Action of surface-active substances on biological membranes. IV. Hemolytic and membrane-perturbing action of homologous series of beta-D-glucopyranosyl-1-alkylphosphates.

The hemolytic action of a homologous series of beta-D-glucopyranosyl-1-alkylphosphates on human erythrocytes has been examined. The agent's affinity for the red cell membrane and the mean number of the agent's molecules which, upon interaction with an erythrocyte, make it undergo hemolysis have been measured. The contribution of the head group and that of a CH2 group of the surfactants to the free energy of the agents' binding to the cell membrane have been estimated. The effect of the surfactants on the red cell volume and the lytic concentrations of the agents have been measured. The contribution of a CH2 group to the free energy of the interaction of the amphiphiles embedded in the membrane bilayer with their environment has been evaluated and is proposed to be used as a measure of the membrane matrix stability.

Investigation of the inside-outside distribution, intermembrane exchange and transbilayer movement of phospholipids in sonicated vesicles by shift reagent NMR.

1. A new NMR approach is described for the investigation of transbilayer asymmetry in phospholipid vesicles consisting of phosphatidylcholine and negatively charged phospholipids. The method makes use of the dependence of the psuedocontact shift of the N-methyl proton resonance induced by paramagnetic ions on the surface concentration of negatively charged phospholipids. When two differently shifting paramagnetic probes are applied from the outside and the inside of a vesicular membrane the transbilayer phospholipid distribution can be estimated without knowledge of the inner and outer radii of the vesicles and the packing density of the phospholipid molecules. 2. The method was employed to study the transbilayer asymmetry in vesicles obtained by cosonication of phosphatidylcholine with phosphatidylserine, phosphatidylglycerol or phosphatidylinositol. The three negative phospholipids were found to distribute with a higher surface concentration in the inner vesicular shell than in the outer one when their total content did not exceed 25 mol%. However, as the amount of negatively charged phospholipids increases the ratio of their inside to outside surface concentrations, i.e., the transbilayer asymmetry of the vesicles, decreases. Prolonged incubation (for several days) does not change the compositional asymmetry of the cosonicated vesicles. 3. By the 'double-probe' technique it was established that spontaneous exchange between separately sonicated phosphatidylcholine and phosphatidylinositol vesicles results in formation of highly asymmetric mixed vesicles with phosphatidylinositol residing only in the outer monolayer. In the presence of antioxidant (alpha-tocopherol) the bilayer asymmetry is preserved for days. However lipid peroxidation induces rapid transbilayer movement (flip-flop) of phospholipids leading to an 'inverted' asymmetry resembling that of cosonicated vesicles. It is suggested that lipid peroxidation promotes phospholipid flip-flop by partially converting the bilayer structure into a non-bilayer configuration. Moderate quantities of lysophosphatidylcholine (up to 15 mol%) induce neither detectable perturbations of the bilayer nor rapid phospholipid flip-flop.

Mechanisms of stabilization of biomembranes by alpha-tocopherol. The role of the hydrocarbon chain in the inhibition of lipid peroxidation.

