Lipid Layer Of Membrane Research Articles

Phosphatidylcholine-Engineered Exosomes for Enhanced Tumor Cell Uptake and Intracellular Antitumor Drug Delivery.

Exosomes derived from non-tumor cells hold great potential as drug delivery vehicles because of their good biosafety and natural transference of bioactive cargo between cells. However, compared to tumor-derived exosomes, efficient delivery is limited by their weak interactions with tumor cells. It is essential to engineer exosomes that improve tumor cellular internalization efficiency. A simple and effective strategy to enhance tumor cell uptake by engineering the exosome membrane lipids can be established by drawing on the role of lipids in tumor exosomes interacting with tumor cells. Amphiphilic phosphatidylcholine (PC) molecules are inserted into the membrane lipid layer of reticulocyte-derived exosomes (Exos) by simple incubation to construct PC-engineered exosomes (PC-Exos). It is demonstrated that PC-Exos showed significantly enhanced tumor cell internalization and uptake rate compared to native Exos, up to a twofold increase. After therapeutic agent loading, PC-Exos remarkably promotes intracellular drug or RNA accumulation in cancer cells, thus showing enhanced in vitro anti-tumor activity. This work demonstrates the crucial role of engineering exosomal lipids in modulating cancer cellular uptake, which may shed light on the design of high-efficiency exosome-based drug delivery carriers.

Клітинні механізми еритродіерезу

Normally erythrodieresis is in a dynamic equilibrium with the process of erythropoiesis, and is therefore one of the factors to providing a relatively constant number of red blood cells in the bloodstream. The physiologically old, damaged and non-viable erythrocytes, as well as the erythrocytes which are produced during stress erythropoiesis, are destroyed. Erythrocyte clearance is a selective process. First of all, the cells that have lost their ability to deform are removed from the bloodstream. The deformability of red blood cells depends on the shape of the cells, the viscosity of the cytoplasm and the mechanical properties of the membrane. Old and altered erythrocytes are quite rigid, and are therefore delayed in the narrow capillaries and venous sinuses of the liver and spleen. In addition, macrophages of the liver and spleen phagocytize erythrocytes, which expose “eat me” signaling molecules on their surface. Exposure of phosphatidylserine on the outer cell surface of erythrocytes and vesicles results in their elimination from the bloodstream by Kupffer cells and other mononuclear phagocytes. During the initiation of erythrophagocytosis, the phosphatidylserine of the outer lipid layer of the erythrocyte plasma membrane directly interacts with the receptors Stabilin-2, TIM-1, TIM-4 or CD300 of macrophages. The macrophage’s integrins avb3 and avb5, as well as the Mer receptor tyrosine kinase indirectly interact with the cell surface-exposed phosphatidylserine through the soluble proteins MFG-E8, Gas 6 and protein S. Clustering of the erythrocyte membrane protein band 3 cau­ses the binding of natural antibodies, and opsonization of erythrocytes with C3b enhances this process and facilitates the recognition of such cells by red pulp macrophages in the spleen. In senescent erythrocytes, the formation of the CD47-SIRPα complex («do not eat me» signal), is suppressed, and this is an additional stimulus for erythrocytes removing by splenic and liver macrophages. The purpose of the review is to describe the mechanisms of erythrophagocytosis and the molecular determinants of erythrocyte senescence and death, including eryptosis and neocytolysis, and to illustrate the substantiated facts and contradictions that exist at the present time of the study of this scientific problem.

Structural and functional properties of erythrocytes in patients with chronic cerebral ischemia.

