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.

Synthesis, surface and antimicrobial properties of some quaternary ammonium homochiral camphor sulfonamides.

A group of homochiral quaternary ammonium sulfonamides bearing hydrophobic camphor derived moieties were synthesized and characterized. The described synthetic procedure is quick and efficient. The novel quaternary ammonium bromides were tested as antimicrobial and antifungal agents. They exhibited strong antimicrobial and also antifungal activity, especially N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfonamido] ethyl}-N,N-dimethyltetradecan-1-aminium bromide 1c. The surface properties of prepared compounds were evaluated by surface tension measurements and critical micelle concentration (CMC) with surface tension at CMC (γCMC) was calculated.

Effect of amphiphilic surfactant LDAO on the solubilization of DOPC vesicles and on the activity of Ca(2+)-ATPase reconstituted in DOPC vesicles.

Solubilization of large unilamellar 1,2-dioleoylphosphatidylcholine (DOPC) vesicles by N-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was studied using turbidimetry. From turbidity data, the LDAO partition coefficient between the aqueous phase and DOPC bilayers was obtained. Using this partition coefficient, the LDAO:DOPC molar ratio in the bilayer was calculated and effects of LDAO on the bilayer stability, bilayer thickness and on the phosphohydrolase activity of sarcoplasmic reticulum Ca(2+) transporting ATPase (SERCA) reconstituted into DOPC were compared at the same LDAO:DOPC molar ratios in the bilayer. The sequence "bilayers in vesicles - bilayer fragments (flat mixed micelles) - tubular mixed micelles - globular mixed micelles" was suggested for the solubilization mechanism of DOPC vesicles from the combined turbidimetric and small-angle neutron scattering (SANS) results. The effective molecular packing parameter delta = 0.5, corresponding to the mixed bilayer - mixed tubular micelle transition, was calculated from fragmental DOPC and LDAO volumes at the molar ratio LDAO:DOPC = 2.00 in bilayers, in the middle of transition region observed earlier experimentally by small-angle neutron scattering (SANS). The bilayer thickness decrease induced by LDAO in DOPC observed by SANS did not result in the SERCA phosphohydrolase activity decrease and this indicates that some other factors compensated this bilayer effect of LDAO. The ATPase activity decrease at higher LDAO concentrations was caused by the bilayer deformation. This deformation resulted in the formation of non-bilayer aggregates in LDAO+DOPC system.

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters.

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.

Effects of N-alkyl-N,N-dimethylamine-N-oxides on the activity of purified sarcoplasmic reticulum Ca(2+)-transporting ATPase.

N-alkyl-N,N-dimethylamine-N-oxides (CnNO, n = 10-20 is the number of alkyl carbon atoms) stimulate the skeletal sarcoplasmic reticulum (SR) Ca(2+)-transporting ATPase activity at low concentrations and inhibit it at high concentrations. The minimum concentration (cmin), at which CnNO inhibits the ATPase, continuously decreases up to n = 16-18 and then increases. The values of Cmin are smaller than the CnNO critical micelle concentration (cmc) for C10NO-C14NO homologs, but larger than cmc for C18NO-C20NO homologs. The ATPase inhibition is caused by the CnNO-induced lipid bilayer structural perturbation in the ATPase annular region, modulated by the partition equilibria of the CnNO molecules between the bilayer and aqueous phase for short alkyl chain (n = 10-16) CnNO homologs, and between the bilayer, micelles and aqueous phase for long alkyl chain (n = 18-20) CnNO homologs.

[Catalytic activity of butyrylcholinesterase in biodegradation of organic ammonium salts in vitro]. / Katalytická aktivita butyrylcholínesterázy v biodegradácii organických amóniových solí in vitro.