The effects of alpha-tocopherol and its homologues with different chain lengths (6-hydroxy-chromanes: C1, C6, C11) on lipid peroxidation in natural membranes (liver microsomes and mitochondria, brain synaptosomes) and liposomes were studied. It was shown that the antioxidant activity of alpha-tocopherol homologues decreased in the order: C1 greater than C6 greater than C11 greater than alpha-tocopherol (C16). Using fluorescent measurements, the possible reasons underlying these differences were investigated: (i) the distribution between the aqueous media and nonpolar phase of the membrane, which predetermines the binding of alpha-tocopherol homologues to membranes; (ii) the incorporation of alpha-tocopherol homologues into lipid bilayer; (iii) non-uniform distribution (formation of the clusters) of tocopherol homologues in the lipid bilayer; and (iv) transbilayer mobility of alpha-tocopherol homologues and accessibility of the inhibitors for radical-generating centres under enzymically and non-enzymically induced lipid peroxidation. It was demonstrated that: (i) binding of C1 with membranes was less efficient than that of longer-chain homologues (C6, C11, C16); (ii) the level of incorporation of alpha-tocopherol homologues into membranes decreased in a succession alpha-tocopherol C11 greater than C6 greater than C1; (iii) all alpha-tocopherol homologues existed in the lipid bilayer not only in a monomeric form but also associated in clusters thus decreasing the efficiency of radical scavenging; (iv) the short-chain alpha-tocopherol homologue, C1, exhibited a high transbilayer mobility whereas the long-chain one, C16, underwent no transbilayer migration within tens of minutes. The inhibiting effect of alpha-tocopherol esters and C1-acetate was predetermined by their hydrolysis in biomembranes; a strong correlation exists between the rate of the ester hydrolysis and their antioxidant activity in the membrane. In liposomes, in which the esterase activity was absent, alpha-tocopherol esters and C1-acetate exhibited very low lipid peroxidation inhibition.

N alpha-acetylcarnosine is a prodrug of L-carnosine in ophthalmic application as antioxidant.

The naturally occurring compound N alpha-acetylcarnosine (NAC) is proposed as the prodrug of L-carnosine (C) resistant to enzymatic hydrolysis by human serum carnosinase. Rabbit eyes were treated with 1% NAC, C or placebo and extracts of the aqueous humor from the anterior eye chamber were analyzed for imidazole content by reverse phase analytical high performance liquid chromatography (HPLC), thin-layer (TLC) and ion-exchange chromatographic techniques. The topical administration of pure C to the rabbit eye did not lead to accumulation of this compound in the aqueous humor over 30 min in concentration exceeding that in the placebo-treated matched eye. NAC showed dose-dependent hydrolysis in its passage from the cornea to the aqueous humor, releasing C after 15. 30 min of ocular administration of prodrug in a series of therapeutical modalities: instillation < or = subconjunctival injection < or = ultrasound induced phoresis. Different treatment techniques showed excellent toleration of 1% NAC by the eye. Once in the aqueous humor, C might act as an antioxidant and enter the lens tissue when present at effective concentrations (5-15 mmol/l). The advantage of the ophthalmic prodrug NAC and its bioactivated principle C as universal antioxidants relates to their ability to give efficient protection against oxidative stress both in the lipid phase of biological membranes and in an aqueous environment. NAC is proposed to treat ocular disorders which have the component of oxidative stress in their genesis (cataracts, glaucoma, retinal degeneration, corneal disorders, ocular inflammation, complications of diabetes mellitus, systemic diseases).

PUVA-induced erythema and changes in mechanoelectrical properties of skin. Inhibition by tocopherols.

Influence of antioxidants on two phototoxic effects of 8-methoxypsoralen (8-MOP) was studied: erythema and changes in mechanoelectrical properties of skin. alpha-Tocopherol and its analogs with shortened lateral hydrocarbon chains at C2-atoms of chromane groups (chromanols) were used as antioxidants. alpha-Tocopherol and its analogs inhibited both phototoxic effects of 8-MOP. Inhibition was observed only if antioxidants were present in skin during irradiation. When applied after irradiation these antioxidants produce no inhibitory effect. The antioxidant antierythemal action depends greatly on their concentration. The protective effects is maximal at antioxidant concentrations 2.5 . 10(-10) - 5 . 10(-9) mol . cm-2 of skin, at concentrations higher than 5 . 10(-9) mol . cm-2 the protective action is decreased. The protective effect of antioxidants depends on the irradiation dose.

Photophysical properties of carborane-containing derivatives of 5,10,15,20-tetra(p-aminophenyl)porphyrin.

