Increasing the Efficiency of Cytochrome P450 3A4 Electrocatalysis Using Electrode Modification with Spatially Ordered Anodic Aluminum Oxide-Based Nanostructures for Investigation of Metabolic Transformations of Drugs.

A new approach for modifying electrodes using porous membranes based on anodic aluminum oxide with pore diameters of 0.1 and 0.2 µm and a membrane-like substance didodecyldimethylammonium bromide (DDAB) was proposed to study the electrocatalytic efficiency of the system. This approach makes it possible to increase the catalytic efficiency of the cytochrome P450 3A4-dependent N-demethylation of erythromycin by 132% when using a membrane with pore diameter of 0.1 µm and by 32% when using a membrane with 0.2 µm pore size. Electrode modification using porous membranes shifted the potential of electrochemical reduction and catalysis of cytochrome P450 3A4 in positive direction by 0.070-0.050 V, which indicates a thermodynamically more favorable process of electron transfer and enzymatic electrocatalysis.

Comparative Analysis of Blood Plasma Proteome in Patients with Renal Cell Carcinoma.

Comparative mass spectrometric analysis of protein composition was carried out in 36 blood plasma specimens from patients with renal cell carcinoma and 20 specimens from donors. Analysis of protein composition of plasma specimens devoid of the major protein fractions showed a 20-50% higher level of protein identifications in patient' specimens. Specimens of the control and experimental series were similar by protein composition, 70-80% identifications in experimental and control series coinciding. High similarity of biological processes with participation of the proteins identified in both series was observed. The greater part of proteins in both series were located extracellularly and were exosomal (specimens from renal cancer patients) or vesicular (specimens from healthy volunteers).

A New Method for Quantitative Determination of Steroid Metabolites of Cytochrome P450-Dependent Reactions Using Fluorescent Spectroscopy.

A method for determination of hydroxylase activity of cytochrome P450 3A4 (CYP3A4) towards its substrate hydrocortisone using fluorescent analysis of the product was developed. 6ß-hydroxycortisol, formed during CYP3A4-dependent electrocatalysis, has a characteristic fluorescent peak at λ = 427 ± 2 nm after treating with the sulfuric acid : ethanol (3 : 1) mixture and excitation at λ = 365 nm, which is different from the substrate (hydrocortisone) fluorescence (λ = 525 ± 2 nm). The limit of detection of 6ß-hydroxycortisol was 0.32 µM. The developed analytical approach was used to determine the kinetic parameters of CYP3A4-dependent hydrocortisone hydroxylation.

[Enzymatic synthesis of high-modified DNA].

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

Quantitative target proteomics of chromosome 13 human blood plasma proteins.

Quantitative proteomic analysis of 50 blood plasma samples of healthy volunteers who underwent a comprehensive medical examination and were found eligible for space flights was performed. As a result of directed mass spectrometric analysis, signals for 128 proteins, which accounted for nearly 40% of the total number of chromosome 13 gene products, were detected. The analysis of interindividual variation of concentrations of chromosome 13 proteins showed the presence of a pool comprising 41 proteins with a low variation (CV < 30%), which can potentially be used as biomarkers.

Electroanalysis of myoglobin based on electropolymerized molecularly imprinted polymer poly-o-phenylenediamine and carbon nanotubes/screen printed electrode.

Electroanalysis of myoglobin as a marker of acute myocardial infarction by means of screenprinted electrodes modified with multiwalled carbon nanotubes and polymeric artificial antibodies is developed. Plastic antibodies to myoglobin (molecularly imprinted polymers, MIPs) based on o-phenylenediamine were produced by electropolymerization. Molecular imprinting technology in biosensor analysis was used as alternative to natural receptors (namely, antibodies) and demonstrated high sensitivity (1.5 × 10(-2) A/nmol of myoglobin) and selectivity.

[Postgenomic Medicine: Alternative to Biomarkers].

In the article relevance of high-performance postgenomic technologies is tackled. The intrinsic problems of the implementation of genomics, proteomics and metabolomics in routine clinical practice are considered. Further development of postgenomic medicine requires severe change in the current research approaches. Avenue for development of such approaches are illustrated by metabolome research of human blood plasma. The postgenomic biomarkers are pictured as molecular iceberg, greater part of which is inaccessible for detection with measurement methods. Due to diversity of protein forms the spectrum of molecular markers will always evaluate in terms of incompleteness and inconsistence regardless of technological development level. These properties of «big data¼ are typical of data intensive domains. Special computational methods are essential for data intensive analytics and hardly suitable for the evidence-based medicine.

