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PubMed1987

Alcohol interactions with lipids: a carbon-13 nuclear magnetic resonance study using butanol labeled at C-1.

The interactions of carbon-13 enriched butanol with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied using C-13 nuclear magnetic resonance. It was found that above the gel to liquid crystal phase transition the resonance from the butanol could be resolved into two signals with similar chemical shifts but different T1 values and line widths. In contrast, only one narrow resonance was observed for ethanol, which has considerably less solubility in the lipids than butanol. Thermodynamic analyses of the effects of butanol on the phase transition temperature predict much greater solubility or butanol when the lipid is above the phase transition temperature than when it is below. It was concluded that the two butanol resonances represent two slowly exchanging populations, the free butanol in the aqueous phase and butanol dissolved in the liquid crystalline region of the lipid. No bound butanol was detected below the gel to liquid crystal phase transition. Relaxation studies were performed on the resonance of the bound butanol in DPPC and DMPC, including measurements of T1, line width, and nuclear Overhauser enhancement. Theoretical analysis of the relaxation parameters indicates that the lipid-bound alcohol has very high mobility within the fluid lipid bilayer. The data are consistent with butanol being present at the aqueous boundary or head group region of the lipid.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

The duplex-hairpin conformational transition of d(CGCGCGATCGCGCG) and d(CGCGCGTACGCGCG): a thermodynamic and kinetic study.

We have studied the duplex-hairpin conformational transition in two perfectly palindromic sequences, d(CGCGCGATCGCGCG)(I) and d(CGCGCGTACGCGCG)(II), by means of UV-melting, electrophoretic and T-jump experiments. Both tetradecamers exhibit biphasic thermal profiles. The lower temperature transition is concentration dependent whereas the higher temperature transition is not. The former transition has been characterized by gel electrophoresis and shows two distinct bands, whose intensity depends on temperature. This behavior is due to the occurrence of a slow premelting interconversion between the duplex and hairpin forms in both tetradecamers. The kinetics of hairpin formation from the duplex is studied by T-jump experiments. Relaxation spectra are well reproduced by a single relaxation time with rate constants characterized by a high temperature coefficient. In 10 mM NaCl, the duplex-hairpin conversion of I is characterized by an apparent activation energy of 96 +/- 6 kcal/mol, a value rather close to the expected denaturation enthalpy. In 1 mM NaCl a value slightly lower has been obtained. The rate of duplex-hairpin interconversion has been found to decrease as the salt concentration is raised. These data suggest that the transformation from the duplex to the hairpin form should imply a transition state with a simultaneous breaking of most base pairs, if not total strand separation.

Base Sequence↗

PubMed2004

Demixing in simple fluids induced by electric field gradients.

Phase separation in liquid mixtures is mainly controlled by temperature and pressure, but can also be influenced by gravitational, magnetic or electric fields. However, the weak coupling between such fields and concentration fluctuations limits this effect to extreme conditions. For example, mixing induced by uniform electric fields is detectable only at temperatures that are within a few hundredths of degree or less of the phase transition temperature of the system being studied. Here we predict and demonstrate that electric fields can control the phase separation behaviour of mixtures of simple liquids under more practical conditions, provided that the fields are non-uniform. By applying a voltage of 100 V across unevenly spaced electrodes about 50 micro m apart, we can reversibly induce the demixing of paraffin and silicone oil at 1 K above the phase transition temperature of the mixture; when the field gradients are turned off, the mixture becomes homogeneous again. This direct control over phase separation behaviour depends on field intensity, with the electrode geometry determining the length-scale of the effect. We expect that this phenomenon will find a number of nanotechnological applications, particularly as it benefits from field gradients near small conducting objects.

Journal Article↗

PubMed2004

Cholesterol modulation of sphingomyelinase activity at physiological temperatures.

Bacillus cereus sphingomyelinase activity was assayed on large unilamellar vesicles composed of sphingomyelin (SM)/cholesterol (Ch) mixtures at varying proportions. Natural (egg) SM was used with a gel-fluid transition temperature at ca. 40 degrees C. When the enzyme was assayed at 37 degrees C, the activity on pure SM was exceedingly low, but a small increase was observed as soon as some Ch was added, and a large enhancement of activity occurred with Ch proportions above 25 mol%. The data were interpreted in terms of sphingomyelinase activity being higher in the cholesterol-induced liquid-ordered phase than in the gel phase. The abrupt increase in activity above 25 mol% Ch would occur as a result of a change in domain connectivity, when the Ch-rich liquid-ordered domains coalesced. In equimolar SM/Ch mixtures, that were in the liquid-ordered state in a wide range of temperatures, sphingomyelinase activity was virtually constant in the 30-70 degrees C range. The results demonstrate that at the mammalian and bird physiological temperatures Ch modulates sphingomyelinase activity, and that this can occur precisely because most SM have a gel-fluid transition temperature above the physiological temperature range. In addition, Ch activation of sphingomyelinase and the strong affinity of Ch for SM allow the rapid, localised and self-contained production of the metabolic signal ceramide in specific microdomains (rafts).

