The fluorescent molecule diphenylhexatriene (DPH) has been often used in combination with fluorescence anisotropy measurements, yet little is known regarding the non-linear optical properties. In the current work, we focus on them and extend the application to fluorescence, while paying attention to the conformational versatility of DPH when it is embedded in different membrane phases. Extensive hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the influence of the phase- and temperature-dependent lipid environment on the probe. Already, the transition dipole moments and one-photon absorption spectra obtained in the liquid ordered mixture of sphingomyelin (SM)-cholesterol (Chol) (2:1) differ largely from the ones calculated in the liquid disordered DOPC and solid gel DPPC membranes. Throughout the work, the molecular conformation in SM:Chol is found to differ from the other environments. The two-photon absorption spectra and the ones obtained by hyper-Rayleigh scattering depend strongly on the environment. Finally, a stringent comparison of the fluorescence anisotropy decay and the fluorescence lifetime confirm the use of DPH to gain information upon the surrounding lipids and lipid phases. DPH might thus open the possibility to detect and analyze different biological environments based on its absorption and emission properties.
- MeSH
- cholesterol chemie MeSH
- difenylhexatrien chemie MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční polarizace MeSH
- lipidové dvojvrstvy chemie MeSH
- molekulární konformace MeSH
- sfingomyeliny chemie MeSH
- simulace molekulární dynamiky MeSH
- tranzitní teplota MeSH
- vztahy mezi strukturou a aktivitou MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
1,6-Diphenyl-1,3,5-hexatriene (DPH) is one of the most commonly used fluorescent probes to study dynamical and structural properties of lipid bilayers and cellular membranes via measuring steady-state or time-resolved fluorescence anisotropy. In this study, we present a limitation in the use of DPH to predict the order of lipid acyl chains when the lipid bilayer is doped with itraconazole (ITZ), an antifungal drug. Our steady-state fluorescence anisotropy measurements showed a significant decrease in fluorescence anisotropy of DPH embedded in the ITZ-containing membrane, suggesting a substantial increase in membrane fluidity, which indirectly indicates a decrease in the order of the hydrocarbon chains. This result or its interpretation is in disagreement with the fluorescence recovery after photobleaching measurements and molecular dynamics (MD) simulation data. The results of these experiments and calculations indicate an increase in the hydrocarbon chain order. The MD simulations of the bilayer containing both ITZ and DPH provide explanations for these observations. Apparently, in the presence of the drug, the DPH molecules are pushed deeper into the hydrophobic membrane core below the lipid double bonds, and the probe predominately adopts the orientation of the ITZ molecules that is parallel to the membrane surface, instead of orienting parallel to the lipid acyl chains. For this reason, DPH anisotropy provides information related to the less ordered central region of the membrane rather than reporting the properties of the upper segments of the lipid acyl chains.
- MeSH
- antifungální látky chemie MeSH
- difenylhexatrien chemie MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční polarizace MeSH
- fosfatidylcholiny chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- itrakonazol chemie MeSH
- lipidové dvojvrstvy chemie MeSH
- povrchové vlastnosti MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.
- MeSH
- algoritmy MeSH
- buněčná membrána chemie metabolismus MeSH
- cholesterol metabolismus MeSH
- difenylhexatrien chemie metabolismus MeSH
- difuze MeSH
- fluorescenční polarizace MeSH
- FRAP MeSH
- HEK293 buňky MeSH
- kinetika MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus MeSH
- receptory thyroliberinu chemie genetika metabolismus MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- zelené fluorescenční proteiny chemie genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers - OS) or completely (dense shell glycodendrimers - DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.
