TMA-DPH Fluorescence Anisotropy Research Articles

The influence of cationic lipoid - 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine - on model lipid membranes

The triesters of phosphatidylcholine as the derivatives of natural phosphatidylcholines are less cytotoxic than the other cationic lipoids, therefore they can be applied in lipofection and in drug delivery. However, a successful and effective use of these compounds requires detailed information of their mechanism of action, which is probably highly complex and multi-stages. However, the first barrier in the way to cell and thus the first side of action of these compounds is the cellular membrane. The aim of this work was to investigate the effect of one cationic lipoid, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) on model POPC/SM/Chol = 1:1:1 membranes. The experiments were performed on monolayer and bilayer systems and they involved the surface pressure measurements, Brewster angle microscopy studies, dynamic light scattering and zeta potential measurements and the experiments with the surfactant solution and steady-state fluorescence anisotropy of DPH and TMA-DPH. Moreover, to perform the studies systematically also the properties of the binary (POPC/EPOPC, SM/EPOPC, Chol/EPOPC) and ternary (POPC/Chol/EPOPC, SM/Chol/EPOPC) model systems were investigated. The obtained results indicated that even low concentration of EPOPC alters properties and organization of model membranes. Namely, EPOPC makes the interactions in model membrane weaker and increases fluidity and permeability of the lipid system. Finally, based on these data it can be proposed that the mechanism of action of EPOPC in lipofection/drug delivery involves the modifications in membrane organization, which facilitates the incorporation of drug or other material into the cell.

St John's wort extract influences membrane fluidity and composition of phosphatidylcholine and phosphatidylethanolamine in rat C6 glioblastoma cells

BackgroundChronic stress, an important factor in the development of depressive disorders, leads to an increased formation of cortisol, which causes a hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, cortisol mediates an adaptive effect on plasma membrane fluidity which may affect signal transduction of membrane-bound receptors and contribute to pathophysiological changes. MethodsMembrane fluidity was measured by fluorescence anisotropy using DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene). Changes in cellular content of phosphatidylcholine species was determined by pulse-chase experiments using deuterated choline and mass spectrometry. Single molecule tracking was used to examine the lateral mobility of β1-adrenoceptors and changes in cAMP formation were measured by ELISA. ResultsChronic exposure (6 – 8 days) of C6 cells to cortisol dose-dependently decreased DPH and TMA-DPH fluorescence anisotropy, reflecting increased membrane fluidity. In contrast, cells pretreated with St. John's wort extract Ze117 showed increased DPH and TMA-DPH fluorescence anisotropy values, indicating a membrane rigidification effect which was mediated at least by the constituents hypericin, hyperforin, quercetin, amentoflavone and biapigenin. The observed membrane fluidizing effect of cortisol could be reversed by cotreatment with Ze117. The membrane rigidification of Ze117 was in line with the in parallel observed decrease in the phosphatidylcholine/phosphatidylethanolamine ratio determined in whole cell lipid extracts. Interestingly, pulse-chase experiments demonstrated, that Ze117 inhibited the incorporation of choline-D9 in phosphatidylcholine species with saturated or monounsaturated fatty acids compared to control cells, while the synthesis of phosphatidylcholine species with polyunsaturated fatty acids was not affected. C6 cells whose membranes have become more rigid by Ze117 showed altered lateral mobility of β1-adrenoceptors as well as reduced cAMP formation after stimulation with the β1-adrenoceptor agonist dobutamine. ConclusionObviously, the signaling of β1-adrenoceptors depends on the nature of the membrane environment. It can therefore be assumed that Ze117 has a normalizing effect not only on the membrane fluidity of “stressed” cells, but also on lateral mobility and subsequently on the signal transduction of membrane-associated receptors.

Lipidic nanovesicles stabilize suspensions of metal oxide nanoparticles

We have studied the effect of adding lipid nanovesicles (liposomes) on the aggregation of commercial titanium oxide (TiO2), zinc oxide (ZnO), or cerium oxide (CeO2) nanoparticles (NPs) suspensions in Hepes buffer. Liposomes were prepared with pure phospholipids or mixtures of phospholipids and/or cholesterol. Changes in turbidity were recorded as a function of time, either of metal nanoparticles alone, or for a mixture of nanoparticles and lipidic nanovesicles. Lipid nanovesicles markedly decrease the NPs tendency to sediment irrespective of size or lipid compositions, thus keeping the metal oxide NPs in suspension. Cryo-electron microscopy, fluorescence anisotropy of TMA-DPH and general polarization of laurdan failed to reveal any major effect of the NPs on the lipid bilayer structure or phase state of the lipids. The above data may help in developing studies of the interaction of inhaled particles with lung surfactant lipids and alveolar macrophages.