[Registration of the protein in the serum with a field-effect nanotransistor biosensor].

A method for detection of cancer-associated protein D-NFATc1 in serum using nanowire (NW) biosensor based on field-effect nanotransistor is developed. Field-effect nanotransistor was fabricated on the basis of «silicon-on-insulator¼ structures. For the biospecific detection of target protein, the NW surface was modified with aptamers against the target protein. Using the 3 um-NW enabled to obtain stable source-drain characteristics and to register D-NFATc1 in serum at concentration of 2.5 x 1014 M in the mode of drain-source current vs. gate voltage characteristics measurements. Data collection in the mode of drain-source current vs. gate voltage characteristics measurements was carried out with the use of high-speed data collection system running TURBO NBS software.

Taurine modulates catalytic activity of cytochrome P450 3A4.

The influence of the biologically active compound taurine on the stability and catalytic properties of the hemoprotein cytochrome P450 3A4 has been investigated. The catalytic properties were analyzed by electrochemical methods (cyclic and square-wave voltammetry) using cytochrome P450 3A4 immobilized on the electrode. Taurine at concentrations in the range 10-70 µM stimulated the electrochemical reduction of cytochrome P450 3A4, and the reduction was the highest (115 ± 3%) in the presence of 50 µM taurine. Taurine pronouncedly attenuated the itraconazol-caused inhibition of the P450 isoenzyme P450 3A4. Taurine protected cytochrome P450 3A4 due to stabilizing it during electrolysis at controlled voltage in the presence of erythromycin as a substrate. This protection was manifested by an increase in the amount of the "residual" reduced form of the hemoprotein (52 ± 5 and 71 ± 8%, respectively).

[Atomic force microscopy monitoring of temperature dependence of cytochrome BM3 oligomeric state].

The change in temperature is one of the factors affecting the activity of enzymes. In this work thermal denaturation and aggregation of cytochrome P450 BM3 were studied by atomic force microscopy. To determine specific temperature transitions the fluorescence analysis was used. In the low melting temperature range, 10-33 degrees C, a decrease in the fluorescence intensity of aromatic residues was observed with an increase in the fluorescence intensity of flavin groups. Protein melting in this range indicated three narrow S-shaped cooperative transitions at temperatures 16, 22 and 29 degrees C. Atomic force microscopy analysis in this temperature range showed that the shape of BM3 molecules remained globular in the form of compact objects (heights h < 7 nm, lateral dimensions d < 50 nm), but protein oligomeric state changed. The first two transitions were accompanied by a decrease in the degree of oligomerization and the third one was accompanied by its increase.

Electrosynthesis and binding properties of molecularly imprinted poly-o-phenylenediamine as artificial antibodies for electroanalysis of myoglobin.

Molecularly imprinted poly-o-phenylenediamine with template myoglobin molecules (i.e., polymeric antibodies to myoglobin, molecularly imprinted polymer, MIP) was synthesized via electropolymerization. Electropolymerization, washing, and the interaction of the polymeric antibodies with myoglobin was examined by square wave voltammetry and microgravimetry. The analysis of myoglobin was carried out through direct electrochemical detection of the reduction peak of Fe(3+) of the hemeprotein on screen-printed graphite electrodes modified by the MIP. According to the electrochemical analysis, MIP surfaces demonstrated remarkably higher ability to bind the protein compared to that of surfaces prepared by the same route under the same conditions but in the absence of myoglobin (surfaces of the non-imprinted polymer, NIP). The imprinting factor I max(MIP)/I max(NIP) was found to be 2-4. The equilibrium dissociation constant K d of the interaction of myoglobin with MIP electrodes was evaluated as (2.4 ± 0.5) × 10(-8) M. The lower detection limit of myoglobin by a MIP sensor was determined as 0.5 × 10(-9) M, the range of detectable concentrations being 10(-9)-10(-5) M.

Electrochemical profiling of poliovirus particles inactivated by chemical method and ionizing radiation.