Treatment of chronic cerebral ischemia is still an important problem, because in most cases you cannot influence the constantly progressive character of the disease. Particularly relevant is the search for relationships between laboratory parameters and clinical manifestations of chronic cerebral ischemia with a subsequent search of the most informative indicators for timely assessment of disease severity and prediction of outcome. The aim of the study was to determine the relationships between neurological and cognitive disorders with representativeness in the membrane of red blood cells proteins and lipids in patients with chronic cerebral ischemia stage I and II treatment. Under constant observation there were 44 patients with a diagnosis of chronic cerebral ischemia stage I or II on the background of hypertensive disease stage II. Already in the early stages of the development of chronic cerebral ischemia are significant changes in the content of proteins that form the structure of erythrocyte membrane, shape and flexibility of the membrane, provide intracellular metabolism. Revealed violations in the content and ratio of lipids in the membrane. Primarily, this refers to the content of membrane glycerophospholipids and sphingomyelins, which are the basis of the double lipid layer of cell membrane and play a major role in regulating protein macromolecules and to the normal metabolism of erythrocytes. There is a relatively large number of significant relationships between laboratory parameters and parameters of the neuropsychiatric status in patients with chronic cerebral ischemia stage I or II before and after treatment.

Characteristics of water and ion exchange of Elodea nuttallii cells at high concentrations of lanthanides

Changes of diffusive permeability of membranes of Elodea nuttallii cells following a short-term (60 min) treatment with high concentrations of lanthanides were recorded by the 1H NMR-diffusometry and conductometry methods. The 1-h infiltration of segments of Elodea nuttallii internodes in 10 mM solutions of nitrates of La, Nd and Lu resulted in the increased leakage of electrolytes from cells, but has no effect on a water diffusive permeability of membranes. In samples subjected to a 30 min pretreatment with a water channel inhibitor HgCl2 the water diffusive permeability of membranes (Pd) drops down under the influence of lanthanides, as well as an outcome of electrolytes. To explain the observed effects the change of spontaneous curvature of membrane lipid layer has been taken into consideration. The interaction of lanthanides with lipids of plasmalemma leads to the negative spontaneous curvature of lipid layer at which membrane channels are unclosed. Blocking of the ionic and water channels by mercury ions compensate the effect of change of spontaneous curvature of lipid layer.

The Flip-Flop Diffusion Mechanism across Lipids in a Hybrid Bilayer Membrane

In this study, we examine the mechanism of flip-flop diffusion of proton carriers across the lipid layer of a hybrid bilayer membrane (HBM). The HBM consists of a lipid monolayer appended on top of a self-assembled monolayer containing a Cu-based O2 reduction catalyst on a Au electrode. The flip-flop diffusion rates of the proton carriers dictate the kinetics of O2 reduction by the electrocatalyst. By varying both the tail lengths of the proton carriers and the lipids, we find the combinations of lengths that maximize the flip-flop diffusion rate. These experimental results combined with biophysical modeling studies allow us to propose a detailed mechanism for transmembrane flip-flop diffusion in HBM systems, which involves the bending of the alkyl tail of the proton carrier as the rate-determining step. Additional studies with an unbendable proton carrier further validate these mechanistic findings.

New Opportunities Offered By Lipid-Modified Electrodes to Interrogate Transmembrane Diffusion Mechanisms and Kinetics