Plasma and mitochondrial membrane perturbation induced by aluminum in human peripheral blood lymphocytes

Aluminum is a redox-inert element that could induce cell damage via activation of oxidative stress. In this work, the effect of aluminum on different cellular compartments of human peripheral blood lymphocytes was studied. The presence of aluminum induced a lipid peroxidation and physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, while the pyrene excimerization coefficient (Kex) increased.Flow cytometry measurements, using JC-1, Rhodamine 123 and H2-DCFDA as fluorescent probes, indicated that aluminum induces a slight mitochondrial membrane depolarization that was associated with a moderate increase in reactive oxygen species production. A significative influence on these parameters was measured only at high aluminum concentration.

Lipid Dynamics of Cardiolipin/DMPC and Cardiolipin/DOPC in Nanodiscs

Cardiolipin is a negatively charged phospholipid that comprises roughly 20% of the lipids in the inner mitochondrial membrane. The dynamics of this membrane can influence interactions between cytochrome c and cardiolipin, which have been shown to initiate the intrinsic pathway of apoptosis. This investigation studies how the dynamics of a model membrane system of varying composition will behave in the presence and absence of cardiolipin. Nanodiscs are composed of varying concentrations of 18:1 cardiolipin and DOPC or DMPC, doped with either NBD-PE or TMA-DPH. (18:1 cardiolipin: 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine DOPC: (Δ9-Cis) PC 1,2-dioleoyl-sn-glycero-3-phosphocholine NBD-PE: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) TMA-DPH: trimethylammonium diphenylhexatriene). Time-Resolved Emission Spectra (TRES) of NBD-PE doped nanodiscs are used to report on head group relaxation and Time-Resolved Fluorescence Anisotropy of TMA-DPH doped nanodiscs are used to report on acyl chain motions. These methods are combined to provide insight into how cardiolipin affects lipid membrane dynamics.

Impact of gender on platelet membrane functions of Alzheimer's disease patients

There are many evidences suggesting that oxidative stress is one of the earliest events in Alzheimer disease (AD) pathogenesis and plays a key role in the development of the AD pathology.The existence of substantial gender-related differences in the clinical features of AD has been recently confirmed (i.e. pathophysiologic features and epidemiologic trends). In addition, study results appear to indicate that the etiopathogenetic mechanisms of AD differ significantly in the 2 sexes. Based on previous results regarding changes in AD platelet plasma membrane, the purpose of the present study was to assess the impact of gender in the same model above reported. In particular we aimed at studying platelets from AD patients (M-AD and F-AD) and matched controls (M-C and F-C), divided into gender, by studying nitric oxide (NO) and peroxynitrite (ONOO−) production, the intracellular Ca2+ concentration ([Ca2+]i), membrane Na+/K+-ATPase activity and fluidity.NO production was significantly elevated in platelets from both F-AD and M-AD compared to matched controls. M-AD showed NO production significantly higher than F-AD and it was the same between M-C and F-C. A similar trend was seen for ONOO−.Platelets of both M-AD and F-AD had intracellular Ca2+ concentrations significantly higher than F-C and M-C, while membrane Na+/K+-ATPase activity showed an opposite trend, but these differences are still significant.M-AD male subjects showed a significantly increased DPH fluorescence anisotropy (r) compared with controls, while for F-AD this discrepancy was not significant. The difference in DHP fluorescence anisotropy remained significant between M-AD and F-AD as well as between M-C and F-C.The TMA-DPH fluorescence anisotropy showed the same trend, but there were no significant differences between M-AD and F-AD, as well as between controls.The results of the current research support the conclusion that F-AD is not at greater risk than M-AD for oxidative stress injuries. Studies on gender differences could lead to a higher probability of improved health outcomes via better-targeted therapies.

In vitro effect of AlCl3 on human erythrocytes: Changes in membrane morphology and functionality

Aluminum belongs to a group of potential toxic elements capable of penetrating the human body. In this paper, the effect of aluminum concentrations on red blood cell membranes using different fluorescent probes able to localize in various parts of the phospholipid bilayer (TMA-DPH, laurdan and pyrene) were studied. Our results confirm that human erythrocytes exposed to aluminum undergo physico-chemical modifications at the membrane level. A decrease in fluorescence anisotropy of TMA-DPH and in the polarity of the lipid bilayer with a concomitant shift toward a gel phase was observed, and the pyrene excimerization coefficient (kex) increased.Furthermore, the presence of aluminum induced lipid peroxidation and reduced the activity of erythrocyte antioxidant enzymes (SOD, CAT and GSHPx). Al-induced morphological changes on the erythrocyte membrane surface were monitored using atomic force microscopy. These results provide further information on the target of action of different aluminum amounts.