Bacillus cereus↗

PubMed1980

The effects of various incubation temperatures, particulate isolation, and possible role of calmodulin on the activity of the base exchange enzymes of rat brain.

The involvement of calmodulin in the choline, ethanolamine, and serine exchange activities of rat brain microsomes was investigated. Calmodulin stimulated choline exchange activity to a greater extent than ethanolamine and serine exchange activities. The three base exchange activities were inhibited by antipsychotic drugs believed to prevent calmodulin interaction, but not by calmodulin-binding protein. The solutions employed for tissue homogenization and subsequent isolation of microsomes greatly influenced the base exchange activities. The process of resuspending isolated microsomes and recentrifugation, or "washing," produced major losses of detectable activity. The base exchange enzyme activities were maximal at 45 degrees, and Arrhenius plots revealed a common transition temperature of 31 degrees. The activation energies for the base exchange reactions decreased at temperatures above the observed transition temperature. Kinetic data, Km and Vmax, for the base exchange activities at 27, 37, and 45 degrees are presented.

Animals↗

PubMed1991

Low-temperature 2H NMR spectroscopy of phospholipid bilayers containing docosahexaenoyl (22:6 omega 3) chains.

Polyunsaturated fatty acids are widely distributed components of biological membranes and are believed to be involved in many biological functions. However, the mechanisms by which they act on a molecular level are not understood. To further investigate the unique properties of omega 3 polyunsaturated phospholipid bilayers, deuterium nuclear magnetic resonance (2H NMR) studies have been made of the liquid-crystalline (L alpha) and gel phases of a homologous series of mixed-chain phosphatidylcholines containing docosahexaenoic acid: (per-2H-n:0)(22:6)PC, where n = 12, 14, 16, and 18. The moments of the 2H NMR lineshapes have been evaluated, and from these the warming and cooling main phase transition temperatures were determined. The transition temperatures of the mixed-chain series were found to be significantly lower than those of the corresponding lipids in the disaturated series, di(per-2H-n:0)PC, with hystereses ranging from 2 to 14 degrees C. Distinct effects of the docosahexaenoyl chain on bilayer order were found, though these effects varied across the mixed-chain series. In evaluating the moment data, an empirical method for normalizing the moments with respect to differences in temperature was applied, in addition to using the reduced temperature method. For the systems studied here, the method of normalization had no significant effect on the interpretation of the moment data.

Deuterium↗

PubMed2002

Spontaneous breaking of time-reversal symmetry in the pseudogap state of a high-Tc superconductor.

A change in 'symmetry' is often observed when matter undergoes a phase transition-the symmetry is said to be spontaneously broken. The transition made by underdoped high-transition-temperature (high-Tc) superconductors is unusual, in that it is not a mean-field transition as seen in other superconductors. Rather, there is a region in the phase diagram above the superconducting transition temperature Tc (where phase coherence and superconductivity begin) but below a characteristic temperature T* where a 'pseudogap' appears in the spectrum of electronic excitations. It is therefore important to establish if T* is just a cross-over temperature arising from fluctuations in the order parameter that will establish superconductivity at Tc (refs 3, 4), or if it marks a phase transition where symmetry is spontaneously broken. Here we report that, for a material in the pseudogap state, left-circularly polarized photons give a different photocurrent from right-circularly polarized photons. This shows that time-reversal symmetry is spontaneously broken below T*, which therefore corresponds to a phase transition.

Journal Article↗

PubMed2004

Discrete and heterogeneous rotational dynamics of single membrane probe dyes in gel phase supported lipid bilayer.

