Fluorescent probing of the ligand-binding ability of blood plasma in the acute-phase response.

The acute-phase response alters the composition of carrier proteins in plasma, which may affect the blood deposition and transport of biomediators and drugs. The effect of the acute-phase response on the ligand binding ability of plasma was studied in leukemic children with and without systemic inflammation (sepsis and septic shock). To target different transport proteins, differentially charged fluorescent dyes were used: anionic ANS (8-anilinonaphthalene-1-sulfonate), uncharged Nile red, and cationic Quinaldine red. Human serum albumin was a principal carrier for ANS and competed for Nile red binding with lipoproteins. The synchro-scan fluorescence spectra of Nile red in plasma distinguished two species of the dye bound to serum albumin and to low-density and/or very low-density lipoproteins. The binding of Quinaldine red did not correlate with albumin and lipoprotein levels, and was probably determined by alpha(1)-acid glycoprotein. Compared with the control group, leukemia increased Quinaldine red binding by 65% and did not significantly affect the binding of other probes. Sepsis and septic shock did not change the binding of Quinaldine red, but progressively decreased ANS binding, finally by about 33%, and shifted Nile red distribution from serum albumin toward lipoproteins. These changes reflected a modified composition of the three principal transport proteins in plasma in the acute-phase response. Simple and rapid fluorescent tests developed in this study can be used to evaluate the acute-phase response and to optimize drug administration protocols in clinical practice.

[Use of excimerization of pyrene for assessing lipid microviscosity in biological membranes]. / Ispol'zovanie éksimerizatsii pirena dlia otsenko mikroviazkkosti lipidov v biologicheskikh membranakh.

A method for estimating the fluidity of natural membranes from the pyrene excimer/monomer fluorescence ratio (Ie/Im) is proposed. The method makes it possible to exclude artefacts such as fluorescence quenching, aggregation, and redistribution of the probe in lipid mains with different microviscosity. It is shown that, upon variation of intramembrane pyrene concentration [pyr], the occurrence of a common crossover point in pyrene fluorescence spectra normalized to the corresponding probe concentration (isoemission or isobestic point) or, as a consequence, the linear dependence of Ie/[pyr] on Im/[pyr] can serve as a criterion of diffusion (fluidity)-controlled excimerization of pyrene. The isobestic point can be used for determining the range of working concentrations of the probe in membrane suspension. It was found from the intensity of pyrene fluorescence in the isobestic point and quenching with potassium iodide that at t < 30 degrees C, the probe is uniformly distributed throughout the membrane, and its excimerization is mainly controlled by the microviscosity of environment.

[Effect of acidosis on membrane potential and calcium transport in rat brain synaptosomes]. / Vliianie atsidoza na membrannyi potentsial i transport kal'tsiia v sinaptosomakh mozga krys.

The influence of acidosis on the transmembrane potential, sodium pump and membranous systems of calcium transport was studied on isolated presynaptic nerve terminals (synaptosomes) from rat brain. It is established that acidic shift causes a decrease of membrane potential, a large inhibition of the sodium pump (by three times at pH 6.0). All the systems controlling both inward- and outward-directed calcium fluxes are partially blocked by low pH. At pH 6.0 the basal influx and calcium pump are reduced two-fold while the voltage-sensitive calcium channels and Na+/Ca2+ exchanger are inhibited by three and four to five times, respectively. We have no found any evidence of acidosis-induced net flux of calcium directed inwards.

Effect of proteolysis on the state of lipid phase in rat brain synaptosomal membranes.

Enzymatic proteolysis of the proteins of synaptic membranes has been found to be accompanied by the promotion of lipid peroxidation probably mediated by the liberation of membrane-bound iron. As fluorescent probes pyrene and diphenylhexatriene show, the microviscosity and micropolarity of membrane lipid phase rise as a result of lipid peroxidation. Different structural changes induced by proteolysis are displayed under inhibition of lipid peroxidation. Thus, the microviscosity of the bulk lipid phase appears to be lowered and the annular lipid microviscosity raised. Another explanation of the fluorescent data for annular lipids is the exclusion of pyrene molecules from this lipid pool, leading to a reduction of the probe local concentration. The changes observed in membrane lipid phase are considered as primary structural effects of proteolysis, not mediated by a phospholipase activation.

