Spin label reduction kinetics,a procedure to study the effect of drugs on membrane permeability: The effects of monosodium urate,dimethyl sulfoxide and amphotericin B

A method is presented to study the effect of drugs on membrane permeability. It is based on the reduction of a spin label trapped in the internal aqueous compartment(s) of membranes by ascorbate ions added to the bulk aqueous phase. The decay of the electron spin resonance signal of the spin label as a function of time gives an indication of the effect of added agents on the permeability of membranes. To demonstrate the technique, the effect on model membranes of egg phosphatidylcholine of the gout-implicated compound monosodium urate, the aprotic solvent dimethyl sulfoxide and the polyene antibiotic amphotericin B were examined. Monosodium urate did not affect the permeability, casting doubt on a proposed mechanism whereby the agent disrupts the membranes via hydrogen bonding. Dimethyl sulfoxide promoted a gradual increase in rate of solute passage across cholesterol-containing model membranes. Amphotericin B had pronounced effect on the permeability of cholesterol-containing membranes, causing nearly total loss of paramagnetism immediately after addition. Some aspects of the mechanism of action of the drugs are discussed as well as the advantages and disadvantages of the method. The experiments also allow the evaluation of the effect of surface charge and cholesterol on the dimensions of model membranes.     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Laurdan fluorescence spectroscopy in the thylakoid bilayer: The effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment

Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach (Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 °C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.     

Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behavior — it has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid–lipid and protein–lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein–lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.     

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, β-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and β-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (> 85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.     

Substitution of chloride by bromide modifies the low-temperature tyrosine Z oxidation in active photosystem II

Chloride is an essential cofactor for photosynthetic water oxidation. However, its location and functional roles in active photosystem II are still a matter of debate. We have investigated this issue by studying the effects of Cl⁻ replacement by Br⁻ in active PSII. In Br⁻ substituted samples, Cl⁻ is effectively replaced by Br⁻ in the presence of 1.2 M NaBr under room light with protection of anaerobic atmosphere followed by dialysis. The following results have been obtained. i) The oxygen-evolving activities of the Br⁻-PSII samples are significantly lower than that of the Cl⁻-PSII samples; ii) The same S₂ multiline EPR signals are observed in both Br⁻ and Cl⁻-PSII samples; iii) The amplitudes of the visible light induced S₁Tyr_Z^• and S₂Tyr_Z^• EPR signals are significantly decreased after Br⁻ substitution; the S₁Tyr_Z^• EPR signal is up-shifted about 8 G, whereas the S₂Tyr_Z^• signal is down-shifted about 12 G after Br⁻ substitution. These results imply that the redox properties of Tyr_Z and spin interactions between Tyr_Z^• and Mn-cluster could be significantly modified due to Br⁻ substitution. It is suggested that Cl⁻/Br⁻ probably coordinates to the Ca²⁺ ion of the Mn-cluster in active photosystem II.     

Enhancement of YD spin relaxation by the CaMn4 cluster in photosystem II detected at room temperature: A new probe for the S-cycle

The long-lived, light-induced radical Y_D^• of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn₄ cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in Y_D^• EPR intensity upon the application of a single laser flash to S₁ state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y_D^• by the OEC in the S₂ state, as a consequence of the single laser flash turnover. The post-flash decay reflected S₂ → S₁ back-turnover, as confirmed by their correlations with independent measurements of S₂ multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y_D^• as a probe.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10⁵ FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm² within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.     

New insights on ChlD1 function in Photosystem II from site-directed mutants of D1/T179 in Thermosynechococcus elongatus

The monomeric chlorophyll, Chl_D1, which is located between the P_D1P_D2 chlorophyll pair and the pheophytin, Pheo_D1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg²⁺ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn₄CaO₅ cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *Chl_D1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of P_D1P_D2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the Chl_D1⁺Pheo_D1^? radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of ¹O₂ generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to Chl_D1 upon Q_A^? formation, indicates that in the T179H-PSII and in the WT*3-PSII, the Chl_D1 itself is the chlorophyll that is first damaged by ¹O₂, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, Chl_D1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of Chl_D1 very likely contribute to the well-known electrochromic shifts observed at ~430?nm during the S-state cycle.     

The tandem of free radicals and methylglyoxal

Methylglyoxal is an alpha-oxoaldehyde inevitably produced from triose-phosphate intermediates of phosphorylating glycolysis, and also from amino acids and acetone. Recently, the attention has been focused on the involvement of free radicals in methylglyoxal toxicity. In this review, a summary of the relationship between methylglyoxal metabolism and free radical production is presented, extending discussion from the possible metabolic routes to the toxicological events by reviewing the role of free radicals in both generation and degradation of this 1,2-dicarbonyl as well as in the modification of biological macromolecules, and focusing on the action of methylglyoxal upon cellular glutathione content. Methylglyoxal-provoked free radical generation involving reactive oxygen species (ROS), reactive nitrogen species (RNS) as well as organic radicals like methylglyoxal radial or crosslinked protein radical as potential risk factors to tissue damage propagation, is thoroughly discussed. Special attention is paid to the potential therapeutic interventions. The paper arrives at the conclusion that a tight junction exists between methylglyoxal toxicity and free radical (particularly ROS) generation, though the toxicity of 1,2-dicarbonyl evolves even under anaerobic conditions, too. The events follow a sequence beginning with carbonyl stress essential for the toxicity, leading to free radical formation and finally ending in either apoptosis or necrosis. Both oxidative and nitrosative stress play important but not indispensable role in the development of methylglyoxal toxicity.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