In order to probe the local dynamics of lipid bilayers in the gel phase, we measured the rotational time trajectories of a membrane probe, diI(3), in supported bilayers of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) using single molecule fluorescence polarization imaging. diI(3) has two hydrocarbon tails that mimic phospholipid tails and has its transition dipole moment lying mostly on the plane of the membrane; hence it is an excellent probe for rotational dynamics in membranes. Above the transition temperature, the probes are laterally mobile and do not display polarized emission. In the gel phase below the transition temperature, lateral mobility is severely reduced and the emission becomes polarized with its polarization direction changing in the milliseconds time scale. Molecule by molecule analysis of the rotational time scales revealed significant heterogeneities among molecules, much larger than would be due to statistical noise. Control experiments using small unilamellar vesicles suggest that the heterogeneities are not caused by surface interactions and are intrinsic to the gel phase membrane. The rotational dynamics is strongly temperature dependent and the thermally activated state for the rotational motion has a large entropic barrier (> 30kB), indicating that relatively large local disorder is required for the rotational motion to occur. Rotational hopping between discrete angles has been observed at the lowest temperatures (approximately 10 degrees C). Our results suggest that the gel phase membrane is not uniform at the microscopic level but is highly dynamic with the rigidity of local environments constantly changing.

Carbocyanines↗

PubMed1979

Relationships between Root Temperature and the Transport of Ammonium and Nitrate Ions by Italian and Perennial Ryegrass (Lolium multiflorum and Lolium perenne).

At root temperature below 14 C the absorption of (15)N from NH(4) (+) greatly exceeded that from NO(2) (-) by tillers of Lolium multiflorum and Lolium perenne under conditions where pH, external concentration, plant N status, and pretreatment temperature were varied. There was a marked increase in the temperature sensitivity of NO(3) (-) transport below 14 C, irrespective of the temperature at which plants were grown previously. A marked increase in the temperature sensitivity was also seen for NH(4) (+) transport, but this occurred at the lower temperature of 10 C. Pretreatment of roots at 8 C lowered this still further to 5 C. Above and below these transition temperatures the Q(10) values for NO(3) (-) and NH(4) (+) transport were similar. Thus, the increased absorption of NH(4) (+) relative to NO(3) (-) at low temperatures seems to be related primarily to the difference in transition temperatures.It seems possible that NO(3) (-) and NH(4) (+) are absorbed through separate regions of the cell membrane differing in lipid composition and phase transition temperatures.

Journal Article↗

PubMed2003

Hydration pressure and phase transitions of phospholipids. II. Thermotropic approach.

It is widely known that dehydration increases the main phase transition temperature of phospholipids. A mathematical analysis now shows that hydration pressure can be calculated by the dehydration-induced shift of the phase transition temperature. The hydration-dependent piezotropic and thermotropic phase transitions were determined by using calorimetry and FT-IR spectroscopy, and the application of our approach gives hydration pressure parameters that agree very well with the values obtained with the osmotic stress method.

Calorimetry, Differential Scanning↗

PubMed2004

Thermoresponsive N,N-dialkylacrylamide copolymer blends as DNA sieving matrices with a thermally tunable mesh size.

In an earlier study we showed that a blend of thermoresponsive and nonthermoresponsive hydroxyalkylcelluloses could be used to create a thermally tunable polymer network for double-stranded (ds) DNA separation. Here, we show the generality of this approach using a family of polymers suited to a wider range of DNA separations: a blended mixture of N,N-dialkylacrylamide copolymers with different thermoresponsive behaviors. A mixture of 47% w/w N,N-diethylacrylamide (DEA)/53% w/w N,N-dimethylacrylamide (DMA) (DEA47; thermoresponsive, transition temperature = 55 degrees C in water) and 30% w/w DEA/70% w/w DMA (DEA30; nonthermoresponsive, transition temperature > 85 degrees C in water) copolymers in the ratio of 1:5 w/w DEA47:DEA30 was used to separate a dsDNA restriction digest (PhiX174-HaeIII). We investigated the effects of changing mesh size on dsDNA separation, as controlled by temperature. We observed good DNA separation performance with the copolymer blend at temperatures ranging from 25 degrees C to 48 degrees C. The separation selectivity was evaluated quantitatively for certain DNA fragment pairs as a function of temperature. The results were compared with those obtained with a control matrix consisting only of the nonthermoresponsive DEA30. Different DNA fragment pairs of various sizes show distinct temperature-dependent selectivities. Over the same temperature range, no significant temperature dependence of selectivity is observed for these DNA fragment pairs in the nonthermoresponsive control matrix. Overall, the results show similar trends in the temperature dependency of separation selectivity to what was previously observed in hydroxyalkylcellulose blends, for the same DNA fragment pairs. Finally, we showed that a ramped temperature scheme enables improved separation in the blended copolymer matrix for both small and large DNA fragments, simultaneously in a single capillary electrophoresis (CE) run.

Acrylamide↗

PubMed1996

Structure, interactions and dynamics of PRD1 virus I. Coupling of subunit folding and capsid assembly.