Assessment of the mode of action of polyhexamethylene biguanide against Listeria innocua by Fourier transformed infrared spectroscopy and fluorescence anisotropy analysis

Polyhexamethylene biguanide (PHMB) is a cationic biocide. The antibacterial mode of action of PHMB (at concentrations not exceeding its minimal inhibitory concentration) upon Listeria innocua LRGIA 01 was investigated by Fourier transformed infrared spectroscopy and fluorescence anisotropy analysis. Fourier transformed infrared spectra of bacteria treated with or without PHMB presented some differences in the lipids region: the CH(2)/CH(3) (2924 cm(-1)/2960 cm(-1)) band areas ratio significantly increased in the presence of PHMB. Since this ratio generally reflects membrane phospholipids and membrane microenvironment of the cells, these results suggest that PHMB molecules interact with membrane phospholipids and, thus, affect membrane fluidity and conformation. To assess the hypothesis of PHMB interaction with L. innocua membrane phospholipids and to clarify the PHMB mode of action, we performed fluorescence anisotropy experiments. Two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivative 1-[4-(trimethyl-amino)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), were used. DPH and TMA-DPH incorporate inside and at the surface of the cytoplasmic membrane, respectively. When PHMB was added, an increase of TMA-DPH fluorescence anisotropy was observed, but no changes of DPH fluorescence anisotropy occurred. These results are consistent with the hypothesis that PHMB molecules perturb L. innocua LRGIA 01 cytoplasmic membrane by interacting with the first layer of the membrane lipid bilayer.

Rho signaling participates in membrane fluidity homeostasis.

Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeast's sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation.

Fusion Peptide of Gp41 Self Associates in the Model Membrane and then Interacts with its Trans-Membrane Domain

We have examined the peptide structure and membrane packing when the fusion peptide (FP) of gp41 was added either in the membrane alone or in the membrane containing gp41 the trans-membrane domain (TMD). Circular Dichroism (CD) measurements showed that FP is mostly in a beta sheet conformation independent of FP concentration. TMD has ∼ 30% helical, ∼25% beta sheet and the complex of TMD and FP has less alpha helix than the TMD itself, which indicates the TMD looses its alpha helical structure upon interacting with FP. DPH and TMA-DPH fluorescence anisotropy revealed that FP alone increased the interior packing of the membrane, but FP in the presence of TMD increased the interior packing at lower concentrations of FP and then decreased it at higher concentrations. FP alone increased membrane surface packing at lower concentrations but increased it at higher concentrations. In the presence of TMD, FP addition decreased surface packing cooperatively. From the lifetime of TMA-DPH in H2O and D2O we documented water penetration into the membrane. FP alone increases water penetration slightly whereas FP in presence of TMD significantly increased water penetration into the interface region of the membrane in a cooperative fashion. The fluorescence lifetime of C6NBDPC revealed that FP alone fills more space than the FP in the presence of TMD. In summary, our results clearly demonstrate that gp41 FP forms a complex with the gp41 TMD to alter both TMD structure and membrane structure. It remains to be seen whether this complex promotes membrane fusion. Supported by NIGMS grant 32707 to BRL.

Effect of amyloid beta peptides Aβ1–28 and Aβ25–40 on model lipid membranes

To investigate the molecular interaction of amyloid beta peptides Aβ1–28 or Aβ25–40 with model lipid membranes differential scanning calorimetry (DSC) and DPH and TMA DPH fluorescence anisotropy approaches were used. The main transition temperature (T m) and enthalpy change (ΔH) of model lipid membranes composed of DMPC/DPPG on addition of Aβ25–40 or Aβ25–40 at 10:1 (w/w) phospholipid/peptide ratio either non-aggregated or previously aggregated were examined. The effect of Aβ1–28 and Aβ25–40 on the membrane fluidity of liposomes made of DMPC/DPPG (98:2 w/w) was determined by fluorescence anisotropy of incorporated DPH and TMA DPH. The results of this study provide information that Aβ1–28 preferentially interacts with the hydrophilic part of the model membranes, while Aβ25–40 rather locates itself in the hydrophobic core of the bilayer where it reduces the order of the phospholipids packing.

