Biomedical subjects

A I Archakov

Publications and source records attributed to A I Archakov.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of human liver cytochromes P450 by combining the biochemical and proteomic approaches.

Highly purified human liver microsomes were processed by a combination of the biochemical and proteomic methods. Microsomes were purified from the morphologically normal liver tissue obtained from the resected and discarded masses of surrounding liver upon surgical treatment for hemangioma (control) or hepatic metastases arising from colon cancer (pathology). Proteins of each sample were separated by two-dimensional (2-DE) and one-dimensional electrophoresis (1-DE); selected gel regions were excised, in-gel digested and analyzed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Analysis of collected fingerprints has revealed a total of 13 microsomal membrane proteins involved in the biotransformation of xenobiotics. These were disulfide isomerase, flavine monooxygenase, NADPH-cytochrome P450 reductase and 10 cytochrome P450 forms, namely: CYPs 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. These same samples were characterized by the enzymatic assays using the marker substrates for CYPs 1A, 2B, 3A4, 2C and 2E1. Correlations between mass spectrometric data and enzymatic activities were investigated to demonstrate the manner in which the functional and structural aspects of proteomics meet each other in the field of cytochromes P450.

Cytochrome P-450 Enzyme System↗

PubMed2006

Two-dimensional electrophoretic proteome study of serum thermostable fraction from patients with various tumor conditions.

One of the problems of plasma proteomics is a presence of large major components. In this work, we use the thermostable fraction as a way to deplete these major proteins. The thermostable fraction of serum samples from patients with ovarian, uterus, and breast cancers and benign ovarian tumor was analyzed using two-dimensional electrophoresis combined with MALDI-TOF(-TOF)-mass spectrometry. Of them, alpha-1-acid glycoprotein and clusterin are expressly down-regulated in breast cancer, whereas transthyretin is decreased specifically in ovarian cancer. Apolipoprotein A-I forms have decreased spot volumes, while haptoglobin alpha1, in contrast, is elevated in several tumors. These data are partly consistent with previous art studies on cancer proteomics, which involve mass-spectrometry-based serum profiling techniques. Serum thermostable fraction may be recommended as a good tool for medium and small protein proteome investigation, in particular, by 2D-electrophoresis.

Adult↗

PubMed2005

Proteomic and biochemical analysis of the mouse liver microsomes.

The efficiency of the proteomic approach for the revelation of proteins, including components of the liver microsomal monooxygenase system (cytochromes b5 and P450) was demonstrated. The liver microsomes and their ghosts (i.e. membranes devoid of "ballast" proteins) were prepared from the control and phenobarbital-treated mice. Microsomes and their ghosts were characterized using the conventional biochemical assay and analysed by one- and two-dimensional electrophoresis (1-DE and 2-DE, respectively) coupled with MALDI-TOF peptide mass fingerprinting procedure. Catalytic activity of cytochromes P450 was measured using specific fluorogenic substrates for CYP1A, CYP2A, CYP2B and CYP2C families. The protein composition of control and phenobarbital-induced ghosts was analysed. The proteomic 2D-based protein separation method enabled us to reveal up to 1005 proteins, the majority of them being soluble. Among the 34 identified proteins, the cytochrome b5-like protein was revealed; however, cytochromes P450 appeared to be undetectable under 2-DE separation conditions. The separation of microsomal ghosts proteins by 1-DE, followed by mass-spectrometric analysis of bands from the 45 to 66 kDa gel range made it possible to identify hydrophobic proteins including cytochromes P450 (CYP2A4 and CYP2A5) and dimethylaniline monooxygenase. The high O-deethylation rate of 7-ethoxycoumarin-a substrate for rodent CYPs 2A and 2B, in particular for CYP2A5-was observed, in agreement with the results of mass-spectrometric identification. Collectively, the data obtained indicate that a combination of enzyme activity assays and various protein separation techniques coupled with mass-spectrometric protein identification allows a more comprehensive insight into the machinery of the cellular detoxifying system.

Animals↗

PubMed2005

Identification of glycosaminoglycan-binding sites within hepatitis C virus envelope glycoprotein E2*.