Organic ammonium salts of N-(2-benzoyloxyethyl)-alkyldimethylammonium bromide (BCHn-1) type are formed by the homological series Ar-COO(CH2)2-N+(CH3)2CnH2a + 1.Br-, whose structure contains a biodegradably labile ester bond, on the basis of which they rank among disinfectants and antiseptics of soft character. They are preferentially biotransformed hydrolytically to produce benzoic acid and substituted choline. The rapidity of enzymatic hydrolysis depends on the chemical structure (the length of the aliphatic chain on the ammonium nitrogen), it increases up to the number of 10 nitrogens of the aliphatic chain, and it rapidly decreases with further prolongation. The paper aimed to demonstrate the catalytic activity of butyrylcholinesterase on the enzymatic hydrolysis of selected organic ammonium salts in the medium of the microsomal fraction of the rat liver on the basis of inhibitory kinetic studies with physostigmine, a cholinesterase inhibitor. The product of enzymatic hydrolysis of BCHn-1, benzoic acid, was determined after extraction with chloroform from the acid medium by means of HPLC analysis with the use of the internal standard p-iodobenzoic acid at the wavelength of 228 nm. Kinetic parameters K(M) and VMAX were evaluated following Lineweaver-Burke using the method of linear regression analysis. The specific activity of butyrylcholinesterase (E.C.3.1.1.8) in the enzymatic hydrolytic process of BCHn-1 was significantly influenced by the presence of physostigmine, which was manifested by increased K(M), KI, and IC50 values in the investigated enzymatic process of selected substrates of the homological series BCHn-1, and by decreased VMAX and rate constants.

[Solubilization of unilamellar egg yolk phosphatidylcholine liposomes by N-alkyl-N,N-dimethylamine N-oxides]. / Solubilizácia unilamelárnych lipozomómov z vajecného fosfatidylcholínu N-alkyl-N,N-dimetylamín N-oxidmi.

The solubilization of extruded (100 nm) unilamellar egg yolk phosphatidylcholine (EYPC) liposomes by a series of N-alkyl-N,N-dimethylamine N-oxides (CnNO, n = 10-14 carbon atoms in the alkyl substituent) was studied using turbidimetry. The solubilizing concentration of CnNO (cS) was estimated as the CnNO concentration causing the half-maximum decrease in turbidance. From the linear cS dependence on EYPC concentration, the lipid--aqueous phase molar partition coefficient (Kp) and the CnNO:EYPC molar ratio in the CnNO + EYPC aggregates (nL:nEYPC) at cS were obtained: Kp = 82 +/- 25 and nL:nEYPC = 0.70 +/- 0.20 for C10NO, Kp = 507 +/- 215 and nL:nEYPC = 0.60 +/- 0.16 for C12NO, and Kp = 12357 +/- 93 and nL:nEYPC = 1.13 +/- 0.01 for C14NO. The value of Gibbs free energy of CnNO alkyl methylene group transfer from the aqueous to the lipid phase calculated from the Kp dependence on n is -1.2 +/- 0.2 RT (R = gas constant, T = absolute temperature), within the experimental error being the same as -1.026 +/- 0.006 RT obtained from the critical micellar concentrations of CnNO. The increased value of nL:nEYPC for C14NO is caused by the decreased hydrophobic mismatch of CnNO and EYPC hydrocarbon chain lengths. This mismatch results in a structural defect in the bilayer hydrophobic core, the primary cause of bilayer destabilization and solubilization.

X-ray diffraction and neutron scattering studies of amphiphile-lipid bilayer organization.

The lipid bilayer thickness d(L), the transbilayer distance of lipid phosphate groups d(pp/inf> and the lipid surface area A(L) of fluid hydrated bilayers of lamellar phases of egg phosphatidylcholine or dipalmitoylphosphatidylcholine containing N-alkyl-N,N-dimethylamine N-oxides (CnNO), 1,4-butanedi-ammonium-N,N'-dialkyl-N,N,N',N'-tetramethyl dibromides (GSn) or mono-hydrochlorides of [2-(alkyloxy)phenyl]-2-(1-piperidinyl)ethylesters of carbamic acid (CnA) were obtained by X-ray diffraction, and the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine vesicles containing C12NO was obtained by the neutron scattering. The values of d(L), d(pp/inf> and A(L) change linearly up to the 1:1 amphiphile:lipid molar ratio. The slopes of these dependencies increase for d(L) and d(pp/inf> and decrease for AL) with an increasing number of carbons n in the amphiphile long hydrocarbon substituent (18> or =n> or =8 for CnNO, 16> or =n> or =9 for GSn, 12> or =n> or =5 for CnA), while the opposite trends are observed for the short substituent (8> or =n>/=6 for CnNO, 9> or =n> or =7 for GSn, 5> or =n> or =3 for CnA). In case of long substituents, the effects on dL), dpp/inf> and AL) are caused by the decrease in the difference between the lipid and amphiphile hydrocarbon chain lengths and by the increase in their van der Waals attraction. The short substituent amphiphiles are mobile and exchange between multiple binding sites in the bilayer, minimizing the bilayer surface area.