Diffusion of small molecules between intra- and extra-cellular spaces across lipid layers is instrumental to the understanding of fundamental biological transport processes and the improvement in drug delivery schemes. In natural systems, organisms develop intricate ion channels and stimuli-responsive gating machineries for signal transduction, cell identification, and toxin removal, while still allowing the selective and proficient uptake of vital nutrients that are critical to survival, growth, and reproduction. Understanding membrane function is a colossal step in developing cutting-edge drug therapies in a rational way. Lipinski’s rule of five describes the molecular properties essential for the bioavailability and pharmacokinetics of candidate drugs inside human bodies and thus serves as a first-order consideration for drug design.Altering the length of an alkyl tail is thus a viable method to tune the lipophilicity of a compound, thus modulating the transport properties of the drug. In this presentation, we report on our efforts to interrogate the “flip-flop” diffusion mechanism of alkyl proton carriers traversing the lipid layer of a hybrid bilayer membrane (HBM) using electrochemical techniques including but not limited to electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and pulse techniques. The 4-nm thick HBM electrochemical platform consists of a lipid monolayer appended on top of a self-assembled monolayer (SAM) containing a dinuclear Cu-triazole complex for O2 reduction reaction (ORR) on a Au electrode prepared using electron-beam evaporation (Scheme 1a). The “flip-flop” diffusion rates of the proton carriers dictate the O2 reduction turnover frequency by the Cu-based electrocatalyst. By varying both the tail lengths of the proton carriers and the lipids, we find that a difference between the lipid tail lengths and the proton carrier tail lengths of approximately 6-10 Å results in a maximum “flip-flop” diffusion rate. We evaluate these empirical findings using a biophysical model that contains parameters considering changes in Coulombic interactions, stress buildup, surface tension, and dielectric constants across the lipid-water interface. The model correctly mimics the relative alterations in the energy barriers associated with the rate-determining step (RDS) for the “flip-flop” diffusion process (Scheme 1b). Studies with a rigid proton carrier further substantiate findings that the RDS involves the bending of the alkyl tail of the proton carrier as it moves across the hydrophobic interior of the lipid layer. We envision that the methodologies developed here will ultimately lead to improved understanding of the mechanismof “flip-flop” diffusion in lipid bilayers and aid in the development of next-generation targeted drug delivery schemes. Acknowledgements. E.C.M.T. acknowledges a Croucher Foundation Scholarship. We thank the US Department of Energy (DE-FG02-95ER46260) for support of this research. Scheme 1 is presented here. Figure 1

Vitamins and functional nutrients for extremely low birth weight infants

While the need for appropriate post natalgrowth provides many studies focused on macronutrients in the nutrition of preterm infants, the concern for the quality of development of micro premies increases the interest in micronutrients, vitamins and trace elements. Moreover, the ongoing challenge to avoid serious complications of extreme prematurity requires investigations on functional nutrients especially those with antioxidant effect or immunomodulatory properties. Despite the lack of defined recommendations, much evidence is available. The antioxidant properties of vitamin A and vitamin E make these elements important in the nutrition of ELBW infants who are born with a strong deficit of these vitamins. Low plasma vitamin A concentrations increases the risk of chronic lung disease and long-term respiratory disabilities in preterm infants. Vitamin E counteracts peroxidation of polyunsaturated fattyacids in lipid layers of cell membranes[1]. Preterm infants are exposed to a special risk for metabolic bone disease also due to maternal deficiency and difficulty achieving adequate enteral intake of calcium, phosphorus, and vitamin D. The link between vitamin D supplementation, serum 25-OH-D concentration and long term outcome inpreterminfants are poorly understood. Some findings supportthe opinion that a vitamin D intake of 800-1000 IU/day is necessary in preterm infants while higher doses are needed in newbornof mothers with vitamin D deficiency [2]. Furthermore, vitamin D plays a rolein physiological processes that affect neuromuscular and immune functions, heart, lung, pancreas, and brain. Many trials reported enhanced visual acuity and better cognitive performance in the short-term assessments of preterm infants fed formulas supplemented with long chain polyunsaturated fattyacids(LCPUFA) [3-5]. However, there are many questions about the appropriate dosages and some doubts on the long-term advantages [6,7]. Among strategies for the prevention of NEC and sepsis through the use of immunonutrients, lactoferrin[8] and probiotics[9] are getting a place in clinical practice. Other studies will be needed to assess the extension of the evidence currently observed, especially for neonatesin low NEC rates settings. To date are available only weak evidence about the efficacy of arginine and glutamineto prevent intestinal mucosal injury in high risk infants [10]. Lutein supplementation for preterm infants raises plasma concentrations to those observed in human milk fed term infants and ensures the benefit of anti oxidant effect of carotenoidson immature retina and developing brain. Larger trials are needed to determine the best methods of administration to affect the retinopathy of prematurity[11].

Human flavin-containing monooxygenase 3 on graphene oxide for drug metabolism screening.

