anti-cancer agent

The anti-cancer drug tamoxifen is a potent inhibitor of lipid peroxidation induced by Fe(III)-ascorbate in ox-brain phospholipid liposomes. Similar anti-oxidant effects, but with varying potencies, are also shown by 4-hydroxytamoxifen, cholesterol, ergosterol and 17-β-oestradiol. We now describe a computer-graphic fitting technique that demonstrates a structural similarity between the five compounds. In addition, we have quantified the differences (relative to cholesterol) between the anti-oxidant activities of the compounds in terms of a novel expression reffered to here as the cholesterol coefficient (C_c) Finally, we discuss how the inhibitory effect of tamoxifen on lipid peroxidation may result from a membrane stabilization that is associated with a decrease in membrane fluidity. This action may be related to the anti-proliferative effect exerted by tamoxifen on cancer and fungal cells.     

Conformations and dynamics of surfactants in micelles and liquid crystals by nuclear magnetic relaxation

The order parameters as well as the rates of overall and internal motions of aggregated surfactants can be obtained from deuteron and carbon-13 nuclear relaxation experiments. The main contribution to the relaxation is generally the quadrupolar coupling (²H) or the short range dipolar interaction with protons (¹³C). In some cases it is convenient to derive the same information from the¹³C relaxation induced by long range dipolar interactions with a paramagnetic probe exchanging rapidly among the polar heads of surfactant molecules. This paper outlines the methods of interpretation of relaxation data by means of a rotational jump model of internal motions, taking into account most of the accessible conformers. The conformational and dynamical parameters are obtained from the magnetic field dependence of the longitudinal relaxation rates (micelles) or from the simultaneous fit of these rates and of the dipolar or quadrupolar splittings (liquid crystals). Some examples of application of these methods are given from recent works on single and double detailed surfactants.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Subcellular Distribution and Developmental Expression of Cholesterol Ester Hydrolases in Fetal Rat Brain Cultures

Abstract: Cholesterol ester hydrolase activities previously have been identified in brain and linked to the production of myelin, which has very low levels of esterified cholesterol. We have studied two cholesterol ester hydrolase activities (termed the pH 6.0 and pH 7.2 activities) in cultures derived from 19- to 21-day-old dissociated fetal rat brains and in developing rat brain. In vivo the levels of both the pH 6.0 and pH 7.2 activities began to increase by about 10 postnatal days, reached maximal levels at 20 days (20 and 1.5 nmol/h/mg protein, respectively), and thereafter remained nearly constant (pH 6.0) or decreased somewhat before becoming constant (pH 7.2). In contrast, in the cultures the pH 6.0 cholesterol ester hydrolase activity was low until 21 days in culture (DIC; 20 nmol/h/mg protein), increased to a peak activity at 31 DIC (60 nmol/h/mg protein), remained high for 24 days, and finally decreased (18 nmol/h/mg protein at 63 DIC); the pH 7.2 cholesterol ester hydrolase activity was very low until 20 DIC, increased to a peak activity at 31 days (3 nmol/h/mg protein), and thereafter decreased to a lower level (2 nmol/h/mg protein) that was maintained for about 24 days before decreasing (0.7 nmol/h/mg protein at 63 DIC). Therefore, (a) the time courses of appearance of both cholesterol ester hydrolase activities were delayed by 10–14 days relative to that seen in vivo, and (b) the specific activities observed in the cultures were transiently two- to three-fold higher than in rat brain, but then declined to levels characteristic of whole brain homogenates. Subcellular fractionation of the cultures demonstrated that the pH 7.2 cholesterol ester hydrolase activity, along with myelin basic protein and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity, was enriched in a membrane fraction collected at an interface between 0.32 M and 0.9 M sucrose; the pH 6.0 cholesterol ester hydrolase activity, in contrast, was enriched in the microsomal fraction.     

缺乏抗坏血酸对豚鼠胆固醇代谢的影响

边缘性缺乏抗坏血酸之豚鼠,于三周内其肝脏及小肠粘膜3-羟-3-甲基戊二酰辅酶A还原酶(HMGR)活力均下降到原有水平的50％,但肝脏胆固醇7α-羟化酶活力尚无显著性改变。坏血病豚鼠(三周内)上述几种酶活力都下降至原有水平的50％左右。豚鼠摄取抗坏血酸不足,其血清总胆固醇浓度显著增加,而血清高密度脂蛋自胆固醇浓度显著减少,其改变程度与抗坏血酸缺乏状况一致。