Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol

The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents.     

Inhibition of Glutamate-Induced Intensification of Free Radical Reactions by Gangliosides: Possible Role in Their Protective Effect in Rat Cerebellar Granule Cells and Brain Synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 M) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na⁺, K⁺-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.     

Lipid-cell interactions: Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and ³¹P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.     

Endothelin-1-induced spreading depression in rats is associated with a microarea of selective neuronal necrosis

Two different theories of migraine aura exist: In the vascular theory of Wolff, intracerebral vasoconstriction causes migraine aura via energy deficiency, whereas in the neuronal theory of Le?o and Morison, spreading depression (SD) initiates the aura. Recently, it has been shown that the cerebrovascular constrictor endothelin-1 (ET-1) elicits SD when applied to the cortical surface, a finding that could provide a bridge between the vascular and the neuronal theories of migraine aura. Several arguments support the notion that ET-1-induced SD results from local vasoconstriction, but definite proof is missing. If ET-1 induces SD via vasoconstriction/ischemia, then neuronal damage is likely to occur, contrasting with the fact that SD in the otherwise normal cortex is not associated with any lesion. To test this hypothesis, we have performed a comprehensive histologic study of the effects of ET-1 when applied topically to the cerebral cortex of halothane-anesthetized rats. Our assessment included histologic stainings and immunohistochemistry for glial fibrillary acidic protein, heat shock protein 70, and transferase dUTP nick-end labeling assay. During ET-1 application, we recorded (i) subarachnoid direct current (DC) electroencephalogram, (ii) local cerebral blood flow by laser-Doppler flowmetry, and (iii) changes of oxyhemoglobin and deoxyhemoglobin by spectroscopy. At an ET-1 concentration of 1 muM, at which only 6 of 12 animals generated SD, a microarea with selective neuronal death was found only in those animals demonstrating SD. In another five selected animals, which had not shown SD in response to ET-1, SD was triggered at a second cranial window by KCl and propagated from there to the window exposed to ET-1. This treatment also resulted in a microarea of neuronal damage. In contrast, SD invading from outside did not induce neuronal damage in the absence of ET-1 (n = 4) or in the presence of ET-1 if ET-1 was coapplied with BQ-123, an ET(A) receptor antagonist (n = 4). In conclusion, SD in presence of ET-1 induced a microarea of selective neuronal necrosis no matter where the SD originated. This effect of ET-1 appears to be mediated by the ET(A) receptor.     

Lipid-cell interactions: A novel mechanism of transfer of liposome-entrapped substances into cells

A new approach has been developed for studying the transfer of liposome-entrapped substances into cells. The cells are incubated with liposomes containing two markers that in the free (non-entrapped) state enter the cells at different rates. Comparison of the ratio of cell-associated markers applied either in free or in liposome-entrapped form permits the evaluation of different pathways of cellular uptake of the intraliposomal substances. When epithelial cell sheets were incubated with egg phosphatidylcholine liposomes containing two different sugars they became cell-associated at a ratio different from their initial ratio inside the liposomes. Since the cell-associated ratio was shifted towards the value observed when the cells were incubated with a mixture of the two sugars in the free state, it is suggested that the liposomes become permeable during incubation and that the liberated substances enter the cells in the free form. On the other hand, cell-liposome interaction was demonstrated by NMR measurement and gel-filtration experiments to result in transformation of small unilamellar liposomes into larger multilayered aggregates. This transformation depends on the contact of the liposomes with the cell sheet. It is supposed that interliposomal aggregation is the underlying mechanism of cell-induced leakage of liposomes.     

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and Age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke)

Effects of 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6′-plastoquinonyl) decylrhod-amine 19 (SkQR1) on rat models of H₂O₂- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H₂O₂ or by ischemia/reperfusion. Higher SkQ1 (125–250 nmol/kg per day for 2–3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2–4 days, whereas one injection of SkQ1 or SkQR1 (1 μmol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 μmol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 12, pp. 1607–1621.     

Evaluation of the Kinetic Parameters of Trickle Bed Vapor Phase Bioreactors

The dependence of toluene elimination capacity on its load was determined in five small-scale reactors filled with glass beads carrying biocatalyst cells. With an increase in the operation time, the calculated maximum elimination capacity was shown to increase in parallel with the biomass density in the biocatalyst bed. A fivefold increase in the trickling rate did not affect the reactor performance. A simplified mathematical model for evaluating the minimal required biocatalyst bed volume at a certain loading was developed based on the experimental curve of elimination capacity versus loading.     

Stimulation by Gangliosides of Viability of Rat Brain Neurons and of Neuronal PC12 Cell Line under Conditions of Oxidative Stress

We studied effect of gangliosides on viability of brain neurons and neuronal PC12 cell line exposed to toxic concentrations of compounds activating free radical reactions. It is found that preincubation of cerebellar granule cells and PC12 cells with micromolar concentrations of ganglioside GM1 increases statistically significantly viability of these cells submitted to inductors of oxidative stress, such as hydrogen peroxide and the Fe²⁺-ascorbate system However, the effect of ganglioside GM1 in the PC12 cells failed to be revealed 1–2 days after treatment of the cells with trypsin, which indicates an importance of interaction of gangliosides with surface proteins for realization of their protective action. GM1, GD1a, and other gangliosides were shown to produce the neuroprotective effect on cerebellar granule cells in the presence of toxic glutamate concentrations. Not only micro-, but also nanomolar concentrations of these gangliosides increased statistically significantly the neuronal viability, although at micromolar concentrations this effect as a rule was more pronounced. The obtained data allow suggesting that the neuroprotective action of gangliosides is determined to a considerable degree by their ability to inhibit free-radical reactions in nerve cells.