Mária Klacsová

DNA condensation and its thermal stability influenced by phospholipid bilayer and divalent cations.

We studied the effect of divalent alkaline earth metal cations Ca²⁺, Mg²⁺ and transition metals Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ on DNA condensation and its protection against thermal denaturation in presence of dioleoylphosphatidylcholine liposomes (DOPC). Experimental results have shown that Ca²⁺ and Mg²⁺ as well as Zn²⁺ mediate DNA condensation. Cu²⁺ causes DNA double helix destabilization, and does not mediate binding between DNA and DOPC liposomes. Co²⁺ and Ni²⁺ can interact with DNA on both ways mentioned above. Static light scattering was use to follow the size of aggregates in DNA condensation process. Phospholipid bilayer and divalent cations protect condensed DNA against thermal destabilization. The highest stabilization effect was found in aggregates with Ca²⁺ and Zn²⁺, whereas in presence of either Co²⁺ or Ni²⁺ some volume fraction of DNA is denatured.

Molecular and component volumes of saturated n-alkanols in DOPC + DOPS bilayers

The volumetric properties of fluid bilayers consisting of dioleoylphosphatidylcholine (DOPC, 96wt%) and dioleoylphosphatidylserine (DOPS, 4wt%) with incorporated saturated n-alkanols (CnOH, n=10-16 is the even number of carbons in alkyl chain) were studied by vibrating tube densitometry. The mixing of DOPC and DOPS was found to be ideal and the molecular volumes of pure lipids V(DOPC) and V(DOPS) are additive in mixed bilayers. The increase of V(DOPS) with temperature was steeper than that of V(DOPC) as quantified by significantly higher coefficient of isobaric thermal expansivity gamma. This difference is supposed to be related to the high thermal expansivity of the serine headgroup. The molecular volumes of lipids and CnOH (V(CnOH)) in fluid DOPC+DOPS bilayers are constant and additive within error limits for n=12 and 16 and for the CnOH mole fractions in the lipid bilayer x(AL)<or=0.5 and x(AL)<or=0.3, respectively. In homologous series of CnOHs incorporated into DOPC+DOPS bilayer, the V(CnOH) volume increases linearly with the CnOH chain length n at x(AL)=0.29. From this dependence obtained at several temperatures, the component volume of the CnOH hydroxyl group V(OH) was calculated and found to decrease between 20 degrees C and 51 degrees C while the component volume of the CnOH methylene group V(CH2) increases with temperature. The excess molecular volume, DeltaV, for the transfer of CnOH into the DOPC+DOPS bilayers display a biphasic dependence on the alkyl length, n, with a minimum for n=12. When calculated with respect to the pure CnOH, DeltaV is negative for homologs with n>8.

Influence of quercetin on the interaction of gliclazide with human serum albumin – spectroscopic and docking approaches

Protein-binding interactions are displacement reactions which have been implicated as the causative mechanisms in many drug-drug interactions. Thus, the aim of presented study was to analyse human serum albumin-binding displacement interaction between two ligands, hypoglycaemic drug gliclazide and widely distributed plant flavonoid quercetin. Fluorescence analysis was used in order to investigate the effect of substances on intrinsic fluorescence of human serum albumin (HSA) and to define binding and quenching properties of ligand-albumin complexes in binary and ternary systems, respectively. Both ligands showed the ability to bind to HSA, although to a different extent. The displacement effect of one ligand from HSA by the other one has been described on the basis of the quenching curves and binding constants comparison for the binary and ternary systems. According to the fluorescence data analysis, gliclazide presents a substance with a lower binding capacity towards HSA compared with quercetin. Results also showed that the presence of quercetin hindered the interaction between HSA and gliclazide, as the binding constant for gliclazide in the ternary system was remarkably lower compared with the binary system. This finding indicates a possibility for an increase in the non-bound fraction of gliclazide which can lead to its more significant hypoglycaemic effect. Additionally, secondary and tertiary structure conformational alterations of HSA upon binding of both ligands were investigated using synchronous fluorescence, circular dichroism and FT-IR. Experimental data were complemented with molecular docking studies. Obtained results provide beneficial information about possible interference upon simultaneous co-administration of the food/dietary supplement and drug.