Human flavin-containing monooxygenase 3 (hFMO3), a membrane-bound hepatic protein, belonging to the second most important class of phase-1 drug-metabolizing enzymes, was immobilized in its active form on graphene oxide (GO) for enhanced electrochemical response. To improve protein stabilization and to ensure the electrocatalytic activity of the immobilized enzyme, didodecyldimethylammonium bromide (DDAB) was used to mimic lipid layers of biological membranes and acted as an interface between GO nanomaterial and the hFMO3 biocomponent. Grazing angle attenuated total reflectance Fourier transform infrared (GATR-FT-IR) experiments confirmed the preservation of the protein secondary structure and fold. Electrochemical characterization of the immobilized enzyme with GO and DDAB on glassy carbon electrodes was carried out by cyclic voltammetry, where several parameters including redox potential, electron transfer rate, and surface coverage were determined. This system's biotechnological application in drug screening was successfully demonstrated by the N-oxidation of two therapeutic drugs, benzydamine (nonsteroidal anti-inflammatory) and tamoxifen (antiestrogenic widely used in breast cancer therapy and chemoprevention), by the immobilized enzyme.

Proton switch for modulating oxygen reduction by a copper electrocatalyst embedded in a hybrid bilayer membrane.

Molecular switches gate many fundamental processes in natural and artificial systems. Here, we report the development of an electrochemical platform in which a proton carrier switches the activity of a catalyst. By incorporating an alkyl phosphate in the lipid layer of a hybrid bilayer membrane, we regulate proton transport to a Cu-based molecular oxygen reduction reaction catalyst. To construct this hybrid bilayer membrane system, we prepare an example of a synthetic Cu oxygen reduction reaction catalyst that forms a self-assembled monolayer on Au surfaces. We then embed this Cu catalyst inside a hybrid bilayer membrane by depositing a monolayer of lipid on the self-assembled monolayer. We envisage that this electrochemical system can give a unique mechanistic insight not only into the oxygen reduction reaction, but into proton-coupled electron transfer in general.

An efficient depyrogenation method for recombinant bacterial outer membrane lipoproteins

Bacterial outer membrane lipoproteins are anchored in the outer membrane lipid layer in close association with lipopolysaccharides (LPS) and with other hydrophobic membrane proteins, making their purification technically challenging. We have previously shown that a thorough delipidation of outer membrane preparations from the Escherichia coli expression host is an important step to eliminate contaminant proteins when purifying recombinant antigens expressed in fusion with the Pseudomonas aeruginosa OprI lipoprotein. Here we report the cloning and expression of three antigens in fusion with OprI (ovalbumin, eGFP and BbPDI) and our efforts to deal with the variable LPS contamination levels observed in different batches of purified lipoproteins. The use of polymyxin B columns or endotoxin removal polycationic magnetic beads for depyrogenation of purified lipoproteins resulted in high protein losses and the use of Triton X-114 or sodium deoxycholate during the course of affinity chromatography showed to be ineffective to reduce LPS contamination. Instead, performing a hot phenol/water LPS extraction from outer membrane preparations prior to metal affinity chromatography allowed the purification of the recombinant fusion lipoproteins with LPS contents below 0.02EU/μg of protein. The purified recombinant lipoproteins retain their capacity to stimulate bone marrow-derived dendritic cells allowing for the study of their immunomodulatory properties through TLR2/1. This is a simple and easy to scale up method that can also be considered for the purification of other outer membrane lipoproteins.