Bacteriophage PRD1, which infects Escherichia coli and Salmonella typhimurium, consists of an icosahedral capsid enclosing a membrane-packaged double-stranded DNA genome. The viral shell has been investigated using time and temperature resolved Raman and ultraviolet-resonance Raman spectroscopy to reveal novel features of the capsid structure and its pathway of assembly from P3 subunits. Raman spectra show that the shell is thermostable to 50 degrees C, and disassembles between 50 and 70 C degrees with only a small change in P3 conformation. However, the products of thermal disassembly depend sensitively upon total protein concentration. Characterization by analytical ultracentrifugation indicates that below 8 mg/ml, the purified shell disassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed. Guanidine hydrochloride (GuHCl) dissociation of the P3 shell yields similar results. Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degrees C. Thus, shell disassembly diminishes P3 thermostability. Both the lower temperature transition (30 degrees C to 50 degrees C) of the trimer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximately 5% of the P3 peptide backbone from alpha-helix to beta-strand. Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell than in the trimer, consistent with the observed non-specific polymerization of trimers at high concentration. Conversely, the exchange of indole 1NH groups shows that approximately 65% of tryptophan residues are protected against exchange in the assembled shell. The results suggest a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chains from solvent access. We propose a capsid assembly model which couples P3 shell formation with the final step in folding of the P3 subunit.

Bacteriophages↗

PubMed2004

Thermodynamics of alpha-lactalbumin-DL-alpha-dipalmitoylphosphatidylcholine interactions and effect of the antioxidant nicotinamide on these interactions.

Differential scanning calorimetry has been used to understand the thermodynamics of the interactions of dl-alpha-dipalmitoylphosphatidylcholine (DPPC) with alpha-lactalbumin and the effect of the antioxidant nicotinamide on these interactions. Nicotinamide decreases the thermal transition temperature of both the lipid and the protein at high concentrations. The thermal unfolding transitions of the protein were two state and calorimetrically reversible. There was no significant change in the shape and thermodynamic parameters accompanying the lipid endotherms, suggesting that nicotinamide did not penetrate the lipid bilayer. The thermal unfoldings of alpha-lactalbumin in the presence of DPPC as cosolute also adhered to two-state reversible mechanism. The changes in the thermodynamic parameters accompanying the thermal transitions were small, indicating no significant interaction of alpha-lactalbumin with DPPC. The changes in the thermodynamic parameters indicate that the lipid bilayer organization, as well as the partitioning of the extrinsic protein alpha-lactalbumin into the bilayer, is not affected in the entire studied concentration range of the lipid. It is observed that the presence of increasing concentration of nicotinamide (as high as 1.0 mol dm(-3)) in the lipid-protein mixture does not affect its partitioning into the lipid bilayer, although nicotinamide preferentially interacts with alpha-lactalbumin. The change in the effect of nicotinamide on lipid transition temperature in the mixture and literature report suggests that nicotinamide may be forming a hydrogen-bonded complex with the protein through its amide functionality. The surface tension data of aqueous nicotinamide in combination with the thermal denaturation results of protein in presence of nicotinamide confirmed that surface tension effect does not have any significant contribution to the effect of nicotinamide on protein.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2005

Preparation and characterization of intelligent core-shell nanoparticles based on poly(D,L-lactide)-g-poly(N-isopropyl acrylamide-co-methacrylic acid).

New thermo-responsive, pH-responsive, and biodegradable nanoparticles comprised of poly(D,L-lactide)-graft-poly(N-isopropyl acrylamide-co-methacrylic acid) (PLA-g-P(NIPAm-co-MAA)) were developed by grafting biodegradable poly(D,L-lactide) onto N-isopropyl acrylamide and methacrylic acid. A core-shell type nano-structure was formed with a hydrophilic outer shell and a hydrophobic inner core, which exhibited a phase transition temperature above 37 degrees C suitable for biomedical application. Upon heating above the phase transition temperature, PLA-g-P(NIPAm-co-MAA) nanoparticle showed a polarity increase of pyrene in either buffer solution or intra-hepato-carcinoma cells as determined by fluorescence measurement, indicating that the structure of nanoparticles caused leakages from outer shell copolymers aggregation and collapsed. The drug loading level of 5-fluorouracil (5-FU) encapsulated in the PLA-g-P(NIPAm-co-MAA) nanoparticles can be as high as 20%. The release of 5-FU from nanoparticles was strongly controlled by the pH in the aqueous solution. Based on these results, PLA-g-P(NIPAm-co-MAA) nanoparticles can be used as a drug carrier for intracellular delivery of anti-cancer drug.

Acrylamides↗

PubMed1982

Gamma-gamma directional correlation study of model membranes.