Osmotic regulation of sodium pump in rat brain synaptosomes: the role of cytoplasmic sodium.

The effect of hypoosmolality of incubation medium on the rat of ouabain-sensitive 86Rb+ transport in rat brain synaptosomes was studied. A decreased osmolality from 310 to 250 mOsm increased the rate of 86Rb+ uptake from 3.72 to 6.23 nmol/mg of protein min. To evaluate the involvement of cytoplasmic sodium in sodium pump stimulation inhibitors of ion channels and transport pathways able to increase [Na+]in were used. Tetrodotoxin (1 microM), amiloride (0.5 mM) and verapamil (0.1 mM) had no influence on the osmotic response of the sodium pump. The decrease of sodium concentration in incubation medium to 15 mM, leading to a practical loss of its transmembrane gradient, did not abolish stimulation of pump. No increase in 22Na+ influx or intrasynaptosomal sodium content was registered at hypotonic conditions. It is suggested that osmotic regulation of Na+,K(+)-ATPase is not connected with an increase of internal sodium through opening of sodium channels, or with activation of other membrane sodium-transporting systems.

Effect of gamma-irradiated carbohydrates on isolated synaptic membranes.

The effect of gamma-irradiated solutions of carbohydrates, mainly glucose, upon Na+, K(+)-ATPase and lipid peroxidation in rat brain synaptosomal membranes was studied. The membrane damage by irradiated glucose was enhanced in the presence of Fe2+ and was diminished when a free-radical scavenger (BHT) or metal chelators (EDTA, EGTA) were present. It is suggested that a key element in the free-radical membrane damage by irradiated carbohydrates is an Fe(2+)-complex of some species of the radiolysis products. Participation of radiotoxins of carbohydrate origin in radiobiological effects is discussed.

[Interaction of bromophenol blue with serum albumin: criteria for using dyes for assessing the state and quantity of protein]. / Vzaimodeistvie bromfenolovogo sinego s syvorotochnym al'buminom: kriterii ispol'zovaniia krasiteliia dliia otsenki sostoianiia i kolichestva belka.

The optical properties of the complexes of the pH-dependent dye bromophenol blue (BPB) with human serum albumin were investigated by the spectrophotometric method. The solvatochromic longwave displacement of bound BPB-2 absorption and BPB-1/BPB-2 redistribution were shown to form the optical signal of complexes. Because of the distortion of the bound BPB-2 signal its quantity was determined as delta A630 = A630 - A660 and the use of lambda max as structural parameter was limited to low pH less than or equal to 3. The conclusion was made that BPB is inapplicable as a structural probe on account of low structural dependence of delta A630 and pH-limitation of lambda max used. The maximal absorption delta Amax = Amax - A660 and its structural independence were obtained in the region of 70-100% occupation of the dye-binding centers of the protein. It is the optimal conditions for the quantitative determination of protein. After maximal dye binding (15-16 molecules of BPB per 1 molecule of albumin) the aggregation and precipitation of the complexes occurred.

[The conflicting action of low and high doses of ozone on the reproductive capacity and respiratory activity of Candida utilis yeast cells]. / Raznonapravlennoe deistvie nizkikh i vysokikh doz ozona na reproduktivnuiu sposobnost' i aktivnost' dykhaniia drozhzhevykh kletok Candida utilis.

Different doses of ozone were shown either to stimulate (1-2 x 10(8) molecules O3/cell) or inhibit (1 x 10(9) molecules O3/cell) respiration and reproduction of C. utilis yeast. The observed changes were preserved for several hours after treatment with ozone. Possible role of structural rearrangements of cell membranes in the ozone-induced effects has been discussed.