Hydroxypropyl Methylcellulose Mediated Precipitation Inhibition of Sirolimus: From a Screening Campaign to a Proof-of-Concept Human Study

The aim of this study was to develop a sirolimus (BCS class II drug substance) solid oral dosage form containing a precipitation inhibitor, which would result in an improved sirolimus absorption in humans compared to the formulation containing nanosized sirolimus without a precipitation inhibitor, i.e., Rapamune. The selection of the precipitation inhibitor was based on the results of a screening campaign that identified two "hit" excipients: HPMC 603 (i.e., Pharmacoat 603) and Poloxamer 407. However, in a confirmatory precipitation inhibitor study using biorelevant media (Fa/FeSSIF) HPMC 603 more effectively inhibited sirolimus precipitation than Poloxamer 407. In the PAMPA assay, HPMC 603, but not Poloxamer 407, significantly increased the flux of the sirolimus across the membrane lipid layer. Additionally, a differential scanning calorimetry (DSC) and an infrared (IR) spectroscopy study revealed that interactions between the sirolimus and HPMC 603 were developed that could lead to the observed precipitation inhibition effect. Based on the above data, two formulations with HPMC 603-coated sirolimus particles were developed, namely, formulation A (d (0.5) = 0.21 μm) and formulation B (d (0.5) = 1.7 μm). A human pharmacokinetic study outlined that significantly higher AUC and Cmax were obtained for formulations A and B in comparison to Rapamune. This result could be attributed to the HPMC 603 (Pharmacoat 603) mediated sirolimus precipitation inhibition resulting in improved sirolimus absorption from the gastrointestinal tract in humans.

Selenium and Vitamin A and E in the Nutrition of Very Low-Birth Weight Preterm Infants

Deficient antioxidant defenses in preterm infants have been implicated in diseases such as bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, periventricular leukomalacia, and intraventricular hemorrhage. The antioxidant properties of selenium, vitamin A, and vitamin E make these elements important in the nutrition of Very Low-Birth Weight (VLBW) infants. Selenium is a component of glutathione peroxidase, an enzyme that prevents the production of free radicals. The decrease in plasma selenium in VLBW infants in the first month after birth makes evident that preterm infants have low selenium store and require supplementation by parenteral and enteral nutrition. A meta-analysis, with only three trials, showed that selenium supplementation did not affect mortality, and the incidence of neonatal chronic lung disease or retinopathy of prematurity, but was associated with a reduction in lateonset sepsis. Most VLBW infants and extremely Low-Birth Weight Infants (ELBW) are born with low vitamin A stores and need vitamin A supplementation by intramuscular or enteral route. Low plasma retinol concentrations increase the risk of chronic lung disease/bronchopulmonary dysplasia and long-term respiratory disabilities in preterm infants. There is evidence that vitamin A supplementation decreases the mortality or oxygen requirement at one month of age, and oxygen requirement at 36 weeks’ postmenstrual age. Vitamin E blocks natural peroxidation of polyunsaturated fatty acids from lipid layers of cell membranes. VLBW infants have a decrease in plasma concentrations in the first month after birth suggesting the need of vitamin E supplementation. A meta-analysis on vitamin E supplementation concluded that vitamin E did not affect mortality, risk of bronchopulmonary dysplasia, and necrotizing enterocolitis but reduced the risk of intraventricular hemorrhage and increased the risk of sepsis. Serum vitamin E concentrations higher than 3.5 mg/dL are associated with a decrease in the risk of severe retinopathy of prematurity, and blindness, but also with an increase in neonatal sepsis. Caution is recommended with the supplementation of high doses of parenteral vitamin E and supplementation that increases serum levels above 3.5 mg/dL. In conclusion: although it is known that preterm infants are deficient in selenium, vitamin A and E, more studies are required to determine the best way to supplement and the impact of supplementation on neonatal outcome

Hyperlipidemia offers protection against Leishmania donovani infection: role of membrane cholesterol

