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Vol 23, No 3 (2020)


CHO cell line selection for development of stable cell line producing recombinant human follicle-stimulating hormone

Monakhova V.S., Pigareva N.V., Simbirtsev A.S.


Follicle-stimulating hormone (FSH) is one of the most important hormones of reproductive system and it is widely used as a treatment in an assisted reproductive technology. Choice of productive cell line for recombinant human FSH (rhFSH) protein production is the major issue of biotechnological process. Study objective. The aim of the study was to generate stable cell lines for rhFSH production using different parental CHO cell lines and compare their characteristics. Material and methods. Two CHO suspension parental cell lines were used for stable cell line generation - CHO-K1 and CHO-DXB 11 adapted to suspension conditions. Cell count and viability were analyzed by trypan blue exclusion method. RhFSH concentration was measured by ELISA, protein identity was confirmed by western blot analysis. Results. Four clones generated from CHO-DXB 11 cell line were selected after 3 steps - pool selection, clone selection and methotrexate amplification. Productivity during 6 days of cultivation for the most productive clone CHO-FSH 11/24 was 7,1±0,6 mg/l. Three clones were chosen after transfection and cell cloning of CHO-K1 cells transfected with plasmids containing of FSH α- and β-chains sequences. The highest productivity was detected for CHO-FSH91 clone and it was 10,8±0,9 mg/l. Molecular weight of rhFSH was identical for rhFSH purified from cell culture of optimal clones and commercial preparation Gonal-F (Merck Serono).
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):3-7
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Modification of the activity, isoenzyme spectrum and subcellular localization of amino acid exchange enzymes in the placenta during its dysfunction

Pogorelova T.N., Gunko V.O., Nikashina A.A., Larichkin A.V.


During prenatal development, various disorders affect in the metabolism of placenta, uniting the organisms of the mother and fetus. In this regard, the aim of the work is to study the activity of enzymes of amino acid metabolism at physiological pregnancy and placental dysfunction, which occupy a leading place among the complications of gestation that lead to fetal pathology. Material studies served as placental tissue in which the activity of aminotransferases, deaminases, and aminosynthetases was determined by spectrophotometric methods. Subcellular fractions of the placenta were isolated by polyacrylamide gel electrophoresis. Quantitative assessment of activity zones on gels leads to densitometric method. Established multidirectional changes in the activity of all studied enzymes in the placenta during its dysfunction. The degree of changes depends on the subcellular localization and the nature of the isoenzyme spectrum, which also differ from the normal parameters of this obstetric pathology. The maximum amount of isoenzymes is typical for the mitochondrial glutamate dehydrogenase fraction. The activity of aminotransferases isoenzymes has polycytostructural organization. In the chains of biochemical signals that lead to placental dysfunction.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):8-12
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Development of a scheme for accelerated identification of Xanthomonas campestris bacteria using bacteriophage in laboratory conditions

Maiorov P.S., Feoktistova N.A., Vasilyev D.A.


The aim of the study was to develop a phage titer rise reaction (PRR) scheme for the indication of Xanthomonas campestris pv. campestris bacteria. Material and methods. The PRR scheme was developed using the bacteriophage Cl34-Ulgau and the indicator culture of X. campestris pv. campestris bacteria Xc2 is based on the methods applied by the staff of the Department of Microbiology of the Ulyanovsk State Agricultural University. Results. The paper presents the developed scheme of PRR. The optimum parameters for the formulation of the PRR. The working dilution of the phage biopreparation Kl34-ULSAU was 104 BOE/ml. The dilution of the indicator culture of bacteria 107 mc/ml. the cultivation time of crops with indicator culture and phage biopreparation was 18 hours. Conclusion. Identification of the bacteria X. campestris pv. campestris based on the developed phage titer rise reaction scheme is 43 hours.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):13-17
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Clinical prospects for using the Ki67 proliferation index in lymphogenic metastases in breast carcinoma

Balkanov A.S., Gaganov L.E., Rozanov I.D., Shirikov E.I.


Вackground. The number of lymphogenic metastases in the axillary region is the most important predictor of parenchymal metastasis in breast cancer (BC). The proliferation index Ki67 (IP) in lymphogenic metastasis cells as a predictor of metastasis to parenchymal organs has not been studied enough to date. Patients and methods. Comparative analysis of IP in primary tumor cells and lymphogenic metastatic cells are done in 58 patients with luminal BC T1-4N1-3. Results. It was found that the discrepancy of IP (IPdis) in the cells of luminal BC and its lymphogenic metastases occurs in 82.8% of cases. It was found that lymphogenic metastases in the group of patients IP(-)dis higher than in patients IP(+)dis. However, the difference was not statistically significant. Much more detected significant IPdis. In 5 cases, a higher IP in lymphogenic metastatic cells may be the cause of diagnosis of aggressive luminal B molecular subtype of BC, followed by prediction of a higher risk of parenchymal metastasis. Conclusions. The results of comparative analysis of IP in primary tumor cells and lymphogenic metastatic cells in patients with luminal BC N1-3 in the long term can be used as a predictor of high metastatic potential and as a consequence of a short period of relapse-free survival.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):18-24
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Method for restoring functional activity of spermatozoa in HIV-infected men

Pervova Y.V., Starikova T.V.