Increased body temperature induces lymphocyte lipid raft aggregation (35.34)

Abstract Previous recent data reveal that mild heating in vitro of CD4+ and CD8+ T cells (purified from murine splenocytes) at 39.5°C induces the aggregation of lipid rafts; approximately 70% of lymphocytes express aggregated lipid rafts compared to only 20% of cells maintained at 37oC as judged by the distribution of the lipid raft marker, fluorescently labeled GM1. Further, we found that the fluidity of the T cell plasma membrane, as measured by fluorescence anisotropy of TMA-DPH, increases when the temperature is raised from 37° to 39.5°C. Based on these in vitro results, we hypothesized that a physiologically relevant increase in body temperature results in an increase in lipid raft aggregation in T cells in situ. We administered whole body hyperthermia (WBH) to raise the temperature of mice to 39.5°C for 6 hours and immediately harvested and fixed cells from spleen and lymph nodes. Utilizing Imagestream flow cytometry, we observed a 3 fold increase in the number of CD8+ T cells with lipid raft aggregates isolated from WBH mice compared to T cells isolated from normothermic control mice. Lymphocytes from spleen and lymph nodes responded similarly to WBH. Overall, these findings may help to understand the role of physiological temperature shifts (e.g., during febrile episodes and inflammation) on costimulation and lymphocyte activation potential. Supported by NIH P01 CA94045 and RO1 CA71599

Liposome Steady-state Anisotropy Of E.coli Total Lipid Extracts: A Tool To Better Understand Antibiotic Translocation In A Bacterial Environment

The outer membrane of Gram-negative bacteria is a protective diffusion barrier preventing the free entry of solutes into the periplasm. At the same time, the embedded proteins (Omp's for Outer Membrane Proteins) fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Since a large number of years, a lot of simple membranar models with synthetic lipids such as POPC, POPG, DMPG….allowed scientists to better understand porin insertions and antibiotic translocation/interaction. As any molecular phenomena, it is better to study it in their own environment. With the availability of well characterized E.coli total lipid extract phospholipids, in vitro model membranes such as liposomes and proteoliposomes can be created and antibiotic translocation investigated in a bacterial environment, as close as possible to the existing membranes.In this study, the effects of porins on the structural order of lipid membranes was investigated by measuring, as a function of temperature, the steady-state fluorescence anisotropy (rS) of TMA-DPH and DPH incorporated into liposomes and proteoliposomes of E.coli total lipid extract phospholipids. DPH, embedded in the bilayer, can give informations about lipid diffusion. TMA-DPH, anchored at the aqueous interface of the phospholipid bilayer, allow us to better understand Omp's/antibiotic interactions.By using those probes, we were able to obtain information on the effects caused by the insertion of Omp's into the core and at the interface regions of the bilayer. This information, correlated with other membrane models, can be of considerable interest in establishing a possible relationship between the lipid composition of the phospholipid bilayer surface and antibiotic translocation.

Fluorescence anisotropy analysis of the mechanism of action of mesenterocin 52A: speculations on antimicrobial mechanism

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, active against Listeria sp. The interaction of Mes 52A with bacterial membranes of two sensitive Listeria strains has been investigated. The Microbial Adhesion to Solvents test used to study the physico-chemical properties of the surface of the two strains indicated that both surfaces were rather hydrophilic and bipolar. The degree of insertion of Mes 52A in phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane. TMA-DPH reflects the fluidity at the membrane surface and DPH of the heart. With Listeria ivanovii CIP 12510, Mes 52A induced an increase only in the TMA-DPH fluorescence anisotropy, indicating that this bacteriocin affects the membrane surface without penetration into the hydrophobic core of the membrane. No significant K(+) efflux was measured, whereas the Delta Psi component of the membrane potential was greatly affected. With Listeria innocua CIP 12511, Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH, indicating that this peptide inserts deeply in the cytoplasmic membrane of this sensitive strain. This insertion led to K(+) efflux, without perturbation of Delta pH and a weak modification of Delta Psi, and is consistent with pore formation. These data indicate that Mes 52A interacts at different positions of the membrane, with or without pore formation, suggesting two different mechanisms of action for Mes 52A depending on the target strain.

Cholesterol Substitution Increases the Structural Heterogeneity of Caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.