Leishmania donovani (LD), the causative agent of visceral leishmaniasis (VL), extracts membrane cholesterol from macrophages and disrupts lipid rafts, leading to their inability to stimulate T cells. Restoration of membrane cholesterol by liposomal delivery corrects the above defects and offers protection in infected hamsters. To reinforce further the protective role of cholesterol in VL, mice were either provided a high-cholesterol (atherogenic) diet or underwent statin treatment. Subsequent LD infection showed that an atherogenic diet is associated with protection, whereas hypocholesterolemia due to statin treatment confers susceptibility to the infection. This observation was validated in apolipoprotein E knockout mice (AE) mice that displayed intrinsic hypercholesterolemia with hepatic granuloma, production of host-protective cytokines, and expansion of antileishmanial CD8(+)IFN- γ (+) and CD8(+)IFN- γ (+)TNF- α (+) T cells in contrast to the wild-type C57BL/6 (BL/6) mice when infected with LD. Normal macrophages from AE mice (N-AE-MΦ) showed 3-fold higher membrane cholesterol coupled with increased fluorescence anisotropy (FA) compared with wild-type macrophage (N-BL/6-MΦ). Characterization of in vitro LD-infected AE macrophage (LD-AE-MΦ) revealed intact raft architecture and ability to stimulate T cells, which were compromised in LD-BL/6-MΦ. This study clearly indicates that hypercholesterolemia, induced intrinsically or extrinsically, can control the pathogenesis of VL by modulating immune repertoire in favor of the host.

Distribution, Orientation and Dynamics of 17β-Estradiol in Lipid Membranes Investigated by Solid-State NMR

17β-Estradiol (E2) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the interaction of E2 with POPC membranes using a combination of solid-state NMR methods. Investigations were conducted on powder-type samples at lipid/E2 ratios of 5:1 and 10:1 and at a temperature of 303K. Information about the chain dynamics and membrane packing properties was obtained using 2H solid-state NMR. The spectra indicate that E2 does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E2 induced alterations of the membrane structure were detected. 1H magic-angle spinning NMR showed well resolved signals from E2 as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E2 and the membrane lipids indicating that E2 is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E2 is broadly distributed in the membrane with a maximum of the E2 distribution function in the upper chain region of the membrane. To further establish the orientation and dynamics of E2 we obtained a variant in which four hydrogens were replaced by deuterium and recorded corresponding 2H NMR spectra. The analysis of these spectra was performed by a model describing the orientation and dynamics of E2 in the membrane. We determined its average orientation in the membrane and conclude that E2 is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.

ZF21 Protein, a Regulator of the Disassembly of Focal Adhesions and Cancer Metastasis, Contains a Novel Noncanonical Pleckstrin Homology Domain

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cells We have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, β-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to β-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.

Rola kwasu dokozaheksaenowego w czynności komórek nerwowych

Docosahexaenoic acid (DHA, C22: 6n-3) is the most abundant polyunsaturated fatty acid in neuronal phospholipids, particularly in the cortex. The main source of DHA for neural cells is food, and hepatic and astroglia DHA synthesis from essential a-linolenic acid (C18: 3n-3). Accretion of DHA in the brain is most intensive during fetal life and the first two years of life. An adequate level of DHA in cell membranes is important for many functions of neural cells and this is presumably the reason for DHA saving in the adult mammalian brain during dietary a-linolenic acid deficiency. DHA-containing phospholipids in membranes are flexible and membranes possessing a high content of them are quite thin, more permeable to ions and small molecules, have looser lipid packing, and finally are more "dynamic" than membranes composed of other fatty acid containing phospholipids. Furthermore, these membranes create an appropriate environment for integral proteins highly condensed in neurons, such as receptors, ion channels, enzymes, and peripheral proteins. The quantity of phosphatidylserine in the inner membrane lipid layer depends on the availability of DHA to neurons. Phosphatidylserine promotes neuronal survival by translocation/activation of kinase Akt and Raf-1/MEK. DHA present in membrane phospholipids facilitates v-SNARE/t-SNARE complex formation, which is necessary for fusion of synaptic vesicles and plasma membranes necessary for transmitter exocytosis, and neurite outgrowth-dependent plasticity. DHA plays an important neuroprotective role. DHA has been shown to inhibit PGE2 synthesis and COX-1 expression in astrocytes, and DHA derivatives, especially neuroprotectins D, can suppress inflammatory responses, preventing neuronal damage or apoptosis. The results of high DHA content in neuronal membranes and formation of DHA derivates, as well as the function of DHA-dependent phosphatidylserine, may explain the promising results supporting beneficial DHA supplementation in neurodegenerative diseases and improvement of brain function.

