[Participation of Sphingolipids in the Pathogenesis of Atherosclerosis].

Lipid metabolism disorders are the most significant risk factor of development of cardiovascular diseases (CVD). In the process of diagnosing ischemic heart disease and other cardiovascular pathologies, levels of total cholesterol, low- and high- density lipoprotein cholesterol, triglycerides are determined. However, in recent years, close attention has been paid to the intersection of the metabolic pathways of the biosynthesis of cholesterol and sphingolipids. Sphingolipids - a group of lipids, which include a molecule of aliphatic alcohol sphingosine. This group includes sphingomyelins, cerebrosides, gangliosides and ceramides, sphingosines and sphingosine-1-phosphate (S-1-P). Ceramides and sphingosines have pro-apoptotic properties, and S-1-P protects cells from apoptosis. Particular attention as inducer CVD attracts ceramide. It has been established that aggregated lipoproteins isolated from atherosclerotic zones are enriched with ceramides. The level of ceramide and sphingosine increases with ischemia/reperfusion of the heart, in the infarction zone and in the blood, and also in hypertensive disease. S-1-P has a pronounced cardioprotective properties. Its content sharply decreases with ischemia and myocardial infarction. S-1-P performs a special function in the structure of high-density lipoproteins (HDL), being one of the main lipid components of these lipoproteins, which determines their multiple functions. Recently, work has been underway to create drugs that can correct the metabolism of S-1-P. The most successful drugs are those that use the S-1-P receptor as a target, since all of its actions are carried out through receptors. Increasing ceramide and sphingosine and reducing blood plasma level of S-1-P can be an important factor in the development of atherosclerosis. It is proposed to use the determination of the level of sphingolipids in blood plasma for early diagnosis of cardiac ischemia and in arterial hypertension. Chromatography-mass spectrometry has been suggested as the main method for testing these lipids.

[The role of sphingolipids in cardiovascular pathologies]. / Rol' sfingolipidov v serdechno-sosudistykh patologiiakh.

Cardiovascular diseases (CVD) remain the leading cause of death in industrialized countries. One of the most significant risk factors for atherosclerosis is hypercholesterolemia. Its diagnostics is based on routine lipid profile analysis, including the determination of total cholesterol, low and high density lipoprotein cholesterol, and triglycerides. However in recent years, much attention has been paid to the crosstalk between the metabolic pathways of the cholesterol and sphingolipids biosynthesis. Sphingolipids are a group of lipids, containing a molecule of aliphatic alcohol sphingosine. These include sphingomyelins, cerebrosides, gangliosides and ceramides, sphingosines, and sphingosine-1-phosphate (S-1-P). It has been found that catabolism of sphingolipids is associated with catabolism of cholesterol. However, the exact mechanism of this interaction is still unknown. Particular attention as CVD inducer attracts ceramide (Cer). Lipoprotein aggregates isolated from atherosclerotic pluques are enriched with Cer. The level of Cer and sphingosine increases after ischemia reperfusion of the heart, in the infarction zone and in the blood, and also in hypertension. S-1-P exhibits pronounced cardioprotective properties. Its content sharply decreases with ischemia and myocardial infarction. S-1-P presents predominantly in HDL, and influences their multiple functions. Increased levels of Cer and sphingosine and decreased levels of S-1-P formed in the course of coronary heart disease can be an important factor in the development of atherosclerosis. It is proposed to use determination of sphingolipids in blood plasma as markers for early diagnosis of cardiac ischemia and for hypertension in humans. There are intensive studies aimed at correction of metabolism S-1-P. The most successful drugs are those that use S-1-P receptors as a targets, since all of its actions are receptor-mediated.

Structural and catalytic polymorphism of human enzymes: Novel potential platforms for biomedical diagnostics.

This review focuses on some intermediate results on the path from the gene and enzyme structure to physiological responses and personalised medicine. Bioinformatics of genetic and protein-structural polymorphisms, theoretical methods of predicting the influence of single amino acid substitutions on the structure and catalytic activity of enzymes are considered. For a large group of enzymes, interrelations between genetic modifications, structural changes of the proteins and the detected physiological and clinical manifestations are discussed. In this respect, highly productive techniques to determine the catalytic activity of an enzyme as well as non-invasive proteomic approaches are of particular interest. A non-invasive proteomic analysis using mass-spectrometric protein identification of human exhaled breath condensate and tear fluids has been chosen.

Improved electrochemical analysis of neuropathy target esterase activity by a tyrosinase carbon paste electrode modified by 1-methoxyphenazine methosulfate.

A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N = 2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r = 0.990, n = 7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.