Heparan sulphate is one of the candidate receptors for hepatitis C virus (HCV). Envelope glycoproteins of HCV have been proposed to be responsible for recognition and binding with cell receptors. They are characterized by great genetic polymorphism. In this study the mapping of regions with glycosaminoglycan-binding properties within HCV envelope proteins has been undertaken. We prepared a set of overlapping peptides corresponding to conserved regions of these envelope proteins and analysed them by solid phase heparin-binding assay. The search for established glycosaminoglycan-binding motifs in the HCV envelope proteins showed the absence of the sites corresponding to the glycosaminoglycan-binding patterns in consensus sequence. We identified one highly conserved and two less conserved heparin-binding sequences within the envelope protein E2 based on solid phase assay results. We did not find any differences in binding efficiency of these peptides with heparin, heparan sulphate or dextran sulphate. Our data supported the specific association between HCV envelope protein E2 and cell surface glycosaminoglycans. We hypothesize that identified regions from E2 can contribute to HCV binding to cell surface glycosaminoglycans.

Amino Acid Sequence↗

PubMed2005

[Methods of experimental validation of potential target proteins for creation of new drugs].

Review is devoted to the description of the main existing and developing technologies for experimental validation of potential targets predicted in silico by comparative genomics and bioinformatics methods. Since this problem has not been solved yet, the description of a wide set of methods, suitable for the validation of potential targets, is given. The following questions have been considered: (1) applications of proteomic technologies (control of potential targets expression and their variability, analysis of protein-protein interactions); (2) use of genomics technologies in experimental validation of targets (inactivation of the target genes, suppression of transcription, inactivation of the target mRNA, suppression of translation); (3) methods of direct inactivation of target proteins (monoclonal antibodies, light-inactivation, one-chain antibodies, intrabodies, aptamers); (4) high-throughput technologies; (5) targets validations in vivo.

Animals↗

PubMed2005

[Using the mass spectrometry analysis for hepatitis C virus typing].

Determination of the hepatitis C virus (HCV) genotype has become the standard procedure in laboratory diagnostics of HCV infection. Genotype elucidation has prognostic value assignment helps in assessing disease prognosis and promotes establishing appropriate duration of treatment. Now 11 major genotypes and more than 70 subtypes of HCV have been identified using the sequence variability within 5' non-coding region (5' NCR). In Russia the most common subtypes are 1a, 1b, 2a, 3a and more rare - 4 and 5 types. While the "gold standard" for testing is nucleic acid sequencing, a variety of other assays, including the line probe assay or type-specific amplification, has been developed to provide more rapid and cheaper forms of testing. The aim of this study was to determine the type-specific single nucleotide polymorphism (SNP) in 5' NCR HCV by the classical three-step minisequencing method with followed MALDI-TOF mass spectrometry detection The fragments of 5'NCR of HCV genomes were amplified by the nested RT PCR. The removal of excess nucleotides and primers was performed. Three oligonucleotide primers were design to detect two sets of type-specific SNP in 5' NCR HCV. The primer extension reaction was performed using modified thermostable DNA polymerase and in the presence of ddNTP. The molecular weights of primers extension reaction products were analyzed using MALDI-TOF mass spectrometry. The HCV genotype was determined according the presence in analyses sample the molecules with expected molecular weights. The suggested method was used to type HCV from 69 HCV-positive sera. The 1a genotype was determine in 4.5% samples, 1b - 48%, 2a - 4.5% 3a - 29%, 4 - 1.5%. The mixes of two genotypes were found in 13% samples. All data confirmed by direct nucleic acid sequence. Thus, the new method for HCV typing has been developed using the minisequencing reaction and mass spectrometry for the determination of nucleic acid molecular weight.

DNA Fingerprinting↗

PubMed2004

Comparative analysis of different typing methods for Helicobacter pylori clinical isolates.

The goal of the present work was to compare different techniques of molecular typing using as an example clinical isolates of Helicobacter pylori obtained from patients in different regions of Russia. DNA-macroarray genome scanning using individual genes was employed to set up our basic classification of isolates that did or did not contain pathogenicity islands. In parallel, DNA of the same isolates was used in the conventional random amplified polymorphic DNA (RAPD) PCR analysis, and the isolates were also genotyped (cagA, vacA, iceA, and babA status) and their proteomic maps were obtained by means of unidimensional SDS polyacrylamide gel electrophoresis (1D-SDS-PAGE) coupled with identification using peptide mass fingerprinting by MALDI-TOF mass spectrometry. A statistically significant correlation (coefficient of correlation r = 0.25, p = 0.005) was observed between the results of genome scanning and 1D-SDS-PAGE. No correlation was found between RAPD-PCR typing and genome scanning.