Electrochemical profiling of formaldehyde-inactivated poliovirus particles demonstrated a relationship between the D-antigen concentration and the intensity of the maximum amplitude currents of the poliovirus samples. The resultant signal was therefore identified as electrochemical oxidation of the surface proteins of the poliovirus. Using registration of electrooxidation of amino acid residues of the capsid proteins, a comparative electrochemical analysis of poliovirus particles inactivated by electrons accelerated with doses of 5 kGy, 10 kGy, 15 kGy, 25 kGy, 30 kGy at room temperature was carried out. An increase in the radiation dose was accompanied by an increase in electrooxidation signals. A significant increase in the signals of electrooxidation of poliovirus capsid proteins was detected upon irradiation at doses of 15-30 kGy. The data obtained suggest that the change in the profile and increase in the electrooxidation signals of poliovirus capsid proteins are associated with an increase in the degree of structural reorganization of surface proteins and insufficient preservation of the D-antigen under these conditions of poliovirus inactivation.

[Electrochemical measurement of intraprorein and interprotein electron transfer].

Intramolecular and intermolecular direct (unmediated) electron transfer was studied by means of electrochemical techniques in flavohemoprotein cytochrome P450 BM3 (CYP102A1 from Bacillius megaterium) and between cytochrome b5 and cytochrome c. Flavohemoprotein cytochrome P450 BM3 was immobilized on a screen printed graphite electrode, modified with a biocompatible nanocomposite material based on the didodecyldimethylammonium bromide DDAB and gold nanoparticles. Analytical characterictics of DDAB/Au/P450 BM3 electrodes were studied with cyclic voltammetry and square wave voltammetry. It was shown that intramolecular electron transfer was realized between diflavin (FAD/FMN) and heme domain of CYP102A1. An electron transport chain of flavohemoprotein P450 BM3 immobilized at nanostructued electrode is realized as: electrode --> FAD --> FMN --> heme. Electron transfer occurs inside the protein, and it is an evidence of functional interaction between diflavin and heme domains. The effect of a substrate (lauric acid) or inhibitors (metyrapone or imidazole) binding on the electrochemical parameters of flavohemoprotein P450 BM3 was also studied. Interprotein electron transfer was analyzed between cytochrome b5 and cytochrome c. Electrochemical analysis revealed that electron transfer takes place in protein-protein complexes with participants possessing different redox potentials.

[International criteria of the research activity of groups and individual scientists in biology and medicine].

Comparison data on results of efficiency of the research institutes (RI) of RAMS by international and own evaluation criteria of the research activity of above institutions in 2011 are presented. The consistency of international and own evaluation results was observed in 21RI (39%), the institutions-leaders found, and in 13 RI (24%) - less effective institutions. Thus, the use of own evaluation criteria only complicates the unbiased assessment of the Russia's position in the international scientific rating.

Perspectives for the creation of a new type of vaccine preparations based on pseudovirus particles using polio vaccine as an example.

Traditional antiviral vaccines are currently created by inactivating the virus chemically, most often using formaldehyde or ß-propiolactone. These approaches are not optimal since they negatively affect the safety of the antigenic determinants of the inactivated particles and require additional purification stages. The most promising platforms for creating vaccines are based on pseudoviruses, i.e., viruses that have completely preserved the outer shell (capsid), while losing the ability to reproduce owing to the destruction of the genome. The irradiation of viruses with electron beam is the optimal way to create pseudoviral particles. In this review, with the example of the poliovirus, the main algorithms that can be applied to characterize pseudoviral particles functionally and structurally in the process of creating a vaccine preparation are presented. These algorithms are, namely, the analysis of the degree of genome destruction and coimmunogenicity. The structure of the poliovirus and methods of its inactivation are considered. Methods for assessing residual infectivity and immunogenicity are proposed for the functional characterization of pseudoviruses. Genome integrity analysis approaches, atomic force and electron microscopy, surface plasmon resonance, and bioelectrochemical methods are crucial to structural characterization of the pseudovirus particles.

Application of AFM and optical biosensor for investigation of complexes formed in P450-containing monooxygenase systems.

Atomic force microscopy (AFM) allows to visualize and count the individual protein molecules and their complexes within multiprotein systems. On the other hand, optical biosensor (OB) provides information on complex formation kinetics as well as complex lifetime (τ(LT)) and affinity. Comparison of complex lifetime τ(LT) with the time required for enzyme's catalytic cycle (τ(cat)) enables to characterize productive complexes and distinguish them from non-productive ones. Both these approaches were applied for the analysis of the three cytochrome P450-containing monooxygenase systems: cytochrome P450 101, cytochrome P450 11A1 and cytochrome P450 2B4. By using AFM, the formation of binary and ternary protein complexes was registered in all the three systems. OB analysis enabled to kinetically characterize these binary and ternary complexes. It was shown that the binary complexes putidaredoxin reductase (PdR)/putidaredoxin (Pd) and Pd/cytochrome P450 101 (P450 101) formed within the P450 101 system and, also, the binary complexes adrenodoxin reductase (AdR)/adrenodoxin (Ad) and Ad/cytochrome P450 11A1 (P450 11A1) formed within the P450 11A1 system are non-productive (deadlock). At the same time, the ternary PdR/Pd/P450 101 and AdR/Ad/P450 11A1 complexes proved to be productive. The binary cytochrome P450 reductase (Fp)/cytochrome P450 2B4 (2B4) complexes and the ternary Fp/2B4/cytochrome b5 (b5) complexes formed within P450 2B4 system were productive.