- MeSH
- dendrimery chemie metabolismus MeSH
- difenylhexatrien chemie MeSH
- diferenciální skenovací kalorimetrie MeSH
- dimyristoylfosfatidylcholin chemie metabolismus MeSH
- fluidita membrány MeSH
- fluorescenční polarizace MeSH
- fosfatidylglyceroly chemie metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- liposomy chemie metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- maltosa chemie metabolismus MeSH
- membránové lipidy chemie metabolismus MeSH
- polypropyleny chemie metabolismus MeSH
- statická elektřina MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We report sphingolipid-related reorganization of gel-like microdomains in the plasma membrane of living Saccharomyces cerevisiae using trans-Parinaric acid (t-PnA) and 1,6-diphenyl-1,3,5-hexatriene (DPH). Compared to control, the gel-like domains were significantly reduced in the membrane of a sphingolipid-deficient lcb1-100 mutant. The same reduction resulted from sphingolipid depletion by myriocin. The phenotype could be reverted when a myriocin-induced block in sphingolipid biosynthesis was bypassed by exogenous dihydrosphingosine. Lipid order of less-ordered membrane regions decreased with sphingolipid depletion as well, as documented by DPH fluorescence anisotropy. The data indicate that organization of lateral microdomains is an essential physiological role of these structural lipids.
- MeSH
- buněčná membrána chemie metabolismus MeSH
- difenylhexatrien chemie MeSH
- fluorescenční polarizace MeSH
- fluorescenční spektrometrie MeSH
- membránové mikrodomény chemie metabolismus MeSH
- mutace MeSH
- nenasycené mastné kyseliny chemie MeSH
- Saccharomyces cerevisiae MeSH
- sfingolipidy biosyntéza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Two thermophilic strains belonging to Geobacillus stearothermophilus and Meiothermus ruber, which naturally do not synthesize ω-alicyclic fatty acids (ω-FAs) were cultivated with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl carboxylic acids. Gas chromatography-mass spectrometry analysis of fatty acid methyl and picolinyl esters showed that both strains are able to synthesize ω-FAs when cultivated with the appropriate precursor. The incorporation of cyclic acids influenced the whole FA composition as well as membrane fluidity. Membrane fluidity of intact cells was studied by measuring the fluorescence polarisation of the probe l,6-diphenyl-1,3,5-hexatriene incorporated into membrane lipid bilayers. Cytoplasmic membrane became more fluid with increasing content of ω-FAs. This is caused by considerable changes in lipid packing within the membrane induced by the presence of ω-FAs not found in the natural environment of Geobacillus and Meiothermus strains.
- MeSH
- buněčná membrána metabolismus MeSH
- difenylhexatrien metabolismus MeSH
- fluidita membrány MeSH
- fluorescenční barviva metabolismus MeSH
- fluorescenční polarizace MeSH
- Geobacillus stearothermophilus metabolismus MeSH
- gramnegativní bakterie klasifikace metabolismus MeSH
- kyseliny karboxylové metabolismus MeSH
- nenasycené mastné kyseliny biosyntéza MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We characterized physical and chemical properties of cell-membrane fragments from Bacillus subtilis 168 (trpC2) grown at pH 5.0, 7.0 and 8.5. Effects of long-term bacterial adaptation reflected in growth rates and in changes of the membrane lipid composition were correlated with lipid order and dynamics using time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. We demonstrate that the pH adaptation results in a modification of a fatty acid content of cellular membranes that significantly influences both the lipid-chain order and dynamics. For cultivation at acidic conditions, the lipid order increases and membrane dynamics decreases compared to pH 7.0. This results in rigid and ordered membranes. Cultivation at pH 8.5 causes slight membrane disordering. Instant pH changes induce qualitatively similar but smaller effects. Proton flux measurements performed on intact cells adapted to both pH 5.0 and 8.5 revealed lower cell-membrane permeability compared to bacteria cultivated at pH optimum. Our results indicate that both acidic and alkalic pH stress represent a permanent challenge for B. subtilis to keep a functional membrane state. The documented adaptation-induced adjustments of membrane properties could be an important part of mechanisms maintaining an optimal intracellular pH at a wide range of extracellular proton concentrations.
The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to "wobble-in-cone" model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.