[Protein damage after treatment with ozone of protoplasts and isolated E. coli membranes]. / Povrezhdenie belkov pri deistvii ozona na protoplasty i vydelennye membrany E. coli.

The features of ozone-induced damage of E. coli plasma membrane proteins are investigated. A conclusion is made that protein fluorescence quenching is connected with modification of amino acid residues in the vicinity of tryptophane residues. Such modification may be a consequence of reaction with either ozone itself or products of its interaction with membrane lipids and/or proteins. The suggestion of Goldstein and McDonagh that ozone has a predilection for more hydrophilical membrane domains is confirmed. The data obtained are in agreement with a supposition about the leading role of proteins in deleterious action of ozone on cells.

[Erythrocyte hemolysis by detergents--a mathematical model and analysis of the concentration and kinetic curves]. / Gemoliz éritrotsitov detergentami--matematicheskaia model' i analiz kontsentratsionnykh i kineticheskikh krivykh.

A mathematical model of erythrocyte lysis by detergents is developed which takes into consideration the kinetics of detergent binding to plasma membrane. Experimentally obtained sigmoidal kinetic and concentration curves of hemolysis are well described by the model. A comparative study is carried out in terms of the model of hemolytic action for five detergents: Triton X-100, sodium dodecylsulfate, sodium deoxycholate, cetyltrimethylammonium bromide, and cetylpyridinium chloride. The amount of detergent which should be bound to an erythrocyte membrane to induce lysis was found to be roughly the same for all detergents studied. However, detergents vary in their affinity to the membrane. Cetylpyridinium displays the highest affinity (and consequently the highest hemolytic activity), whereas deoxycholate has the least one.

[Intercellular interactions as a reason for the influence of the erythrocyte concentration on the parameters of detergent hemolysis]. / Mezhkletochnye vzaimodeistviia kak prichina vliianiia kontsentratsii éritrotsitov na parametry gemoliza detergentami.

Possible reasons are discussed for the abnormal erythrocyte concentration influence on the course of detergent hemolysis curves. The data obtained lead to a conclusion that erythrocyte plasma membrane properties and, as a consequence, the parameters of erythrocyte-detergent interaction are dependent on cell concentration. The dependence is more sharply expressed for concentrations 5.10(7) cell/ml. In the concentration range (5-20) X 10(7) cell/ml the state of erythrocyte membrane remains apparently unchanged.

[Characteristics of the heterogeneity of the physical properties of the lipid phase of synaptic membranes from the rat brain using the fluorescent probe pyrene]. / Kharakteristika geterogennosti fizicheskikh svoistv lipidnoi fazy sinapticheskikh membran iz mozga krys s pomoshch'iu fluorestsentnogo zonda pirena.

A theoretical analysis of the dependence of pyrene excimerization on its concentration in biological membranes was carried out. It was shown that in synaptic membranes the concentration dependencies of pyrene excimerization parameter upon direct stimulation appear as nonlinear, thus being reflective of the heterogeneity of physical properties of the lipid phase. Upon pyrene excitation at the expense of the energy transfer from tryptophanyl residues the dependence is linear, which points to the homogeneity of the anular lipid pool. It was assumed that a comparison of microviscosity of the anular and bilayer lipids requires independent measurements of the coefficient of the probe distribution between the lipids or of the anular lipid content in the membrane.

[A model of cooperative rearrangements of biological membranes without changes in the affinity of receptor binding centers]. / Model' kooperativnykh perestroek biologicheskikh membran bez izmeneniia srodstva retseptornykh tsentrov sviazyvaniia.

A mathematical model is proposed for cooperative rearrangements induced by specific ligand in certain biomembrane domains. They are considered as the N-valent receptors undergoing rearrangement when n less than or equal to N ligand-binding receptor sites are occupied. The model predicts distinct sigmoidal dependence for change of some structural parameter on ligand concentration when the receptor site-ligand affinity remains constant as binding rises (positive cooperativity is absent).