Effect of affinity sorbent on proteomic profiling of isatin-binding proteins of mouse brain.

Use of small molecules for isolation of particular sub-proteomes is often complicated by the need for chemical modification of a parent compound for affinity sorbent preparation. Isatin (indoledione-2,3) is an endogenous indole that exhibits a wide spectrum of biological activities. Using 5-aminocaproylisatin for proteomic profiling of fractionated rodent brain homogenates, we previously identified more than sixty individual proteins. However, proteins tested in an optical biosensor study for validation of their isatin-binding capacity demonstrated different affinity for immobilized 5-aminocaproylisatin and 5-aminoisatin. In this study, we comparatively evaluated proteomic profiles of isatin-binding proteins separated using both isatin analogs as the affinity ligands. The total number of identified proteins was higher with the shorter isatin analog (88 versus 66), and only 22 proteins were identical in the two proteomic profiles. Thus, proteomic profiling of brain isatin-binding proteins is significantly influenced by the length of the spacer between the amino group used for affinity ligand coupling to Sepharose and the isatin moiety. This suggests that the actual number of brain proteins interacting with endogenous (unmodified) isatin still remains underestimated due to different affinity of proteins for the isatin analogs used for the affinity-based proteomic profiling.

Analysis of polymorphisms in T(H)2-associated genes in Russian patients with atopic bronchial asthma.

BACKGROUND: Bronchial asthma is a chronic respiratory disorder characterized by airway inflammation, airway hyperresponsiveness, and periodic reversible airway obstruction. Subtype 2 helperT cell (T(H)2) cytokines play an important role in the development of allergic airway inflammation in patients with bronchial asthma. OBJECTIVE: To investigate whether the single-nucleotide polymorphisms (SNPs) Ile75Val and Gln576Arg in the IL4RA gene, -33C>T in the IL4 gene, and Gly237Glu in the FCER1B gene contribute to the development and severity of atopic bronchial asthma in Russian patients from Moscow. METHODS: We analyzed DNA samples from 224 patients with atopic bronchial asthma and 172 healthy individuals. Genotyping was performed by primer extension followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: We observed a moderate association between the Arg/Arg genotype of Gln576Arg and protection against asthma (odds ratio [OR], 0.16; P < .012) and a strong association between the T allele and TT genotype of -33C> and atopic bronchial asthma (OR, 1.91 and 4.65, respectively; P < .0001). Carriers of the C allele had a reduced risk of asthma (OR, 0.53; P < .0001). Furthermore, we found that the TT genotype of -33C>T correlated with higher concentrations of total serum immunoglobulin E and interleukin 4 than the CC and CT genotypes. CONCLUSION: We found an association between atopic bronchial asthma and the SNPs Gln576Arg in IL4RA and -33C>T in IL4. IL4RA and IL4 seem to be involved in the pathogenesis of asthma.

['Translational medicine as a way from fundamental biomedical science to public health services].

Increasing distance between practical public health services and collecting of theoretical information in the field of biomedical researches reflects the necessity of professional contact between clinicians and scientists in many areas associated with medicine for active carrying over ("translation") of the modern basic researches in which mechanisms of basic metabolic processes and possibilities of their correction are detected, to effective medical help to individual patient, i.e., personified medicine. Such approach was called transmitting medicine. Examples of the personified medicine in which biomedical researches together with the anamnesis morbi of individual patient that are responsible for treatment strategy including doses and regimens are discussed.

[Personalized medicine: state-of-the-art and prospects].

A review describes general trends and directions of personalized medicine. These include, but not limited to, prediction of disease based on genomic data, diagnostics and therapy monitoring using genomic and postgenomic technologies as well as therapeutic drug monitoring and regenerative cell technologies. The personalized medicine is considered in terms of results of the Human Genome Project and succeeding Human Proteome Project. An importance of personalized approach for modern medicine is emphasized.