Bacterial Typing Techniques↗

PubMed2004

[Structural-functional motifs of sterol 14-alpha demethylases (CYP51)].

CYP51 family of cytochromes P450 (sterol 14-alpha-demethylases) comprises the representatives from different kingdoms of living world, thus positioning itself as the most ancient member of the superfamily. In the course of the present research the collection of 36 full-length CYP51 amino acid sequences was submitted to cluster analysis. Each node of the clustering dendrogram corresponds to the groups of proteins, located on the branches descending from the node. By making the multiple alignment of each group of protein sequences we obtained the node-specific consensus sequences. The informational content of the consensus was defined as the presence of the compact conserved sites, the motifs. The assessment of informational content was computed using Sherman's non-parametric statistical criterion. The high informational content was observed for the 100% conserved consensus sequences of the following CYP51,s groups: fungi, animal+plant, plant+protista and bacteria. These selected consensus sequences were next aligned all together to get the final consensus for the whole family. To enrich the informational content of the CYP51 consensus the level of its conservation was dropped to 75%. Regions of statistically significant conservation were unraveled in the CYP51 consensus sequence. These regions (motifs) were then correlated with the information on secondary structure elements and substrate recognition sites reported for CYP51 from Mycobacterium tuberculosis. Seven motifs appeared to be obligatory for every CYP51 protein. The motifs thus obtained were searched for among all the known cytochrome P450 proteins. Some motifs were found to be absolutely specific for 14-alpha-demethylases, whereas others were common to different species of cytochromes P450.

Amino Acid Motifs↗

PubMed2003

Comparative analysis of proteome maps of Helicobacter pylori clinical isolates.

The gram-negative bacterium Helicobacter pylori is found in human gastric mucosa. A widely distributed H. pylori infection is associated with chronic gastritis, gastric and duodenal ulcers, and malignant neoplasms. In this study proteome maps of four H. pylori clinical isolates derived from patients of two Russian regions (Moscow/Moscow Region and Novosibirsk) were obtained using 2D-electrophoresis and MALDI-TOF-mass-spectrometry. Variability of some H. pylori proteins and the level of their expression have been evaluated. These four isolates could be easily subdivided into two equal groups characterized by the close proteome profiles and the isolate from Moscow Region and the isolate from Novosibirsk constituted one group. The present study demonstrates the potential of proteome technology, which can be employed together with genome and transcriptome studies for the multiparameter typing of clinical isolates of pathogenic microorganisms.

Cell Extracts↗

PubMed2002

Production of carbon monoxide by cytochrome P450 during iron-dependent lipid peroxidation.

Carbon monoxide (CO) formation was studied in the process of lipid peroxidation in phenobarbital-induced rabbit liver microsomes. The reaction was NADPH-dependent and required Fe(2+), which occurs in microsomes as being protein bound and is not a consequence of heme destruction. Zn-protoporphyrin IX, an inhibitor of the heme oxygenase activity, proved to have no effect on CO production, suggesting that heme oxygenase is not involved into the CO generation reaction. At the same time, the addition of cytochrome P450 typical inhibitors SKF 525A and metyrapone to the reaction mixture had an inhibitory effect on the CO formation rate. Antioxidants such as alpha-tocopherol and desferal inhibited lipid peroxidation in phenobarbital-induced rabbit liver microsomes, and in this case the CO production was not registered. Thus, on the basis of the results presented here it is possible to assert that the process of NADPH, Fe(2+)-dependent carbon monoxide formation in microsomes is a result of lipid peroxidation with cytochrome P450 2B4 participation.

Animals↗

PubMed2002

Proteomic technologies in modern biomedical science.

This review highlights modern technologies employed in proteomics. Methods of sample preparations are discussed with special emphasis on the requirements for preparation of biological material, which may seriously influence the results of proteomic studies. Methods of solubilization, electrophoresis, chromatographic protein separation, and visualization of protein spots in gels are described. Modern methods of mass spectrometry used in proteomic studies include combination of protein chips with mass spectrometry. The review also describes approaches of functional proteomics, i.e., interactomics, and also bioinformatic resources used in proteomics for image analysis of 2D-gel-electrophoresis and for identification of protein sequences by mass spectra.

Biomedical Research↗

PubMed2002

Analysis of bispecific monoclonal antibody binding to immobilized antigens using an optical biosensor.