Bioelectrochemical analysis of neuropathy target esterase activity in blood.

Bioelectrochemical analysis of neuropathy target esterase (NTE) and its inhibitors is based on the combination of the NTE-catalyzed hydrolysis of phenyl valerate and phenol detection by a tyrosinase carbon-paste electrode. The use of the tyrosinase electrode improves 10-fold the sensitivity of NTE detection in comparison with a spectrophotometric method. The tyrosinase electrode was found to be suitable for measurements in whole human blood where spectrophotometric detection is considerably restricted. The specificity of NTE in blood for mipafox and di-2-propyl phosphorofluoridate was close to that for neuronal NTE. The NTE-like activity in blood was determined to be 0.19 +/- 0.02 nmol/min/mg of protein.

Structural and functional properties of Langmuir films of antibodies based on amphiphilic polyelectrolytes.

We optimized the procedure for the formation of Langmuir films of antibodies based on amphiphilic polyelectrolytes and studied the physicochemical and immunochemical properties of the films obtained. Their immunochemical properties were compared with the immunochemical activity of antibodies in Langmuir films without amphiphilic polyelectrolytes and with antibodies adsorbed on the surface of polystyrene and graphite. The efficiency of immune adsorption by the films based on amphiphilic polyelectrolytes was shown to be greater; the affinity of antibodies and surface concentration of their active conformation depended on the type of amphiphilic polyelectrolytes used to obtain the films. We investigated the structure of these films at the surface of highly oriented pyrolytic graphite using the method of atomic force microscopy. Changes in the structure of the films under study caused by the increase of surface pressure were demonstrated.

A new approach for determination of neuropathy target esterase activity.

Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.

[Potentiometric electrodes for determining choline, butyrylcholine and cholinesterase inhibitors]. / Potentsiometricheskie élektrody dlia opredeleniia kholina, butirilkholina i ingibitorov kholinésteraz.

Potentiometric choline electrodes were developed on the basis of the mediator-free bioelectrocatalysis. The electrodes made of a composite carbon-polymer material contain choline oxidase and peroxidase coimmobilized on the surface of the electrode. The rate of the potential increase was shown to be proportional to the choline concentration within a broad range of variation. Coupling of choline-sensitive electrodes with butyrylcholinesterase makes possible both the direct detection of butyrylcholine and analysis of butyrylcholinesterase inhibitors.

Kinetic behavior of a receptor-enzyme system: a model for drug addiction.

A theoretical kinetic model describing the behavior of a receptor-enzyme system during formation of drug addiction is considered in this article. The model assumes concomitant action of narcotic on at least two targets with opposite effects. Theoretical kinetic principles that the system must satisfy for the development of drug addiction are formulated. These kinetic principles are the slow inactivation of receptor-enzyme system and the divergence of characteristic times of dynamic concentration of product and enzyme.

STM studies of LB films of amphiphilic polyelectrolytes with antibodies and enzymes.

The structure of LB films of protein-polyelectrolyte complexes transferred onto the pyrographite surface was studied by STM. The images of the films obtained at various protein concentrations in the water subphase and different values of surface pressure were captured. The topology of the surface covered by one or three layers of the films studied was investigated. It is shown that at a protein concentration of 1 mg/ml in the water subphase, the films are composed of aggregated protein molecules, and their structure has an insular character. An increase in the number of transferred layers up to three results in a virtually complete covering of the surface. A decrease in the protein concentration in the water subphase to 1 microgram/ml enabled us to prepare films consisting of individual non-aggregated protein molecules.

Langmuir films from antibodies based on amphiphilic polyelectrolytes.

A technique for forming Langmuir films from antibodies based on an amphiphilic polyelectrolyte was developed. The physicochemical and immunochemical properties of the Langmuir films obtained were studied. The interaction of HBsAg with the films was found to be described by a model with one binding site, whereas that of HBsAg with antibodies adsorbed on a polystyrene plate, by a model with a positive cooperativity. The use of the novel Langmuir films from antibodies increases the sensitivity of the immunoenzyme assay.

[The use of atomic-force microscopy for the analysis of microbiological objects]. / Ispol'zovanie atomno-silovoi mikroskopii dlia analiza mikrobiologicheskikh ob''ektov.

