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BBA - Biomembranes (v.1669, #1)
Synthesis and membrane behavior of a new class of unnatural phospholipid analogs useful as phospholipase A2 degradable liposomal drug carriers by Thomas L. Andresen; Kent Jørgensen (pp. 1-7).
A new and unnatural type of lipid analogs with the phosphocholine and phosphoglycerol head groups linked to the C-2 position of the glycerol moiety have been synthesized and the thermodynamic lipid membrane behavior has been investigated using differential scanning calorimetry. From the heat capacity measurements, it was observed that the pre-transition was abolished most likely due to the central position of the head groups providing better packing properties in the low temperature ordered gel phase. Activity measurements of secretory phospholipase A2 (PLA2) on unilamellar liposomal membranes revealed that the unnatural phospholipids are excellent substrates for PLA2 catalyzed hydrolysis. This was manifested as a minimum in the PLA2 lag time in the main phase transition temperature regime and a high degree of lipid hydrolysis over a broad temperature range. The obtained results provide new information about the interplay between the molecular structure of phospholipids and the lipid membrane packing constrains that govern the pre-transition. In addition, the PLA2 activity measurements are useful for obtaining deeper insight into the molecular details of the catalytic site of PLA2. The combined results also suggest new approaches to rationally design liposomal drug carries that can undergo a triggered activation in diseased tissue by overexpressed PLA2.
Keywords: Abbreviations; DPPC; 1,2-Dipalmitoyl-; sn; -glycero-3-phosphocholine, 1,2-dihexadecanoyl-; sn; -glycero-3-phosphocholine; DPPG; 1,2-Dipalmitoyl-; sn; -glycero-3-phosphoglycerol, 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoglycerol; 1-; O; -DPPC′; (; S; )-1-; O; -hexadecyl-3-hexadecanoyl; -; glycero-2-phosphocholine; 1-; O; -DPPG′; (; S; )-1-; O; -hexadecyl-3-hexadecanoyl-glycero-2-phosphoglycerol; 1-; O; -DPPC; 1-; O; -hexadecyl-2-hexadecanoyl-; sn; -glycero-3-phosphocholine; 1-; O; -DPPG; 1-; O; -hexadecyl-2-hexadecanoyl-; sn; -glycero-3-phosphoglycerol; PLA2; phospholipase A2; DSPE-PEG; 2000; 1,2-distearoyl-; sn; -glycero-3-phosphoethanolamine poly(ethylene glycol); 2000Liposomes; Lipid synthesis; Ripple phase formation; Phospholipase A2; Enzyme lipid interaction; Liposomal drug carrier; Lipid membrane heterogeneity; Calorimetry; Fluorescence
Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures by Céline Laroche; Hélène Simonin; Laurent Beney; Patrick Gervais (pp. 8-16).
Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water–glycerol media. A phase transition occurred at 12 °C in water, at 16.5 °C at Aw=0.75, and at 19.5 °C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 °C in pure water and at 27 °C in binary water–glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 °C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 °C in water, to 64 °C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.
Keywords: Phase transition; Osmotic pressure; S. cerevisiae; Phospholipid; Fourier Transform Infrared spectroscopy
Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics by Akira Shibata; Ayako Sakata; Satoru Ueno; Tsuguhide Hori; Kazushi Minami; Yoshinobu Baba; Naoki Kamo (pp. 17-25).
Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation� in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation
Keywords: Oriented blue membrane; Cationic amine anesthetic; Proton pump regeneration; Proton pump inhibition; Biphasic effect; Binding site
Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells by Lisa Fischer; Silke Haag-Diergarten; Erwin Scharrer; Thomas A. Lutz (pp. 26-33).
The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 μmol/l). ATPγS (10 μmol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 μmol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPγS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.
Keywords: Glucagon; Hepatocyte; Hyperpolarization; Leukotriene receptor; Purinergic receptor
Epithelial transport and barrier function in occludin-deficient mice by J.D. Schulzke; A.H. Gitter; J. Mankertz; S. Spiegel; U. Seidler; S. Amasheh; M. Saitou; S. Tsukita; M. Fromm (pp. 34-42).
This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model.Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium ( Re, epithelial resistance). Conductance scanning differentiated transcellular ( Gc) and tight junctional conductance ( Gtj). The pH-stat technique quantified gastric acid secretion.In occludin+/+ mice, Re was 23±5 Ω cm2 in jejunum, 66±5 Ω cm2 in distal colon and 33±6 Ω cm2 in gastric corpus and was not altered in heterozygotic occludin+/− or homozygotic occludin−/− mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6±1.0 and 0.3±0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin−/− mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8±0.8 kΩ cm2 in urinary bladder and 33±6 Ω cm2 in stomach and not altered in occludin−/− mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin−/− mice.Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.
