Reconversion of Cytochrome P420 into P450 and Reactivation of Hydroxylases in Microsomal Membranes Reconstituted by Self-Assembly

Membrane vesicles formed by means of dialysis of microsomal proteins and lipids, solubilized by cholate, contained an equal quantity of cytochromes P420 and P450. Addition of albumin and phosphatidylcholine aids reconversion of cytochrome P420 into cytochrome P450. In the presence of albumin the reconstituted membranes contained less lysophosphatidylcholine than those reconstituted without it. Comparative analysis does not reveal· marked differences in the phospholipid and protein composition or specific activity and content of NAD(P)H-dependent redox carriers of original and reconstituted membranes. In contrast to original membranes, the reconstituted ones do not possess aniline hydroxylase activity. The NADH-specific dimethylaniline demethylase activity was higher than that of the original membranes. NADPH-dependent demethylase of this substrate could be reconstituted to 50-800/o of its initial activity. The reconstituted membranes differ from original membranes by their higher non-enzymatic lipid peroxidase activity. Thus, it appears that dialysis in the presence of albumin and phosphatidylcholine is the most effective mode of microsomal membrane reconstitution

Reconversion of Cytochrome P420 into P450 and Reactivation of Hydroxylases in Microsomal Membranes Reconstituted by Self-Assembly

Membrane vesicles formed by means of dialysis of microsomal proteins and lipids, solubilized by cholate, contained an equal quantity of cytochromes P420 and P450. Addition of albumin and phosphatidylcholine aids reconversion of cytochrome P420 into cytochrome P450. In the presence of albumin the reconstituted membranes contained less lysophosphatidylcholine than those reconstituted without it. Comparative analysis does not reveal· marked differences in the phospholipid and protein composition or specific activity and content of NAD(P)H-dependent redox carriers of original and reconstituted membranes. In contrast to original membranes, the reconstituted ones do not possess aniline hydroxylase activity. The NADH-specific dimethylaniline demethylase activity was higher than that of the original membranes. NADPH-dependent demethylase of this substrate could be reconstituted to 50-800/o of its initial activity. The reconstituted membranes differ from original membranes by their higher non-enzymatic lipid peroxidase activity. Thus, it appears that dialysis in the presence of albumin and phosphatidylcholine is the most effective mode of microsomal membrane reconstitution

The limit of mass determination with an AFM cantilever-based system

This study was performed in the framework of the Program for Basic Research of State Academies of Sciences for 2013-2020. Shumov I.D. is a recipient of Russian Federation President scholarship for young scientists for 2016-2018 (project identificator SP-4280.2016.4)

Magnetron sputtering deposition of ultra-thin tungsten coatings onto amorphous graphite for enhancement of horseradish peroxidase adsorption

The present study was performed in the framework of the Program for Basic Research of State Academies of Sciences for 2013-2020

Antithrombotic Therapy in Patients with Paroxysmal Atrial Fibrillation after Catheter Treatment

Aim. To study the efficacy and safety of antithrombotic therapy in patients with paroxysmal atrial fibrillation (AF) after catheter treatment during 36 months of follow-up.Material and methods. The retrospective observational study included 592 patients (283 men) who underwent catheter treatment of AF, aged 26 to 86 years (median age was 61.0 [55; 67]) with paroxysmal AF, treated in cardiac arrhythmias department of the Institute of Cardiology of Tomsk National Research Medical Center from 01.01.2017 to 31.12.2019. All patients were retrospectively divided into 2 groups: the first group consisted of patients with effective AF ablation, the second - with ineffective AF ablation. During follow-up after 12, 24 and 36 months, patients' complaints, documented arrhythmia recurrences, adherence to the prescribed treatment, and adverse clinical events were taken into account.Results. In patients with paroxysmal AF, the effectiveness of catheter treatment was 73.1% after 12 months of follow-up, 69.3% – after 24 months, 71.6% – after 36 months. The analysis of our data showed that during the follow-up period of 36 months, the incidence of ischemic stroke against the background of anticoagulant therapy and effective catheter treatment of paroxysmal AF was significantly lower than in patients with unsuccessful ablation (0.3% (n=1) and 3.7% (n=4), respectively), even despite the fact that not all patients from the first group received prescribed medication.Conclusion. The use of anticoagulant therapy in patients with paroxysmal AF after interventional treatment is safe, since the invasive strategy in combination with anticoagulant therapy does not increase the risk of major and minor bleeding, and in the case of effective intervention allows statistically significantly reduce the risk of ischemic stroke and almost completely eliminate the likelihood of other thromboemolic complications

Gene-centric coverage of the human liver transcriptome: QPCR, Illumina, and Oxford Nanopore RNA-Seq