- MeSH
- 2-naftylamin analogy a deriváty MeSH
- buněčná membrána metabolismus účinky léků MeSH
- cetomakrogol farmakologie MeSH
- difenylhexatrien MeSH
- difuze MeSH
- financování organizované MeSH
- fluorescenční barviva MeSH
- fluorescenční spektrometrie MeSH
- krysa rodu rattus MeSH
- laurany MeSH
- mozek metabolismus účinky léků MeSH
- oktoxynol farmakologie MeSH
- proteiny vázající GTP metabolismus MeSH
- receptory GABA-B metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Brains of Alzheimer disease patients in early stages of dementia contain an increased 24(S)-hydroxycholesterol (cerebrosterol)/cholesterol ratio when compared to controls. In this study, effects of amyloid beta peptides and of racemic 24-hydroxycholesterol were evaluated in vitro on undepleted or cholesterol-depleted hippocampal synaptosomes of young and old rats via a high-affinity choline transport and membrane anisotropy measurements. Depletion of membrane cholesterol decreased the transport of [3H]choline, increased the specific binding of [3H]hemicholinium-3 and decreased membrane anisotropy. However, less alterations were found in old when compared to young brains. 500 nM nonaggregated peptides were ineffective but aggregated fragment 1-42 evoked marked drops in the transport and anisotropy values on depleted synaptosomes. 50 microM 24-hydroxycholesterol inhibited choline transport on depleted synaptosomes but it did not influence membrane anisotropy. Peptides eliminated the actions of oxysterol on choline carriers in young but not in old rats. On the other hand, oxysterol eliminated the effects of peptides on membrane anisotropy. Our study suggests a possible role of membrane cholesterol in the regulation of choline carriers and supports data reporting a protective role of membrane cholesterol against toxic effects of amyloid beta peptides. Moreover, via Raman spectroscopy we demonstrate for the first time that peptides form a complex with 24-hydroxycholesterol.
- MeSH
- amyloidní beta-protein chemie toxicita MeSH
- anizotropie MeSH
- cholinergní látky farmakologie MeSH
- difenylhexatrien MeSH
- financování organizované MeSH
- fluidita membrány účinky léků MeSH
- fluorescenční barviva MeSH
- hemicholinium 3 farmakologie MeSH
- hipokampus metabolismus účinky léků MeSH
- hydroxycholesteroly chemie toxicita MeSH
- interpretace statistických dat MeSH
- krysa rodu rattus MeSH
- membránové transportní proteiny metabolismus MeSH
- membrány metabolismus účinky léků MeSH
- potkani Wistar MeSH
- proteiny přenášející neurotransmitery metabolismus MeSH
- Ramanova spektroskopie MeSH
- stárnutí fyziologie MeSH
- synaptozomy metabolismus účinky léků MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
It is suggested that amyloid beta peptides (Abeta) play a role in the pathogenesis of Alzheimer disease but their physiological function is still unknown. However, low pM-nM concentrations mediate a hypofunction of a basal forebrain cholinergic system without marked signs of neurotoxicity. In this study, we compared in vitro effects of soluble nonaggregated human Abeta 1-40 and 1-42 either on synaptosomal hemicholinium-3 sensitive choline carriers or on membrane fluidity in hippocampi of male and female Wistar rats aged 7 and 14 days or 2-3 months. The results indicate age- and sex-dependent effects mediated by peptides at nM concentrations but no significant differences between both fragments. Namely, opposite actions were observed in 14-day (the increase in the choline uptake and membrane fluidity) when compared to 7-day old and adult males (the mild drops). Lineweaver-Burk plot analysis revealed that the enhancement of the high-affinity choline transport in 14-day old males occurs via alterations in K (M )and the change was accompanied by a mild increase in the specific binding of [3H]hemicholinium-3. On the other hand, no age-dependent differences were found in females. Rat Abeta 1-40 mediated similar effects on 14-day old rats as the corresponding human fragment. Moreover, higher levels of soluble peptides were detected in immature when compared to mature male brains by means of competitive ELISA. Our study indicates that Abeta could play a role in postnatal sexual differentiation of hippocampal cholinergic system.
- MeSH
- amyloidní beta-protein farmakologie fyziologie MeSH
- cholin metabolismus MeSH
- difenylhexatrien MeSH
- ELISA MeSH
- financování organizované MeSH
- fluorescenční barviva MeSH
- fluorescenční polarizace MeSH
- hipokampus metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- membránové transportní proteiny metabolismus MeSH
- peptidové fragmenty farmakologie fyziologie MeSH
- potkani Wistar MeSH
- radioligandová zkouška MeSH
- sexuální faktory MeSH
- synaptozomy metabolismus účinky léků MeSH
- techniky in vitro MeSH
- věkové faktory MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