Measurements of the perturbed gamma-gamma directional correlation were made for the 173-247 keV cascade in 111Cd at the In ion site in dipalmitoylphosphatidylcholine liposomes. A time-dependent electric quadrupole interaction was observed. Below the order in equilibrium fluid phase transition temperature the exponential damping coefficient was lambda 2 = 0.033 +/- 0.007 ns-1. Above the transition temperature the coefficient was lambda 2 = 0.015 +/- 0.006 ns-1 indicating a twofold increase in molecular mobility in the disordered phase. Absolute values for the motional correlation time, tau c, were estimated to be on the order of a few nanoseconds. The presence of cholesterol was found to cause no significant difference in the molecular mobility of the phosphatidylcholine head groups in the ordered and fluid states.

Cadmium↗

PubMed1978

Effect of liposomal model membrane composition on immunogenicity.

We have examined the effect of composition on the immunogenicity in mice of liposomal model membranes sensitized with dinitrophenyl-epsilon-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE) derivatives. Neither cholesterol content nor incorporation of exogenous charged amphiphile (dicetylphosphate, stearylamine) exerted a significant influence on the in vivo anti-DNP response as measured by the appearance of direct plaque-forming cells in the spleen. Similarly, the nature of the fatty acids (saturated vs unsaturated) present in DNP-Cap-PE had no effect. In contrast, the nonpolar region of the basic phospholipids comprising the liposomal bilayers played an important role as revealed by a comparative study of model membranes prepared with beef sphingomyelin (SM), egg phosphatidylcholine (PC), and synthetic distearoyl-, dimyristoyl-, dilauroyl-, and dioleoyl-phosphatidylcholines (DSPC, DMPC, DLPC, DOPC). Thus, liposomes with a large content of phospholipids possessing a high transition temperature (e.g., beef SM, DSPC) were more immunogenic than those containing phospholipids of low transition temperature (e.g., egg PC, DOPC). This correlation held for both unsonicated and sonicated liposomes. These findings may have a bearing on the phenomenon of membrane-localized antigen expression.

Animals↗

PubMed1981

Characterization of immunogenic properties of haptenated liposomal model membranes in mice. V. Effect of membrane composition on humoral and cellular immunogenicity.

This paper describes the effect of altering liposomal membrane composition on humoral and cellular immunogenicity of haptenated liposomes in mice. Antibody formation was determined by enumeration of direct, plaque-forming cells in the spleen and delayed-type hypersensitivity (DH) was measured with a footpad swelling test. Humoral immunogenicity of haptenated liposomes was strongly influenced by membrane phospholipid, cholesterol and charged amphiphile composition. Haptenated liposomes prepared from phospholipids with a low (dioleoyl- and dilauroyl-phosphatidylcholine) or high (distearoyl phosphatidylcholine) phase-transition temperature were less immunogenic than those prepared from phospholipids with an intermediate phase-transition temperature (dipalmitoyl phosphatidylcholine and sphingomyelin). In general, increasing the amount of liposomal membrane cholesterol induced a higher humoral response. These results are discussed in relation to liposomal membrane fluidity. Induction of an optimal DH with haptenated liposomes did not occur in the absence of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA). When DDA was used, alterations in membrane composition did not influence cellular immunogenicity. From these results it was concluded that 'intermediate' liposomal membrane fluidity is the most important requirement for induction of optimal antibody formation with haptenated liposomes and that a certain physicochemical configuration of the antigen, provided by the adjuvant DDA, is a prerequisite for induction of DH.

Adjuvants, Immunologic↗

PubMed2003

Effect of hydrostatic pressure on closed-loop phase behavior of block copolymers.

The effect of hydrostatic pressure (P) on closed-loop phase behavior of deuterated polystyrene-block-poly(n-pentyl methacrylate) copolymers [dPS-PnPMA] was investigated by using small-angle neutron scattering and birefringence. For P<20.7 bar, dPS-PnPMA exhibited a lower disorder-to-order transition temperature (T(LDOT)) at 175 degrees C, and then an upper order-to-disorder transition temperature (T(UODT)) at 255 degrees C. With increasing pressure both T(LDOT) and T(UODT) were markedly changed, where dT(LDOT)/dP was 725 degrees C/kbar and dT(UODT)/dP was -725 degrees C/kbar. These are consistent with predictions by the Clausius-Clapeyron equation using measured values of the volume and enthalpy changes of both transitions. The large pressure coefficients imply that the closed-loop phase behavior observed for PS-PnPMA is an entropic-driven phase transition.

Journal Article↗