The problem of male infertility especially affects HIV-infected men, so sperm motility decreases with highly active antiretroviral therapy. The aim of the study was to characterize the biochemical characteristics of sperm in HIV-infected men in order to restore their functional activity. Material and Methods. We observed 22 patients aged 25 to 45 years. This group has an undetectable viral load (40-50 copies / ml) and is receiving antiretroviral therapy. The studies were carried out using a Nikon Eclipse E 200 phase-contrast microscope. Determination of biochemical parameters of sperm plasma after standard sample preparation on a Roche Cobas integra 400+ automatic biochemical analyzer using Roche reagents and control materials. Results. The content of albumin, which determines the tonicity of the medium in the blood and the fulfillment of multiple vital functions, is almost 10 times lower than in blood plasma. It should be noted that the indicators of total protein and albumin in the sperm plasma of HIV-infected men are higher than in a healthy group of men and subfertile patients. The activity of aspartate-aminotransferase (ASAT) exceeds the blood plasma data. Moreover, the activity of ASAT is significantly higher in HIV-infected men. The activity of gamma-glutamyl transferase (GGT) is more than 200 times higher than in blood plasma. Further, as an experiment, we placed the "washed" spermatozoa in biological media (sperm plasma of a healthy donor, a woman's cervical fluid during ovulation) and obtained the following results. The percentage of progressively motile spermatozoa increased in the sperm plasma of healthy donors to 12%. We also observed an increase in sperm motility in the cervical fluid of a healthy woman during ovulation up to 7%. Conclusions. Thus, we have demonstrated that the use of sperm plasma from a healthy donor helps to restore sperm activity to 12%. The percentage of progressively motile spermatozoa increased in the sperm plasma of healthy donors. We observed an increase in sperm motility in the cervical fluid of a healthy woman during ovulation up to 6%.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):25-29
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Influence of multiple vitamin deficiency on the mineral status and some rat blood indicators

Beketova N.A., Vrzhesinskaya O.A., Kodentsova V.M., Kosheleva O.V., Shevyakova L.V., Leonenko S.N., Soto S.J., Sokolnikov A.A.


Insufficient intake of vitamins of group B and vitamin D is typical for the population of Russia. Data on disturb of mineral status and redistribution of trace elements in rat organs and tissues under deficiency of B vitamins had been appeared. The aim of the study was to characterize the effect of multiple vitamin deficiency in growing rats on the mineral status and some blood parameters. Material and methods. Multiple vitamin deficiency in Wistar male rats (n = 12) with an initial body weight (51.4 ± 0.5) was caused by a 5-fold decrease in the vitamin content in the vitamin mixture of a semi-synthetic diet for 23 days. Animals of the control group (n = 12) received a full semi-synthetic diet. The concentration of vitamins A in blood plasma and lyophilized liver and the whole brain of rats was determined by HPLC, vitamins B1 and B2 in the liver, brain, urine and 4-pyridoxic acid in urine fluorimetric method. The content of minerals in the liver and brain was determined by the atomic absorption method. Results. Multiple vitamin deficiency was accompanied by a decrease copper by 6.8 times and magnesium by 26.9%, in the brain, and increase of 11.6% in calcium, an increase of the blood plasma concentration of calcium, protein and phosphorus in the by 2.9-6.9%, creatinine - by 20.3%, a 2.2-fold increase of the ratio of y- and α-tocopherols (due to an increase of 1.5 times the concentration of y-tocopherol), an increase in the De Ritis ratio by 32.9%. Conclusions. Multiple vitamin deficiency affects the redistribution of minerals in organs, which justifies the need to maintain the vitamin status at the optimal level and confirms the benefits of using vitamin-mineral complexes containing not only a complete set of vitamins, but also deficient minerals and trace elements.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):30-36
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Development of approaches to standardization of fresh fruits of Crataegus submollis Sarg

Kurkin V.A., Shaikhutdinov I.K., Pravdivtseva O.E., Avdeeva E.V., Kurkina A.V., Stenyaeva V.V., Varina N.R., Zhdanova A.V.


