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).

[Monitoring of brightness temperature fluctuation of water in SHF range].

The purpose of the research consisted in detection of fluctuation of brightness temperature (TSHF) of water in the area of the temperature Т = 42°Ð¡ (that is critical for human) during its evaporation by SHF radiometry. Methods: Monitoring of the changes in brightness temperature of water in superhigh frequency (SHF) range (3.8-4.2 GHz) near the phase transition temperature of water Т = 42°Ð¡ during its evaporation in the cone dielectric cell. The brightness temperature measurements were carried out using radiometer. Results: Fluctuation with maximum of brightness temperature was detected in 3.8-4.2 GHz frequency range near at the temperature of water Т = 42°Ð¡. It was characteristic for these TSHF fluctuations that brightness temperature rise time in this range of frequencies in ~4°Ð¡ temperature range with 0.05-15°Ð¡/min gradient and a sharp decrease during 10 s connected with measuring vapor conditions. Then nonintensive fluctuation series was observed. At that, the environment temperature remained constant. Conclusion: The significant increasing in brightness temperature of water during its evaporation in SHF range near the temperature of Т ~42°Ð¡ were detected. It was shown that for water, ТSHF pull with the amplitude DТSHF ~4°C are observed. At the same time, thermodynamic temperature virtually does not change. The observed effects can be used in the development of the systems for diadnostics of pathologies in human and analytical system.

[AFM fishing of proteins under impulse electric field]. / ASM-fishing belka v impul'snom élektricheskom pole.

A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.

Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen.

A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

Detection of microwave radiation of cytochrome CYP102 A1 solution during the enzyme reaction.

Microwave radiation at 3.4-4.2 GHz frequency of the cytochrome P450 CYP102 A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10-8 and 10-9 Ðœ. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment.

[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.

SHF radiation from albumin solution upon external excitation.

The purpose of the research consisted in the measurement of nonequilibrium radiation in superhigh frequency (SHF) range from aqueous solution of albumin upon its mechanical stimulation. Methods: the monitoring of change in the ratio between brightness temperatures TSHF and TIR values after the mechanical stimulation of aqueous solution of albumin in the measuring cell at 35-39°Ð¡. The measurements of brightness temperatures were carried out with use of radiothermometer. SHF frequency range corresponded to 3.4-4.2 GHz, SHF frequency range corresponded to 8-13 mm. Results: It was found that mechanical stimulation of aqueous solution of albumin at the temperature ~39°Ð¡, the change in the ratio between TSHF and TIR occurs. This corresponds to emergence of nonequilibrium SHF radiation from the solution near the phase transition with ~39°Ð¡ temperature. Conclusion: The effect of emergence of nonequilibrium SHF radiation from protein solution near its temperature 39°Ð¡ was found. This temperature corresponds to the temperature of human organism upon a number of pathological states connected with inflammatory processes. The discovered effect can be used in the development of novel non-invasive methods of disease diagnostics.

Monitoring of brightness temperature of suspension of follicular thyroid carcinoma cells in SHF range by radiothermometry.

The purpose: The purpose of this research consisted in monitoring of brightness temperature of the suspension of follicular thyroid carcinoma cells during the necrosis of these cells in superhigh frequency (SHF) range. Methods: The monitoring of the change in the ratio between brightness temperatures TSHF and TIR values during the necrosis of these cells. The object of study was follicular thyroid carcinoma cells suspension prepared with use of Versene solution and 0.25% trypsin solution. The cells were precipitated by centrifugation and re-suspended in culture medium. The measurements of brightness temperatures were carried out with use of radiothermoimeter. SHF range was 3.4-4.2 GHz, and infrared (IR) range was 8-13 mm. The temperature of the suspension was maintained at 37.5°Ð¡. Results: It was found that upon the necrosis in the suspension of cells, an increase in brightness temperature in 3.4-4.2 GHz range (SHF range) occurs, while brightness temperature of the medium in the IR range does not change. Conclusion: The monitoring of necrosis of follicular thyroid carcinoma cells was carried out by SHF-radiothermometry. It was shown that during the necrosis the change of non-equilibrium state of cell medium occurs, that results in the change in the ratio between TSHF and TIR. During the necrosis, the brightness temperature in SHF range (TSHF) increases.

Monitoring of microwave emission of HRP system during the enzyme functioning.

Monitoring of microwave emission from aqueous solution of horseradish peroxidase (HRP) in the process of the enzyme functioning was carried out. For the monitoring, a system containing HRP, luminol and Н2О2 was employed. Microwave emission measurements were carried out in the 3.4-4.2 GHz frequency range using the active and passive modes (active-mode and passive-mode measurements). In the active mode, excitation of the solution in the pulsed electromagnetic field was accomplished. In the passive mode, no excitation was induced. It appears that the passive-mode measurements taken in the course of the peroxidase reaction in the enzyme system have shown a 0.5 °Ð¡ increase of the microwave signal. Upon the active-mode measurements, taken in the same reaction conditions, the forced excitation of the solution has also led to the increase (by 2 °Ð¡) of the level of the microwave signal - i.e. to its 4-fold enhancement compared to the signal obtained in passive-mode measurements.