Keywords: Abbreviations; AC; alternating current; I; SC; short-circuit current; R; t; total tissue resistance; R; e; resistance of the epithelial layer; R; sub; resistance of the subepithelial tissues; G; t; total tissue conductivity; G; se; conductivity of the surface epithelium; G; cry; conductivity of the crypts; G; c; conductivity of the transcellular pathway; G; tj; conductivity of the paracellular (i.e. tight junctional) pathway; NS; not significantTight junction; Occludin; Impedance analysis; Conductance scanning; Gastric acid secretion
Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions by Diksha Narhar Dani; Jayashree Krishna Sainis (pp. 43-52).
A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000× g, followed by 90,000× g and finally at 150,000× g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin–NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000× g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000× g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO2 fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.
Keywords: Calvin cycle enzymes; Cyanobacteria; Electron transport system; Photophosphorylation; Thylakoid membrane
Actin cytoskeleton disassembly affects conductive properties of stretch-activated cation channels in leukaemia cells by Alexander Staruschenko; Yuri A. Negulyaev; Elena A. Morachevskaya (pp. 53-60).
Mechanosensitive channels in various eucaryotic cells are thought to be functionally and structurally coupled to the cortical cytoskeleton. However, the results of electrophysiological studies are rather controversial and the functional impact of cytoskeleton assembly–disassembly on stretch-activated channel properties remains unclear. Here, the possible involvement of cytoskeletal elements in the regulation of stretch-activated Ca2+-permeable channels was studied in human leukaemia K562 cells with the use of agents that selectively modify the actin or tubulin system. F-actin disassembly resulted in a considerable reduction of the amplitude of stretch-activated currents without significant change in channel open probability. The effects of treatments with cytochalasins or latrunculin were principally similar, developed gradually and consisted a strong decrease of single channel conductance. Microtubule disruption did not affect stretch-activated channels. The data presented here are in principal agreement with the general conclusion that mechanosensitive channel functions are largely dependent on the integrity of the cortical actin cytoskeleton. Specifically, changes in conductive properties of the pore may provide an essential mechanism of channel regulation underlying functional modulation of membrane currents. Our results allow one to speculate that microfilament organization may be an important determinant in modulating biophysical characteristics of stretch-activated cation channels in cells of blood origin.
Keywords: Abbreviations; MS; Mechanosensitive; SAC; Stretch-activated channel; ENaC; Epithelial Na; +; channel; CytB or D; Cytochalasin B or D; LatB; Latrunculin B; P; o; Channel open probabilityStretch-activated channel; Actin cytoskeleton; Cytochalasin; Latrunculin
Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation by Jan B. Koenderink; Giovanni Zifarelli; Li Yan Qiu; Wolfgang Schwarz; Jan Joep H.H.M. De Pont; Ernst Bamberg; Thomas Friedrich (pp. 61-68).
The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic α-subunit (four isoforms) and an ancillary β-subunit (three isoforms). Mutations in the α2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase α2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane.86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase α2-isoform.
Keywords: Human Na; +; /K; +; -ATPase α; 2; -subunit; ATP1A2; Familial hemiplegic migraine type 2; Pump current; Surface expression; ATP hydrolysis
Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels by Kanae Ichikawa; Tomoya Hikita; Noriyuki Maeda; Sei Yonezawa; Yoshito Takeuchi; Tomohiro Asai; Yukihiro Namba; Naoto Oku (pp. 69-74).
For the improvement of therapeutic efficacy in photodynamic therapy (PDT) by using a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), we previously prepared polyethylene glycol (PEG)-modified liposomes encapsulating BPD-MA (PEG-Lip BPD-MA). PEGylation of liposomes enhanced the accumulation of BPD-MA in tumor tissue at 3 h after injection of it into Meth-A-sarcoma-bearing mice, but, unexpectedly, decreased the suitability of the drug for PDT when laser irradiation was performed at 3 h after the injection of the liposomal photosensitizer. To improve the bioavailability of PEG-Lip BPD-MA, we endowed the liposomes with active-targeting characteristics by using Ala-Pro-Arg-Pro-Gly (APRPG) pentapeptide, which had earlier been isolated as a peptide specific to angiogenic endothelial cells. APRPG-PEG-modified liposomal BPD-MA (APRPG-PEG-Lip BPD-MA) accumulated in tumor tissue similarly as PEG-Lip BPD-MA and to an approx. 4-fold higher degree than BPD-MA delivered with non-modified liposomes at 3 h after the injection of the drugs into tumor-bearing mice. On the contrary, unlike the treatment with PEG-Lip BPD-MA, APRPG-PEG-Lip BPD-MA treatment strongly suppressed tumor growth after laser irradiation at 3 h after injection. Finally, we observed vasculature damage in the dorsal air sac angiogenesis model by APRPG-PEG-Lip BPD-MA-mediated PDT. The present results suggest that antiangiogenic PDT is an efficient modality for tumor treatment and that tumor neovessel-targeted, long-circulating liposomes are a useful carrier for delivering photosensitizer to angiogenic endothelial cells.
Keywords: Photodynamic therapy (PDT); Targeting; Polyethylene glycol (PEG); Liposome; Angiogenesis