Aggregation Properties of Sodium Hyaluronate with Alkanediyl-alpha,omega-bis(dimethylalkylammonium Bromide) Surfactants in Aqueous Sodium Chloride Solution.

Aggregation properties of sodium hyaluronate (NaHA) with alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide) surfactants (referred to as dimeric surfactants) in aqueous sodium chloride solutions have been studied as a function of surfactant chemical structure. Surface tension measurements indicate the unusual parabolic dependence of surface tension vs log surfactant concentration with a surface tension minimum at concentration c(min). The increase of surface tension above c(min) may be related to the formation of clusters consisting of NaHA chain and dimeric surfactants at the air-water interface and in the bulk. From light scattering measurements, molecular weight, hydrodynamic radius, and second virial coefficient have been calculated. The simple calculation of the ratio of positive charge of dimeric surfactant unit per one negatively charged hyaluronate disaccharidic unit in NaHA-surfactant complex reveals that there is a slight excess of positive surfactant charges per one negatively charged disaccharidic unit in the region around c(min) and the NaHA-surfactant complex is not far from electroneutrality. The nonlinear behavior of viscosity vs surfactant concentration in the NaHA-dimeric surfactant system depends on surfactant chemical structure. The behavior is concerned with the size increase due to complex growth and with the size shrinkage above c(min). A model describing the behavior of NaHA-surfactant complex in the bulk and at the interface is suggested. Copyright 2000 Academic Press.

[Inter-organ variability in enzymatic hydrolysis of ammonium salts in rats in vitro]. / Medziorgánová variabilita enzýmovej hydrolýzy organických amóniových solí u potkana in vitro.

The study evaluated the rate and kinetics of enzymatic hydrolysis of N-(2-benzoyloxyethyl)-alkyl-dimethylammonium bromides, potential easily biodegradable disinfectants of soft character. The products of enzymatic hydrolysis of the substrates under study, catalysed by microsomal esterase, included substituted substrates choline and benzoic acid which, as a hydrolytic product, was essayed by HPLC. The effect of the length of the alkyl chain of the individual homologues on the rate of enzymatic hydrolysis and their affinity to microsomal esterase of the rat liver and lung in vitro was examined. The structural modification (varying length of the aliphatic chain on the ammonium nitrogen of these compounds) was found to significantly influence the kinetics of enzymatic hydrolysis of the esteric bond. From the viewpoint of the rate of enzymatic hydrolysis no significant inter-organ variability was observed: in the liver as well as the lung the dependence of the rate of enzymatic hydrolysis on the length of the aliphatic chain possesses the shape of a falling hyperbole with the maxima for BCH2 > BCH4 > BCH8 = BCH10. The specific activity of both esterases ranges within 0.2-3.5 nmol.min-1.mg-1. From the viewpoint of affinity, a marked inter-organ difference was manifested by 10 times higher affinity of the substrates to the lung microsomal esterase in comparison with the liver one. The effect of the length of the alkyl of the individual homologues on the affinity is of a non-linear character also in this case. In both organs, a certain correlation was found between the rate of enzymatic hydrolysis and affinity to microsomal esterases.

Cytoxicity of 1-alkylperhydroazepine N-oxides and quantitative structure-activity relationships.

A new class of nonaromatic amine oxides was tested for cytotoxic activity. The main aim of the present investigation was to screen a series of 1-alkylperhydroazepine N-oxides for in vitro cytotoxicity and to find out whether there is a quantitative structure-activity correlation (QSAR) between cytotoxic effect and structure (as a structural parameter the number of carbon atoms m in the alkyl chain was used). Cytotoxicity was determined here by inhibition of incorporation of [14C]adenine into nucleic acid or [14C]valine into proteins in Ehrlich ascites carcinoma (EAC) cells. On the basis of primary screening, one of the most active compounds, namely 1-tetradecylperhydroazepine N-oxide (TPNO), was chosen for further biochemical study. The drug inhibited the incorporation rate of [14C] labeled precursors (adenine, thymidine, uridine, valine) into appropriate macromolecules of Ehrlich cells. The extent of inhibition was dependent on both time and drug concentration. The lengthening of the alkyl chain in 1-alkylperhydroazepine N-oxides positively affected their cytotoxic activity in EAC cells. For these compounds the optimal m value is 12-14.