[SOI-nanowire biosensor for the detection of D-NFAT 1 protein].

The nanowire (NW) detection is one of fast-acting and high-sensitive methods allowing to reveal potentially relevant protein molecules. A NW biosensor based on the silicon-on-insulator (SOI)-structures was used for biospecific label-free detection of NFAT 1 (D-NFAT 1) oncomarker in real time. For this purpose, SOI-nanowires (NWs) were modified with aptamers against NFAT 1 used as molecular probes. It was shown that using this biosensor it is possible to reach the sensitivity of ~10(-15) M. This sensitivity was comparable with that of the NW biosensor with immobilized antibodies used as macromolecular probes. The results demonstrate promising approaches used to form the sensor elements for high-sensitive disease diagnostics.

[Atomic force microscopy fishing of gp120 on immobilized aptamer and its mass spectrometry identification].

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

[AFM-based technologies as the way towards the reverse Avogadro number].

Achievement of the concentration detection limit for proteins at the level of the reverse Avogadro number determines the modern development of proteomics. In this review, the possibility of approximating the reverse Avogadro number by using nanotechnological methods (AFM-based fishing with mechanical and electrical stimulation, nanowire detectors, and other methods) are discussed. The ability of AFM to detect, count, visualize and characterize physico-chemical properties of proteins at concentrations up to 10(-17)-10(-18) M is demonstrated. The combination of AFM-fishing with mass-spectrometry allows the identification of proteins not only in pure solutions, but also in multi-component biological fluids (serum). The possibilities to improve the biospecific fishing efficiency by use of SOMAmers in both AFM and nanowire systems are discussed. The paper also provides criteria for evaluation of the sensitivity of fishing-based detection systems. The fishing efficiency depending on the detection system parameters is estimated. The practical implementation of protein fishing depending on the ratio of the sample solution volume and the surface of the detection system is discussed. The advantages and disadvantages of today's promising nanotechnological protein detection methods implemented on the basis of these schemes.

[Microwave emission from water in bioanalytical systems].

Mechanical stimulation of aqueous protein solution was found to be accompanied by electromagnetic radiation in the superhigh frequency range. This radiation was observed at solution temperatures near the phase transitions of water. This effect may occur in various bioanalytical systems, associated with injection of aqueous solutions in these systems, and may have impact on the results of bioalytical measurements. The discovered effect of generation of nonequilibrium microwave emission of water medium (which is the main component of the organism) in the 38-39°C temperature range, i.e. in the range of elevated temperature of the organism (which accompanies pathological condition in humans upon inflammatory diseases, i.e., infactious etc.) may be used for development of new non-invasive methods of disease diagnostics.

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.

Analytical nanobiotechnology for medicine diagnostics.

The review is concerned with the state-of-the-art and the prospects of development of nanotechnologies in clinical proteomics. Nanotechnology in clinical proteomics is a new medical research direction, dealing with the creation and application of nanodevices for performing proteomic analyses in the clinic. Nanotechnological progress in the field of atomic force microscopy makes it possible to perform clinical studies on the revelation, visualization and identification of protein disease markers, in particular of those with the sensitivity of 10(-17) M that surpasses by several orders the sensitivity of commonly adopted clinical methods. At the same time, implementation of nanotechnological approaches into diagnostics allows for the creation of new diagnostic systems based on the optical, electro-optical, electromechanical and electrochemical nanosensoric elements with high operating speed. The application of nanotechnological approaches to creating nanopore-based devices for express sequencing of the genome is discussed.

The use of the resonant mirror biosensor to detect point mutations, as demonstrated with synthetic oligonucleotides.

The possibility of using the resonant mirror biosensor to detect point substitutions in oligonucleotides was demonstrated with a fragment of the Helicobacter pylori 23S rRNA gene, point mutations in which are responsible for clarythromycin resistance. Conditions were optimized for the interaction of a probe immobilized on the sensing surface with targets containing various nucleotide substitutions. A probe allowing reliable discrimination of mutant targets was selected. The mismatch position in the probe was shown to affect the kinetic parameters (response) of hybridization with mutant targets, reporting not only the position, but also the character (G or C) of a substitution.

Protein-protein interactions: mechanisms and modification by drugs.

Protein-protein interactions form the proteinaceous network, which plays a central role in numerous processes in the cell. This review highlights the main structures, properties of contact surfaces, and forces involved in protein-protein interactions. The properties of protein contact surfaces depend on their functions. The characteristics of contact surfaces of short-lived protein complexes share some similarities with the active sites of enzymes. The contact surfaces of permanent complexes resemble domain contacts or the protein core. It is reasonable to consider protein-protein complex formation as a continuation of protein folding. The contact surfaces of the protein complexes have unique structure and properties, so they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations have been undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or on the other hand, induce protein dimerization.