Actuality. The fruits of Crataegus submollis Sarg. (family Rosaceae) are a popular food product, but are not currently used in medical practice. Previously, the possibility of using of the fresh fruits of Crataegus submollis Sarg. as a source of antidepressant and diuretic drugs has been shown. One of the new drugs may be a juice based on fresh fruits of Crataegus submollis Sarg. In еру fresh fruits of the Crataegus submollis Sarg. predominate the reduced forms of flavonoids, namely procyanidins. An earlier analysis of the juice from fresh fruits of Crataegus submollis Sarg. with the using of spectrophotometry showed that the maximum absorption in the UV spectrum is 282 nm, which coincides with the maximum absorption of the solution of catechin, a biogenetic precursor of procyanidins. The aim is the development of the method for quantitative determination of the total procyanidins in of the fresh fruits of Crataegus submollis Sarg. Material and methods. The object of the study was fresh fruits of Crataegus submollis Sarg. harvested in September 2019 on the territory of Samara and Samara region. The method of analysis was direct spectrophotometry at a wavelength of 282 nm calculated on catechin. Results. As a results of the study the method of quantitative determination of the total procyanidins (calculated on catechin) in fresh fruits of Crataegus submollis Sarg. by direct spectrophotometry at an analytical wavelength of 282 nm was developed. It was determined that the content of the total procyanidins in fresh fruits of Crataegus submollis Sarg. varies from 0,52±0,03% to 0,97±0,06% (calculated on catechin). The error of a single determination with a 95% confidence probability is ±5,81 %. Conclusion. The highest content of procyanidins can be noted in the sepals and the skin of еру fresh fruits of Crataegus submollis Sarg. The fresh fruits of Crataegus submollis Sarg. are promising medicinal plant raw materials.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):37-42
pages 37-42 views

The component composition of essential oil of the new peppermint (Mentha*pipertita L.) Variety «Kazachka»

Bykova O.A., Anikina A.Y., Tropina N.C., Thaganov R.R., Morozov A.I., Khazieva F.M.


A new variety of peppermint «Kazachka» have been created by the method of clone selection. In the article described the characteristics of new variety for economic and valuable characteristics in comparison with the zoned variety «Kubanskaya 6». The yield of air-dry leaf of the «Kazachka» variety is 1.98-2.26 t / ha, the yield of rhizomes is 12.0-12.6 t / ha, which exceeds the zoned variety «Kubanskaya 6» by 18% and 12%, respectively. The essential oil content is 2.2-2.5%, the menthol content in the oil is up to 48 %. The study of the component composition of the essential oil of the new variety «Kazachka» and the variety «Kubanskaya 6» showed its identity in many components, with the exception of Y-terpinen, which is not found in the grade «Kubanskaya 6». The main components of essential oil in both varieties are menthol (47.8 and 41.8 %), mentone ketones (10.4 and 13.7%) and iso-mentone (21.9 and 23.52%), which allows us to attribute the essential oil of the studied varieties to the menthol chemotype. The ratio of these components in the varieties «Kazachka» and «Kubanskaya 6» is 1.145; 0.758; 0.935, respectively. In the essential oil of the «Kazachka» variety, there is a predominance over the «Kuban 6» variety in the components pulegon (4,313), sabinen (2,479), trans-karyophylene (2,351), germacrene D (2,090), /so-Menthyl acetate (1,778). The «Kubanskaya 6» variety has an increased content of β-myrcene and piperitone components, the ratio of which over the «Kazachka» variety is 5.074 and 3.705 times, respectively. The maximum inter-port differences were found in the content of such a component of essential oil as limonene - in the oil of the «Kazachka» variety, it is 28 times more than in the «Kubanskaya 6» variety. The new variety «Kazachka» is not affected by powdery mildew and rust; is suitable for mechanized cleaning; it has been recommended for cultivation on a pharmacy leaf.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):43-49
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Peculiarities of reproduction of Potentilla alba L. and Potentilla megalantha L. in vitro

Polyakov N.A., Kalashnikova E.A., Kirakosyan R.N., Khazieva F.M.


The results of cultivation of Potentilla alba L. and Potentilla megalantha L. in vitro are presented. These species are valuable medicinal plants whose cells synthesize different classes of phenolic compounds, in particular tannins with high biological activity. Natural resourcesare not meeting the needs of the pharmaceutical industry. The search for alternative ways of reproduction in vitro of Potentilla plants and preservation of their biodiversity is an urgent task. The aim of this work was to study the reproduction characteristics of Potentilla alba L. and Potentilla megalantha L. in vitro. To achieve this goal, various methods of pretreatment of seeds were used: mechanical processing, heat treatment, soaking and stratification. As a result, of the conducted research, it was found that when introduced into the culture in vitro Potentilla alba L. and Potentilla megalantha L. it is necessary to apply seed stratification and carry out their germination on a hormone-free medium. Adding BAP to the nutrient medium at the concentration of 1 mg / l leads to the greatest increase in the biomass of P. megalantha and P.alba, as a result of the formation of the maximum number of adventive microbeads.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(3):50-56
pages 50-56 views

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