Effect of N-lauryl-N,N-dimethylamine N-oxide on dimyristoyl phosphatidylcholine bilayer thickness: a small-angle neutron scattering study.

Small-angle neutron scattering on large extruded unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes was used to determine the DMPC bilayer thickness dL and its change in the presence of N-lauryl-N,N-dimethylamine N-oxide (LDAO). At 36 degrees C, the values of dL are dL = 3.44 +/- 0.10 nm and dL = 2.90 +/- 0.10 nm in pure DMPC bilayers and in bilayers at DMPC:LDAO = 2:1 molar ratio, respectively. Using the specific volumes of DMPC and LDAO and supposing that the molecular volumes and surface areas in the bilayer are additive, the surface areas of DMPC (ADMPC) and of LDAO (ALDAO) were found to be at 36 degrees C: ADMPC = 0.644 +/- 0.018 nm2 and ALDAO = 0.25 +/- 0.05 nm2.

Effect of N,N-dimethylalkylamine N-oxides on the activity of purified sarcoplasmic reticulum (Ca-Mg)ATPase.

Amphiphilic N,N-dimethylalkylamine N-oxides (abbreviation CnNO, n is the number of carbon atoms in the alkyl substituent) modulate the activity of the purified sarcoplasmic reticulum (Ca-Mg)ATPase. The phase of insensivity or slight stimulation of the activity at lower homologue concentrations is followed by the inhibition phase at higher concentrations. The potency to inhibit at high concentrations is maximal for the homologue with the alkyl chain length n = 16. The inhibition could be caused by the binding of homologues to binding sites at the protein/lipid bilayer interface.

QSAR and CAMM study of amphiphilic antimicrobially active 2,2'-bipyridyl monoammonium salts.

Preparation of 2,2'-bipyridyl monoammonium salts is described as well as their conformation study using computer aided molecular modelling (CAMM) methods and quantitative relations between structure, aggregation properties and antimicrobial activity (QSAR) of these derivatives. It was found that using the applied synthetic route the monoammonium salt is prepared free of bis-ammonium salt. While in the case of the unsubstituted 2,2'-bipyridyl the energy difference between s-cis and s-trans conformers is minor and the transition from one state into the other one is possible with s-trans state apparently being preferred, after quaternisation the exclusive conformer is s-cis that is in this state fixed except of steric hindrance between the alkyl substituent bonded to the N+ atom and the hydrogen bonded to 3'C also by a weak hydrogen interaction C-H ... N between the hydrogen of the first carbon of the alkyl chain and the nitrogen of the adjacent ring. This finding is supported also by the results of calculation of point electric charges, dipole moments, 2C-2'C distance and torsion angles of non-quaternised as well as bipyridinium cations. It follows from quantitative dependencies between lipophilicity (expressed by means of aggregation properties-by critical concentration of micelle formation ck, and chromatographic factor RM), structure (length of alkyl chain m) and antimicrobial activity (minimum inhibition concentration, MIC) that the maximum of activity is achieved with compounds of chain length m = 13 to 16 with ck about 1.10(-3) mol/l. It follows from the comparison with simple alkylpyridinium salts that the mechanism of biological activity at the bacterial level will not differ in 2,2'-dipyridyl derivatives from the mechanism of activity of other ammonium salts.

Cut-off effects in biological activities of surfactants.