Absorption, fluorescence emission, fluorescence excitation spectra and fluorescence decay kinetics of carborane derivatives of 5,10,15,20-tetra(p-aminophenyl)porphyrin have been investigated. Carborane derivatives are prepared by acylation of the amino groups of 5,10,15,20-tetra(p-aminophenyl)porphyrin by 9-carboranyl acetyl chloride. From the analysis of the absorption and fluorescence spectra, it is concluded that covalent linking of carborane molecules to the tetraphenylporphyrin molecule significantly changes the self-conjugated pi-system of the porphyrin macrocycle: positions of maxima of absorption and fluorescence spectra shift to the red region by 3-8 nm; the halfwidths of these bands are broadened by 2.5-5.0 nm; the relative intensity of the bands I-IV also changes. The fluorescence decay kinetics of the carborane derivatives are biexponential. According to the experimental data and model simulation, it is concluded that the intramolecular electron transfer proceeds from the porphyrin excited part of the molecule to carboranyls with a rate constant of 415 ps(-1) and efficiency of 0.16-0.8. Recombination of separated charges occurs within 1.4 ns.

[Methods of searching for antigenic determinants for proteins with known primary structure]. / Metody poiska antigennykh determinant dlia belkov s izvestnoi pervichnoi strukturoi.

Theoretical and experimental methods for locating antigenic determinants of proteins with known amino acid sequences are discussed. These methods are systematized on the basis of the theoretical approaches applied, and the efficiency of various predictive methods is compared. Some examples of experimental epitope determination for a number of proteins are given.

[Allosteric regulators of reversible oxygenation of hemoglobin]. / Allostericheskie reguliatory obratimoi oksigenatsii gemoglobina.

Properties of regulators of reversible haemoglobin oxygenation, especially of a natural regulator, 2,3-diphosphoglyceric acid (DPG), are reviewed. DPG provides effective deoxygenation of haemoglobin, helps to maintain its functional properties preventing its transformation into inactive derivatives. Correlation between organism metabolic requirements and haemoglobin oxygen affinity is established by erythrocyte DPG. Several functional analogues of DPG, their structure--activity relationship and possible medical application are discussed.

[A bioregulator of lipid nature--a platelet activating factor (PAF)]. / Bioreguliator lipidnoi prirody--faktor aktivatsii trombotsitov (PAF).

The review concerns metabolism, immunological and antihypertensive action of platelet activating factor (PAF), a bioregulator of lipid nature. Major synthetic approaches to PAF and its analogues are described. The effects of structural modification on the physiological activity of PAF are considered.

[Synthesis of thioanalogs of platelet activating factor (PAF)]. / Sintez tioanalogov faktora aktivatsii trombotsitov (FAT).

Synthesis of thioalkyl, thioacetyl and phosphothionyl PAF analogues was carried out starting with corresponding monoalkyl glycerol ethers. The synthetic route was based on preparation of racemic phosphatidylcholines and subsequent hydrolysis with phospholipase A2 to afford isomers having natural configuration.

[Biosynthetic paths of acyclic lipoxygenase metabolites of polyenic acids]. / Puti biosinteza atsikicheskikh lipksigenaznykh metabolitov polienovykh kislot.

The review deals with the basic features of the fatty acid lipoxygenation resulting in the formation of leukotrienes, hydroxy fatty acids, lipoxins, hepoxylins. The biological role, prospects for the further studies of this class of bioregulators are discussed.

[Synthesis of triads based on deuteroporphyrin IX, naphthoquinone, and aromatic amino acids]. / Sintez triad na osnove deiteroporfirina IX, naftokhinona i aromaticheskikh aminokislot.

Two triad systems were synthesized from deuteroporphyrin IX by tethering 1,4-naphthoquinone derivatives and aromatic amino acids to its propionic groups using the method of mixed anhydrides. Physicochemical characteristics of the triads were studied, and the pi-electron systems of their chromophores were shown to interact. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Synthesis of 1-(1-alkenyl)-2-acetyl-sn-glycero-3-phosphocholines, the aldehyded analogs of platelet activating factor]. / Sintez 1-(1-alkenil)-2-atsetil-sn-glitsero-3-fosfokholinov, al'degirovannykh analogov faktora aktivatsii trombotsitov.