The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K(ass)) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K(ass) of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k(diss)) for anti-HRP shoulder of Babs was 21 times higher than k(diss) for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody-enzyme conjugates in the case of binding of bivalent Mabs.

Antibodies, Monoclonal↗

PubMed2002

Mapping and characterization of B cell linear epitopes in the conservative regions of hepatitis C virus envelope glycoproteins.

Forty-eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV-infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera obtained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immunodominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP.

Adolescent↗

PubMed2002

Virus-specific antibody titres in different phases of hepatitis C virus infection.

This study aimed to examine anti hepatitis C virus (HCV) antibody titres, their changes and differences in acute, chronic and past HCV infection and to examine them after IFN-alpha-therapy. Ninety five patients were studied in a cross-sectional investigation and 18 of them were followed long-term. Titres of IgM and IgG antibodies against core, NS3, NS4 (A + B), NS5A proteins were determined by the third generation enzyme immunoassays. Patients with acute hepatitis C developed IgG antibodies against core protein in titres 1/5-1/800 and against individual NS proteins at the same titres. During the first to second month of acute hepatitis C IgG antibody titres to HCV proteins were very low, but they had risen considerably by the fourth to sixth month. Anti-HCV IgM antibodies were found in half the acute hepatitis serum samples, titres were 1/5-1/40. Sixty individuals with chronic hepatitis C showed IgG antibodies against core in titres 1/800-1/40,000 and against individual NS proteins in titres 1/5-1/20,000. Eight patients with chronic hepatitis C had invariable anti-HCV IgG antibodies over 2-3 years. About 81.7% of chronically infected patients had anti-HCV IgM antibodies in titres 1/5-1/160. Patients with resolution of HCV infection showed only anti-core IgG antibodies (titres 1/5-1/200) or no virus-specific antibodies. Individuals with different response to IFN-alpha-therapy showed two distinct patterns of anti-HCV antibody titres. Acute and chronic HCV infection may be distinguished by anti-core titres.

Acute Disease↗

PubMed2001

Construction and characterization of bioelectrocatalytic sensors based on cytochromes P450.

Semisynthetic flavocytochromes RfP450 1A2, RfP450 2B4 and RfP450scc--molecular conjugates of protein with riboflavin--could be reduced on rhodium-graphite screen-printed thick film electrodes as was confirmed by cyclic voltammograms of immobilized enzymes. Amperometric enzyme electrodes for direct measurement of organic pollutants were developed. The efficiency of controlled potential electrolysis for the reduction of flavocytochromes P450 was comparable with traditional reduction by pyridine nucleotides. The rate constants for substrates conversion obtained by electrochemical methods were close to those obtained using NAD(P)H as an electron source.

Aminopyrine↗

PubMed2001

Revelation of ternary complexes between redox partners in cytochrome P450-containing monooxygenase systems by the optical biosensor method.

Formation of binary and ternary complexes in the water-soluble cytochrome P450cam (P450cam)-containing as well as in the membrane P4502B4(2B4)- and the mixed P450scc-containing monooxygenase systems was investigated in real time by the 'resonant mirror' optical biosensor method. It was shown that the inter-protein electron transfer occurs not only during complex formation but also upon random collision--as was the case with the d-Fp/d-b5 pair (2B4 system). Binary complexes may be either facilitative to electron transfer (electron-transfer complexes) or prohibitive to it (non-productive complexes). Although the binary PdR/Pd and P450cam/Pd complex formation (within the P450cam-system) as well as the binary AdR/Ad and P450scc/Ad complex formation (within the P450scc-system) does occur, the lifetimes of these complexes formed are several orders of magnitude higher than the time required for realization of a complete hydroxylation cycle. At the same time, the lifetimes of the ternary PdR/Pd/P450cam and AdR/Ad/P450scc complexes are sufficient to permit the realization of a complete hydroxylation cycle in either of these systems. For the membrane P450 2B4 system, the formation of both the binary (Fp/2B4 and 2B4/b5) and ternary (Fp/2B4/b5) complexes was registered. The lifetimes of the binary Fp/2B4 and the ternary Fp/2B4/b5 complexes are sufficient for realization of a complete hydroxylation cycle in each of them.

Biosensing Techniques↗

PubMed2001

Molecular recognition in the p450cam monooxygenase system: direct monitoring of protein-protein interactions by using optical biosensor.

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.

Binding Sites↗

PubMed2001

Effect of microsome-liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes.

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of phi=505-828 micros) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.

Animals↗