The lipid-mediated hypothesis of fumonisin B1 toxicodynamics tested in model membranes

The disruption of lipidic metabolism was considered a good candidate to explain FB1 toxicity mechanism. In the present work we investigated molecular organizational changes induced by FB1–biomembrane interaction possibly involved in mycotoxic effects. FB1 was self-aggregated with a critical micellar concentration of 1.97 mM. FB1 (0–81.4 μM), decreased in a dose-dependent manner, the fluorescence anisotropy of TMA-DPH (from 0.349 ± 0.003 to 0.1720 ± 0.0035) in dpPC bilayers, whilst no differences were registered with DPH. At 5.6 μM in the subphase, FB1 increased the lateral surface pressure ( π) of a Langmuir film to an extent that depended on the monolayer composition (Δ π dpPC:DOTAP 3:1 > Δ π dpPC:dpPA3:1 > Δ π dpPC), the molecular packing (Δ π decreased linearly as a function of the initial π) and the subphase pH (Δ π pH 2.6 > Δ π pH 7.4 and maximal π allowing the drug penetration π cut-off was 34.3 and 27.7 mN/m at pH 2.63 and 7.4, respectively). FB1 increased the surface potential of dpPC and dpPC:DOTAP monolayers and decreased that of dpPC:dpPA. This suggested that FB1 acquired different orientations and/or foldings depending on the surface electrostatics and the toxin charge state. Moreover, FB1–lipid interactions were transduced into long-range effects at the mesoscopic level affecting the lipidic self-separated lateral domains shape and density.

A Fluorescence Spectroscopy Study on the Interactions of the TAT-PTD Peptide with Model Lipid Membranes

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.

A Static Magnetic Field Reduces lipopolysaccharide-induced Proinflammatory Cytokine Levels of Fibroblasts by Altering Membrane Fluidity

Lipopolysaccharide (LPS) is one of the major substances that initiate an immune host response in microbial infections which results in cytotoxicity. In terms of the treatment of the immune response, much research on endotoxin tolerance that can reduce LPS-induced damages has been conducted. In this experiment, cultured fibroblasts were challenged with LPS in order to initiate an inflammatory reaction. Cell numbers and various proinflammatory cytokine levels were compared between a static magnetic field (SMF)-exposed group and an unexposed group. Our results showed that with LPS challenge, fibroblasts exposed to a 4000-G SMF demonstrated a higher cell density (8.28±0.14 10^4 cells/ml) when compared to the unexposed (control) group (4.5±0.16 10^4 cells/ml, p＜0.05). Meanwhile, levels of LPS-induced interleukin-1β and tumor necrosis factor-α in the SMF-exposed groups were significantly lower at 32.9% and 61.4% than those of the unexposed counterparts, respectively (p＜0.05). The change in the fluorescence anisotropy of TMA-DPH increased when cells were exposed to SMF. These results indicate that a static magnetic field may decrease LPS-induced proinflammatory cytokine levels by altering the membrane fluidity.

Fluorescence Study of the Interaction between the TAT‐PTD Peptide and the Lipid Bilayer

The mechanism of internalization of cationic peptides into the cells is unknown. We used fluorescence spectroscopy to study the interaction between Protein Transduction Domain of the HIV-1 TAT protein (TAT-PTD; residues 47–60 of TAT, fluorescently labeled with tryptophan) and the lipid bilayer in the form of small unilamellar lipid vesicles (SUV). The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and increase in anisotropy upon the interaction with SUV, which was proportional with the negative charge density in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. Kd1 was independent of the negative charge density and accounted for 24% of the interaction, whereas Kd2, contributing 76% to the interaction, decreased linearly with the density of negative charge in the membrane, suggesting an electrostatic nature of the latter interaction. The former could not be inhibited by high salt, suggesting its van der Waals or hydrophobic nature. TAT-PTD did not dissipate membrane potential (165 mV, negative inside), nor did it affect fluorescence anisotropy of the membrane fluorescence probes TMA-DPH or DPH, indicating preserved membrane integrity upon TAT-PTD binding. When the pyrene-labeled phosphatidyl glycerol was included in the membrane, TAT-PTD induced pyrene excimer formation, but only at the temperature above the melting point of the lipid. Thus, the two prerequisites for a strong binding of TAT-PTD to the lipid membrane are the presence of the negative charge and the liquid crystalline phase of the lipid bilayer.

Incorporation of fluorescent probes into PAMAM dendrimers

Interactions of two fluorescent probes 1-(trimethylammoniumphenyl)-6-phenyl-1,3,5 hexatriene p-toluenesulfonate (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) with polyamidoamine (PAMAM) dendrimers were studied. Changes in fluorescence intensity and steady-state fluorescence anisotropy of TMA-DPH and 12-AS were monitored. It was found that 12-AS molecules incorporated into dendrimer cavities whereas TMA-DPH molecules aggregated on the surface of polymer. Dendrimer size had not significant impact on its host properties.