Properties of Chlorophyll and Derivatives in Homogeneous and Microheterogeneous Systems

Chlorophyll (Mg-Chl) and its derivatives, zinc chlorophyll (Zn-Chl), copper chlorophyll (Cu-Chl), pheophytin (Pheo), pheophorbide (Pheid), and zinc chlorophyllide (Zn-Chld), were studied as to their acid-base equilibrium properties, hydrophobicity, stability, binding, and relative localization in neutral surfactant micellar systems. The stability order of metalochlorophyll (pH(M)) in acidic medium was found to be Cu-Chl > Zn-Chld > Zn-Chl > Mg-Chl. The apparent pK(a) for protonation of porphyrin ring nitrogens was around 1.0 for all derivatives. The pK(a) for protonation of carboxylate phorbide was 5.9 for Pheid and 2.4 for Zn-Chld. This difference was attributed to complexation of carboxylate with zinc. The hydrophobicity of chlorophyll in relation to the ability of partitioning the cell membrane lipid layer was estimated in the octanol/water biphasic system. Pheo, a more hydrophobic molecule, presented the highest partition coefficient (K(P)) in the organic phase, followed by Cu-Chl, Mg-Chl, Zn-Chl, Pheid, and Zn-Chld. The hydrophobic character was the key to relative drug location in the micellar systems. All studied derivatives interacted strongly with Tween 80 micellar systems, and particularly with P-123. For both surfactants, the order followed by binding constant (K(b)) was Zn-Chld > Pheo > Cu-Chl > Mg-Chl > Zn-Chl > Pheid, while binding constants estimated for the Chl containing the phytyl group correlated with K(P). Fluorescence quenching studies have shown that phorbides are located in a less hydrophobic region than the phytyl chain-containing derivatives, which are located preferentially in a deeper micellar microenvironment. Thus, the association of the chlorophylls with specific binding sites of micellar systems is strongly modulated by the presence of phytyl chains and metal coordinated to the porphyrinic ring.

On the local anaesthetic effect of barbiturates

Abstract Controversy exists about whether the free acid (non-ionized) or the anionic (ionized) form of barbiturates abolish the excitation of nerves. The experiments made showed that sodium pentobarbitone and sodium phenobarbitone are more effective at pH 6·8 than 8·8 in blocking the action potential in the desheathed frog nerve. The experimental procedure employed excludes the possibility that this difference in activity is due only to a more effective rate of penetration of the non-ionized form across the nerve membrane. In the same preparation these barbiturates at both pH 6·8 and 8·8 did not interfere with the uptake and release of radiocalcium. These data suggest that barbiturates block the action potential by increasing the surface pressure of the lipid layer of the excitable membrane and do not interfere with the calcium binding to sites which govern the increased membrane conductance during excitation.

Investigating the membrane orientation and transversal distribution of 17β-estradiol in lipid membranes by solid-state NMR

17beta-Estradiol (E(2)) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E(2) using a combination of solid-state NMR methods. (2)H NMR results indicate that E(2) does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E(2) induced alterations of the membrane structure were detected. (1)H magic-angle spinning NMR showed well resolved signals from E(2) as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E(2) and the membrane lipids indicating that E(2) is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E(2) is broadly distributed in the membrane with a maximum of the E(2) distribution function in the upper chain region of the membrane. We conclude that E(2) is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.

Reactive oxygen species and sperm cells

There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Excessive reactive oxygen species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced. The essential steps of lipid peroxidation have been listed as well as antioxidant substances of semen. A variety of detection techniques of lipid peroxidation have been summarized together with the lipid components of sperm membranes that can be subjected to stress. It is unsolved, a threshold for ROS levels that may induce functional sperm ability or may lead to male infertility.