Atomic force microscopy (AFM) was used for the analysis of rickettsiae and viruses. The specificity of interaction was evaluated on the basis of the adsorption of the analyzed antigen on the polymer-antibody film. The film was formed and transferred onto highly oriented pyrolytic graphite (HOPG) by the method of Langmuir-Schaefer with the use of amphiphilic polymers, alkylated polyethyleneimines. According to the data of AFM, polymer-antibody film was 10-30 nm thick and had ruptures, uneven surface. AFM images of Coxiella burnetii, rotavirus and Venezuelan equine encephalitis virus, immunoadsorbed on the antibody film, were obtained. C.burnetii, due to their size equal to (700-900) x (300-500) x Z = 200 nm, were clearly visible on the underlying surface and could be directly counted. Individual virus particles (60-80 nm) cold not be identified on such surface. To analyze such preparations, the program of image analysis was developed. The program classified the registered image with a certain standard. This program determined the presence of virus antigen on the underlying affinity surface with a high degree of precision.

Potentiometric biosensors for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis.

A potentiometric method for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis has been developed. The method includes coimmobilization of three enzymes, butyrylcholinesterase, choline oxidase and peroxidase, on composite carbon electrodes. Catalytic hydrolysis of butyrylcholine and subsequent catalytic oxidation of choline result in the formation of hydrogen peroxide leads to a shift in the electrode potential. The detection limit for trichlorfon analysis is 2 x 10(-13) M. Electrodes remain stable for at least 4 weeks when stored at 277 K.

Glucose potentiometric electrodes based on mediatorless bioelectrocatalysis. A new approach.

A potentiometric method has been developed for analysis of glucose on the basis of glucose oxidase and peroxidase co-immobilized on the surface of an electrode made of composite carbonic material. Catalytic oxidation of glucose results in the formation of hydrogen peroxide. Mediatorless peroxidase catalysis of electroreduction of hydrogen peroxide leads to a shift in the electrode potential. The rate of the increase of the electrode potential is proportional to glucose concentration and calibration curve is linear at 0.025-2.0 mM. Electrodes permit at least 100 measurements without any loss in the activity. Electrodes remain stable for 90 days when stored at 277 K.

Affinity biosensors based on preconcentration/voltammetric analysis. Detection of phenothiazine drugs at Langmuir-Blodgett films of tyrosine hydroxylase.

A new route for operating affinity biosensors based on the voltammetric monitoring of the accumulated guest (analyte) is described. High sensitivity and selectivity accrue from the coupling of the specific receptor binding process and the inherent sensitivity of the preconcentration/pulse-voltammetric scheme. The redox (measurement) process results in dissociation of the receptor-guest complex, thus allowing multiple analytical determinations. The receptor layer also serves as an effective barrier that excludes interfering species. The new concept of preconcentration/voltammetric affinity biosensors is illustrated in connection with the detection of phenothiazine drugs using Langmuir-Blodgett films of their receptor, the enzyme tyrosine hydroxylase. The effect of various experimental variables upon the sensor performance is described.

[Receptor binding oscillations]. / Kolebaniia retseptornogo sviazyvaniia.

A kinetic analysis of the delta-opiate receptor agonist, [3H] DADLE, binding to cell (NG108-15) suspensions has led to the discovery of a new periodic biological phenomenon, namely, receptor activity oscillations. The absence of oscillations for the antagonist binding to the receptors suggests that oscillations are generated only as a result of receptor signal transformation. The correlation between the quantitative characteristics of the kinetic curves and the experimental conditions points to the fact that receptor binding oscillations may be due either to the receptor binding to the G-protein or the interaction of the receptor (or G-protein) with microtubules.

Receptor binding on whole cells can oscillate.

The study of the kinetics of binding of the opiate receptor agonist [3H]DADLE with NG108-15 cell suspensions has revealed a new periodic biological phenomenon, i.e., oscillations of the cellular receptor activity. The absence of oscillations for binding of the receptor antagonist shows that oscillations occur as a result of the transformation of the receptor signal only.

The 65-kDa protein from pig heart. A new substrate for Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3).

In the pig heart sarcolemma, a 65 kDa protein is found to be ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3). ADP-ribosylation of this protein is regulated by guanyl nucleotides and cytosol factor in a fashion similar to that for other C3 substrates. The new exoenzyme C3 substrate was partially purified. This protein is supposed to be a GTP-binding one.

[Tyrosine hydroxylase activity in two-dimensional monomolecular films]. / Aktivnost' tirozingidroksilazy v dvukhmernykh monomolekuliarnykh plenkakh.

The activity of a neurospecific enzyme of tyrosine hydroxylase (TH) in monomolecular films formed onto solid surface was studied. The data obtained show that the formation of two-dimensional films onto negative-charge surfaces by the Langmuir-Schafer technology does not lead to the inactivation of the enzyme. Neuroleptic trifluoperazine increased the activity of TH. Monomolecular films of TH may be used as a sensitive element of biosensors for primary monitoring of neuroleptic-like compounds.