1-(1-Hexadecenyl)- and 1-(1-octadecenyl)-2-acetyl-sn-glycero-3-phosphocholines were synthesized. To this goal, synthetically prepared racemic phosphatidal-cholines were hydrolyzed with phospholipase A2, and lysophosphatidal-cholines obtained were acetylated with acetic anhydride.

[Biospecific chromatography of lipolytic enzymes and methods for determining their activity]. / Biospetsificheskaia khromatografiia lipoliticheskikh fermentov i metody opredeleniia ikh aktivnosti.

The review concerns application of affinity chromatography for isolation of phospholipases and lipases, as well as the methods for determining their activities. Main emphasis is laid on the preparation of biospecific supports with lipid ligands as well as on development of new methods for assaying lipolytic activity.

[Synthesis of arginine-containing peptides and their conjugates with protohemin IX and tetraphenylporphyrin]. / Sintez argininsoderzhashchikh peptidov i ikh kon''iugatov s protogeminom IX i tetrafenilporfirinom.

Arg-containing peptides and their conjugates with protohemin IX were synthesized by the solid phase method using Merrifield resin. The conjugates of Arg-containing peptides with tetraphenylporphyrin were obtained by using phosphorus trichloride as an activating agent.

[Synthesis of leukotriene A4 methyl ester via acetylene intermediates]. / Sintez metilovogo éfira leikotriena A4 na osnove atsetilenovykh soedinenii.

Rac-leukotriene A4 methyl ester has been synthesized from propargylic alcohol and 1-heptyne. The synthetic strategy involves the assembly of carbon chain by acetylenide anion condensations and the introduction of (Z)-double bonds by the triple bond hydrogenation.

[Synthesis of 5,8,11,14,17-eicosapentaenic acid]. / Sintez 5,8,11,14,17-éikozopentainovoi kisloty.

A synthetic route to 5,8,11,14,17-eicosapentaynoic acid, an acetylenic precursor of 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid, has been developed based on condensation of a propargyl synthon with omega-acetylenic fragments. Physico-chemical constants and spectral characteristics (IR, 1H-NMR, and mass-spectrometry) of the target and intermediate compounds are given.

[Preparation of affinity sorbents for the isolation of phospholipase A2]. / Poluchenie affinnykh sorbentov dlia vydeleniia fosfolipazy A2.

The preparation of biospecific organo-silica supports with different concentrations of covalently attached sn-glycero-3-phosphocholine was described. Sorbtion of phospholipase A2 on the hydrophobic analogues of biospecific supports was studied with the aim of elucidating the role of the enzyme-matrix hydrophobic interactions. A comparative characterization of the prepared sorbents was carried out.

[Interaction of hemoglobin with phospholipid vesicular membranes of different composition]. / Izuchenie vzaimodeistviia gemoglobina s fosfolipidnymi vezikuliarnymi membranami razlichnogo sostava.

Incorporation of hemoglobin into vesicles and its oxidation were studied as a function of composition of the vesicular membrane containing erythrocyte membrane lipids as main components. The addition of negatively charged lipids such as phosphatidylserine and phosphatidic acid, was shown to considerably increase the extent of hemoglobin binding, while sphingomyelin did not produce such an effect. Cholesterol and dipalmitoylphosphatidylcholine stabilized oxyHb.

[Synthesis and analysis of glycol bis-phosphates, regulators of reversible hemoglobin oxygenation]. / Sintez i isledovanie bisfosfatov glikolei, reguliatorov obratimoi oksigenatsii gemoglobina.

Bisphosphates of glycols, ethyleneglycol and 1,2-propanediol, were synthesized. These structural analogues of 2,3-diphosphoglyceric acid were shown to reduce significantly the oxygen affinity of stripped hemoglobin.