It has been shown that the best coverage of the HepG2 cell line transcriptome encoded by genes of a single chromosome, chromosome 18, is achieved by a combination of two sequencing platforms, Illumina RNA-Seq and Oxford Nanopore Technologies (ONT), using cut-off levels of FPKM > 0 and TPM > 0, respectively. In this study, we investigated the extent to which the combination of these transcriptomic analysis methods makes it possible to achieve a high coverage of the transcriptome encoded by the genes of other human chromosomes. A comparative analysis of transcriptome coverage for various types of biological material was carried out, and the HepG2 cell line transcriptome was compared with the transcriptome of liver tissue cells. In addition, the contribution of variability in the coverage of expressed genes in human transcriptomes to the creation of a draft human transcriptome was evaluated. For human liver tissues, ONT makes an extremely insignificant contribution to the overall coverage of the transcriptome. Thus, to ensure maximum coverage of the liver tissue transcriptome, it is sufficient to apply only one technology: Illumina RNA-Seq (FPKM > 0)

The Size of the Human Proteome: The Width and Depth

This work discusses bioinformatics and experimental approaches to explore the human proteome, a constellation of proteins expressed in different tissues and organs. As the human proteome is not a static entity, it seems necessary to estimate the number of different protein species (proteoforms) and measure the number of copies of the same protein in a specific tissue. Here, meta-analysis of neXtProt knowledge base is proposed for theoretical prediction of the number of different proteoforms that arise from alternative splicing (AS), single amino acid polymorphisms (SAPs), and posttranslational modifications (PTMs). Three possible cases are considered: (1) PTMs and SAPs appear exclusively in the canonical sequences of proteins, but not in splice variants; (2) PTMs and SAPs can occur in both proteins encoded by canonical sequences and in splice variants; (3) all modification types (AS, SAP, and PTM) occur as independent events. Experimental validation of proteoforms is limited by the analytical sensitivity of proteomic technology. A bell-shaped distribution histogram was generated for proteins encoded by a single chromosome, with the estimation of copy numbers in plasma, liver, and HepG2 cell line. The proposed metabioinformatics approaches can be used for estimation of the number of different proteoforms for any group of protein-coding genes

RNA-Seq gene expression profiling of HepG2 cells: The influence of experimental factors and comparison with liver tissue

© Tyakht et al.; licensee BioMed Central. Background: Human hepatoma HepG2 cells are used as an in vitro model of the human liver. High-throughput transcriptomic sequencing is an advanced approach for assessing the functional state of a tissue or cell type. However, the influence of experimental factors, such as the sample preparation method and inter-laboratory variation, on the transcriptomic profile has not been evaluated. Results: The whole-transcriptome sequencing of HepG2 cells was performed using the SOLiD platform and validated using droplet digital PCR. The gene expression profile was compared to the results obtained with the same sequencing method in another laboratory and using another sample preparation method. We also compared the transcriptomic profile HepG2 cells with that of liver tissue. Comparison of the gene expression profiles between the HepG2 cell line and liver tissue revealed the highest variation, followed by HepG2 cells submitted to two different sample preparation protocols. The lowest variation was observed between HepG2 cells prepared by two different laboratories using the same protocol. The enrichment analysis of the genes that were differentially expressed between HepG2 cells and liver tissue mainly revealed the cancer-associated gene signature of HepG2 cells and the activation of the response to chemical stimuli in the liver tissue. The HepG2 transcriptome obtained with the SOLiD platform was highly correlated with the published transcriptome obtained with the Illumina and Helicos platforms, with moderate correspondence to microarrays. Conclusions: In the present study, we assessed the influence of experimental factors on the HepG2 transcriptome and identified differences in gene expression between the HepG2 cell line and liver cells. These findings will facilitate robust experimental design in the fields of pharmacology and toxicology. Our results were supported by a comparative analysis with previous HepG2 gene expression studies

Combination of atomic force microscopy and mass spectrometry for the target protein in the serum samples of children with autism spectrum disorders

Показана возможность детекции целевых белков, ассоциированных с развитием аутистических расстройств у детей, с помощью комбинированного метода АСМ/МС. Метод основан на комбинации аффинного обогащения белков из биообразцов, их АСМ-визуализации и МС-анализе с количественной детекцией целевых белков.Possibility of detection of target proteins associated with development of autistic disorders in children with use of combined AFM/MS method is demonstrated. The proposed method is based on the combination of affine enrichment of proteins from biological samples, visualization of these proteins by AFM and MS analysis with quantitative detection of target proteins.Работа выполнена при поддержке гранта РНФ 14-25-00132. Шумов И.Д. является получателем стипендии Президента РФ молодым ученым и аспирантам, осуществляющим перспективные научные исследования и разработки по приоритетным направлениям модернизации российской экономики на 2016-2018 годы (СП-4280.2016.4)