In the homologous series of long hydrocarbon chain surface active compounds, their various biological activities increase progressively with increasing chain length up to a critical point, beyond which the compounds cease to be active. The paper reviews several hypotheses of this cut-off effect in biological activities and experimental evidences supporting them. It is suggested that the lateral expansion of the phospholipid bilayer of biological membranes caused by the intercalation of long-chain amphiphile molecules between the phospholipid molecules and the mismatch between their hydrocarbon chain lengths results in the creation of free volume in the bilayer hydrophobic region. The elimination of the free volume via the hydrocarbon chain trans-gauche isomerisation or interdigitation results in the bilayer thickness change or in its destabilisation and formation of non-bilayer phase(s). In combination with the partition and ionisation equilibria of amphiphiles in the lipid/aqueous phase systems, the free volume predicts similar chain length and pH dependencies as observed in biological experiments. It is suggested that the free volume mechanism, in combination with other mechanisms, could be responsible for the cut-off effects in biological activities of amphiphiles.

Immunomodulatory effect of some amphiphilic detergents on the human promyelocytic HL-60 cells.

The effect of two anionic (sodium dodecyl sulfate (SDS) and sodium laurate), two cationic (didodecyldimethylammonium bromide (DDAB) and [(1-methyldodecyl)-trimethylammonium bromide (2-ATDBr)] and a nonionic [(1-methyldodecyl)-dimethyl-amine N-oxide (2-ATDNO)] amphiphilic detergents on the metabolic activation of phagocytes derived from human promyelocytic leukemia cell line HL-60 was investigated. SDS in the concentrations of 10(-4) M significantly stimulated the INT-reduction and superoxide production by HL-60 cells which were preincubated with phorbol 12-myristate 13-acetate (PMA) for a macrophage-like differentiation. This effect was not significant in the case of cells differentiated along monocyte-like and granulocyte-like pathways. A similar influence was observed by sodium laurate, but this effect was significant (P < 0.01) by INT-reduction only. Concentrations higher than 5 x 10(-5) M DDAB, 2-ATDBr and 2-ATDNO inhibited INT-reduction and superoxide generation in HL-60 cells differentiated along all three ways. Our results indicated that differentiated HL-60 cells may be used for in vitro testing of immunotoxic effects on respiratory burst in phagocytes.

Effects of 1-tetradecylperhydroazepine and 1-pentadecylpiperidine N-oxides on Ehrlich ascites mitochondria.

Non-aromatic amine oxides are widely known and used compounds. A great number of amine oxides occurring in nature, or prepared synthetically, are biologically active compounds (antimetabolites and chemotherapics, cancerostatic compounds, etc.). From seven series of newly synthesized amine oxides (63 compounds), 1-alkylperhydroazepine N-oxides (PHNO) and 1-alkylpiperadine N-oxides (PINO) have been chosen for further investigation. The effects of 8 derivatives of PHNO and 8 derivatives of PINO on state 3 and 4 respiration of Ehrlich ascites mitochondria (EAM) have been studied. Derivatives with longer side-chain significantly affected respiration of EAM according to the substrates used. To elucidate the mode of action, the most potent amine oxides from each series, 1-tetradecylperhydroazepine N-oxide (tPHNO) and 1-pentadecylpiperidine N-oxide (pPINO) have been chosen for further study. Both amine oxides stimulated state 4 respiration with glutamate-malate and succinate as substrates. The effect on state 3 respiration depended on the substrates used. Both tPHNO and pPINO were able to release respiration of EAM previously inhibited by oligomycin, both decreased the level of ATP in EAM. ATPase activity was significantly stimulated by both drugs only in higher concentrations. A possible mode of action of amine oxides on oxidative phosphorylation and the relationship between chemical structure are discussed.

Suppression of virulence factors of pseudomonas aeruginosa by subinhibitory concentrations of quaternary ammonium salts.

The effect of subinhibitory concentrations (sub-MICs) of two homologous series of "soft" quaternary ammonium salts (QATs) upon the expression of phospholipase C (Pseudomonas aeruginosa strain 72/92), elastase, proteinase and permeability activity (P. aeruginosa 9/92) was studied. Both strains were isolated from the patients suffering from nosocomial infections. Phospholipase C was the most markedly inhibited enzyme. The inhibitory effect was directly proportional to the concentration of substances but the degree of inhibition in the two homologous series was different. In the second strain of P. aeruginosa its production of elastase, permeability factor and mainly its proteinase activity were less affected by the series of tested QATs. A significant inhibition of production of these factors was manifested only in higher concentrations particularly by the substances with longer alkyls. The effect of QATs on the production of virulence factors could be attributed to their influence on the metabolic activity of organisms.