Design,synthesis and biological evaluation of benzoylacrylic acid shikonin ester derivatives as irreversible dual inhibitors of tubulin and EGFR

In this study, a series of shikonin derivatives combined with benzoylacrylic had been designed and synthesized, which showed an inhibitory effect on both tubulin and the epidermal growth factor receptor (EGFR). In vitro EGFR and cell growth inhibition assay demonstrated that compound PMMB-317 exhibited the most potent anti-EGFR (IC₅₀ = 22.7 nM) and anti-proliferation activity (IC₅₀ = 4.37 μM) against A549 cell line, which was comparable to that of Afatinib (EGFR, IC₅₀ = 15.4 nM; A549, IC₅₀ = 6.32 μM). Our results on mechanism research suggested that, PMMB-317 could induce the apoptosis of A549 cells in a dose- and time-dependent manner, along with decrease in mitochondrial membrane potential (MMP), production of ROS and alterations in apoptosis-related protein levels. Also, PMMB-317 could arrest cell cycle at G2/M phase to induce cell apoptosis, and inhibit the EGFR activity through blocking the signal transduction downstream of the mitogen-activated protein MAPK pathway and the anti-apoptotic kinase AKT pathway; typically, such results were comparable to those of afatinib. In addition, PMMB-317 could suppress A549 cell migration through the Wnt/β-catenin signaling pathway in a dose-dependent manner. Additionally, molecular docking simulation revealed that, PMMB-317 could simultaneously combine with EGFR protein (5HG8) and tubulin (1SA0) through various forces. Moreover, 3D-QSAR study was also carried out, which could optimize our compound through the structure-activity relationship analysis. Furthermore, the in vitro and in vivo results had collectively confirmed that PMMB-317 might serve as a promising lead compound to further develop the potential therapeutic anticancer agents.     

Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the P_D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P_680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA₁ and psbA₂ genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT^?) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA₃ gene. The O₂-evolving activity of His-tagged PSII isolated from WT^? was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA₁ is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT^?. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P₆₈₀, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to P_D1 is not an important determinant of the redox and spectroscopic properties of P₆₈₀ in T. elongatus.     

Impact of apolipoprotein A1- or lecithin:cholesterol acyltransferase-deficiency on white adipose tissue metabolic activity and glucose homeostasis in mice

High density lipoprotein (HDL) has attracted the attention of biomedical community due to its well-documented role in atheroprotection. HDL has also been recently implicated in the regulation of islets of Langerhans secretory function and in the etiology of peripheral insulin sensitivity. Indeed, data from numerous studies strongly indicate that the functions of pancreatic β-cells, skeletal muscles and adipose tissue could benefit from improved HDL functionality. To better understand how changes in HDL structure may affect diet-induced obesity and type 2 diabetes we aimed at investigating the impact of Apoa1 or Lcat deficiency, two key proteins of peripheral HDL metabolic pathway, on these pathological conditions in mouse models. We report that universal deletion of apoa1 or lcat expression in mice fed western-type diet results in increased sensitivity to body-weight gain compared to control C57BL/6 group. These changes in mouse genome correlate with discrete effects on white adipose tissue (WAT) metabolic activation and plasma glucose homeostasis. Apoa1-deficiency results in reduced WAT mitochondrial non-shivering thermogenesis. Lcat-deficiency causes a concerted reduction in both WAT oxidative phosphorylation and non-shivering thermogenesis, rendering lcat^?/? mice the most sensitive to weight gain out of the three strains tested, followed by apoa1^?/? mice. Nevertheless, only apoa1^?/? mice show disturbed plasma glucose homeostasis due to dysfunctional glucose-stimulated insulin secretion in pancreatic β-islets and insulin resistant skeletal muscles. Our analyses show that both apoa1^?/? and lcat^?/? mice fed high-fat diet have no measurable Apoa1 levels in their plasma, suggesting no direct involvement of Apoa1 in the observed phenotypic differences among groups.     

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage

Mitochondrial impairment and calcium (Ca⁺⁺) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca⁺⁺-ATPase pumps are impaired causing cytoplasmic Ca⁺⁺ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser⁶⁵, and reduced calpain-cleavage of Parkin. Treatment with the Ca⁺⁺ ionophore A23187, which facilitates Ca⁺⁺ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca⁺⁺ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca⁺⁺-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.