Partial volumes of cholesterol and monounsaturated diacylphosphatidylcholines in mixed bilayers

Dispersions of multilamellar liposomes prepared from monounsaturated diacylphosphatidylcholines having 18-24 carbons and from varying amounts of cholesterol were studied by densitometry. Ideal mixing of the studied phosphatidylcholines with cholesterol in the fluid phase was observed. The temperature dependence of partial volumes of both phosphatidylcholines and cholesterol was determined. A slight decrease in the partial volume of cholesterol with the lengthening of the acyl chain of the host phosphatidylcholine was observed. By measuring the density of multilamellar liposomes of dinervonoylposphatidylcholine and cholesterol below the main phase transition temperature, the phase boundary between the solid ordered phase and the area of coexistence of the solid ordered and liquid ordered phases was detected.

Molecular and component volumes of N,N-dimethyl-N-alkylamine N-oxides in DOPC bilayers

The volumetric properties of fluid bilayers formed of dioleoylphosphatidylcholine (DOPC) with incorporated N,N-dimethyl-N-alkylamine N-oxides (CnNO, n=6, 10-18 is the even number of carbons in alkyl chain) were studied by vibrating tube densitometry in the temperature interval from 20°C to 50°C. It was found that the DOPC and CnNO mixed ideally in the investigated composition range and hence the molecular volumes of DOPC (VDOPC) and incorporated CnNO (VCnNO) were constant and additive within error limits. From the temperature dependencies of the molecular volumes of DOPC and CnNO their coefficients of isobaric thermal expansivities in the investigated temperature interval were obtained. The VCnNO volumes of CnNO incorporated into DOPC bilayers showed linear dependencies on the CnNO alkyl chain length at all measured temperatures. This allowed to calculate the component volume of the CnNO methylene group (VCH2) at several temperatures and its coefficient of isobaric thermal expansivity. Using the assumption that the component volume of the CnNO methyl group VCH3 = 2VCH2 we also calculated the component volume and the coefficient of isobaric thermal expansivity of the hydrophilic group of CnNO (VNO). We found that the VCH2 volume increases in the whole temperature interval whereas the VNO volume decreases.

Study of interaction of long-chain n-alcohols with fluid DOPC bilayers by a lateral pressure sensitive fluorescence probe.

The excimer 1,2-dipyrenedecanoyl-sn-glycero-3-phosphatidylcholine (dipy10PC) fluorescence probe was used to determine effects of aliphatic alcohols (CnH2n+1OH, n = 12-18 is the even number of carbons in alkyl chain) on fluid dioleoylphosphatidylcholine (DOPC) +dioleoylphosphatidylserine (DOPS) bilayers in multilamellar vesicles at molar ratio DOPC/DOPS = 24.7. The excimer to monomer fluorescence intensity ratio increases with the increase of CnH2n+1OH/DOPC molar ratio and decreases with the CnH2n+1OH alkyl chain length n at a constant CnH2n+1OH/DOPC = 0.4 molar ratio. These effects indicate changes in the bilayer lateral pressure on the level of pyrenyl moieties location.

DOPC-DOPE composition dependent Lα-HII thermotropic phase transition: SAXD study

The structural polymorphism and parameters of lyotropic phases formed in the mixed dioleoylphosphatidylcholine-dioleoylphosphatidylethanolamine (DOPC-DOPE) system upon heating and varying DOPC:DOPE composition were studied by means of small-angle X-ray diffraction (SAXD). In the temperature range 5-80°C a sequence of fluid lamellar Lα - inverse hexagonal HII - inverse cubic QII phases was detected at DOPE mole fractions XDOPE≥0.65. A superposition of two bicontinuous cubic QII phases of Pn3m and Ia3d space groups was identified. The Lα to HII phase transition temperature, the onset of the QII phase formation, as well as the lattice spacings of the Lα and HII phases were found to decrease with rising DOPE content. Moreover, evidence of structural rearrangement during the Lα to HII phase transition is given and change of transition mechanism with varying XDOPE is suggested.

Small Angle X‐ray Diffraction Study on DOPC+DOPE Liposomes containing Long 1‐Alkanols

The effect of 1‐alkanols (abbreviation CnOH, n = 8–18 is the even number of alkyl carbons) on the transition from lamellar Lα to inverted hexagonal HII phase of multilamellar dioleylphosphatidylcholine (DOPC)+dioleylphosphatidylethanolamine (DOPE) vesicles at full hydration was studied using small angle X‐ray diffraction. For DOPC:DOPE = 1:9 molar ratio the phase transition temperature TBH was observed at ∼40° C. The CnOHs studied caused a significant decrease of TBH. The hexagonal phase was observed at presence of shorter alkanols (C8OH–C14OH), at 0.4:1 alkanol:lipid molar ratio, even at 5° C. For C16OH and C18OH the offset of La→HII transition was detected.