Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.040
V. Pletnov
Aim. To evaluate the activity of NO-synthase isoforms, the concentration of peroxynitrites and nitrosothiols in the soft tissues of the periodontium of rats under the conditions of modeling chronic stress against the background of lipopolysaccharide-induced inflammation. Methods. Experimental studies were performed on 24 male Wistar rats weighing 190–240 g. The animals were divided into 4 groups: 1 — control, 2 — chronic stress (ChrStr group), animals were kept above water for 1 hour every day for 30 days, 3 — animals that were intraperitoneally injected with 0.4 μg/kg of bacterial LPS of S. typhi (pyrogenal) (LPS group); 4 — animals that were simultaneously simulated chronic stress as in group 2 and administered LPS as in group 3 (ChrStr+LPS). The activity of inducible NO-synthase (iNOS), constitutive NO-synthase (cNOS), the concentration of nitrosothiols (S-NO), and concentration of peroxynitrites of alkali and alkaline earth metals (ONOO) were studied in the homogenate of the periodontal soft tissues of rats. Results. The activity of NOS in the soft periodontal tissues of rats under chronic stress simulation conditions, LPS administration, combined exposure to chronic stress and LPS was increased compared to control group. The concentration of ONOO- in the soft periodontal tissues of rats under chronic stress simulation conditions, LPS administration, combined exposure to chronic stress and LPS was increased compared to the control group. The concentration of nitrosothiols in the soft periodontal tissues of rats under the conditions of chronic stress simulation, LPS administration, combined exposure to chronic stress and LPS was decreased compared to the control group. Conclusions. The combined effect of bacterial lipopolysaccharide and chronic stress leads to increased production of nitrogen monoxide from inducible NO-synthase and elevates concentration of reactive forms of nitrogen, which creates possibility for development of nitrosative stress in the soft periodontal tissues.
{"title":"ROLE OF NO IN SOFT PERIODONTAL TISSUES OF RATS DURING STRESS AND INFLAMMATION","authors":"V. Pletnov","doi":"10.15407/biotech16.02.040","DOIUrl":"https://doi.org/10.15407/biotech16.02.040","url":null,"abstract":"Aim. To evaluate the activity of NO-synthase isoforms, the concentration of peroxynitrites and nitrosothiols in the soft tissues of the periodontium of rats under the conditions of modeling chronic stress against the background of lipopolysaccharide-induced inflammation. Methods. Experimental studies were performed on 24 male Wistar rats weighing 190–240 g. The animals were divided into 4 groups: 1 — control, 2 — chronic stress (ChrStr group), animals were kept above water for 1 hour every day for 30 days, 3 — animals that were intraperitoneally injected with 0.4 μg/kg of bacterial LPS of S. typhi (pyrogenal) (LPS group); 4 — animals that were simultaneously simulated chronic stress as in group 2 and administered LPS as in group 3 (ChrStr+LPS). The activity of inducible NO-synthase (iNOS), constitutive NO-synthase (cNOS), the concentration of nitrosothiols (S-NO), and concentration of peroxynitrites of alkali and alkaline earth metals (ONOO) were studied in the homogenate of the periodontal soft tissues of rats. Results. The activity of NOS in the soft periodontal tissues of rats under chronic stress simulation conditions, LPS administration, combined exposure to chronic stress and LPS was increased compared to control group. The concentration of ONOO- in the soft periodontal tissues of rats under chronic stress simulation conditions, LPS administration, combined exposure to chronic stress and LPS was increased compared to the control group. The concentration of nitrosothiols in the soft periodontal tissues of rats under the conditions of chronic stress simulation, LPS administration, combined exposure to chronic stress and LPS was decreased compared to the control group. Conclusions. The combined effect of bacterial lipopolysaccharide and chronic stress leads to increased production of nitrogen monoxide from inducible NO-synthase and elevates concentration of reactive forms of nitrogen, which creates possibility for development of nitrosative stress in the soft periodontal tissues.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43684211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.050
A. V. Udovenko
The aim of the study was to develop a method for determination the activity of thrombin, which is based on the turbidimetry curve of the formation and dissolution of a blood plasma clot. Methods. Donor blood samples were collected in 3.8% sodium citrate (1 part of sodium citrate and 9 parts of blood, pH 7.4). Plasma was separated from blood cells within 1 hour after blood collection by centrifugation the latter at 1200 g for 20 minutes. Aliquots of plasma were stored at -20 °C. Results. To determine the concentrations of thrombin and plasmin, TDCs of the formation and dissolution of blood plasma clots, initiated by the APTT reagent, were used. Based on the values of τ obtained, a calibration curve was constructed in the coordinates 1/τ – [Thr] (the rate of protofibrils formation in s-1 vs thrombin concentration in NIH units in 1 ml). Conclusion. The proposed methods to determine the activity of thrombin and plasmin made it possible to quantitatively calculate the rate of prothrombin activation in the lag period, the concentration and activity of thrombin based on the rate of fibrin and protofibrils formation as well as the activity and concentration of plasmin at the point of the complete clot dissolution,
{"title":"DETERMINATION OF THROMBIN AND PLASMIN ACTIVITY IN HUMAN BLOOD PLASMA USING THE TURBIDIMETRIC CURVE OF CLOT FORMATION AND DISSOLUTION","authors":"A. V. Udovenko","doi":"10.15407/biotech16.02.050","DOIUrl":"https://doi.org/10.15407/biotech16.02.050","url":null,"abstract":"The aim of the study was to develop a method for determination the activity of thrombin, which is based on the turbidimetry curve of the formation and dissolution of a blood plasma clot. Methods. Donor blood samples were collected in 3.8% sodium citrate (1 part of sodium citrate and 9 parts of blood, pH 7.4). Plasma was separated from blood cells within 1 hour after blood collection by centrifugation the latter at 1200 g for 20 minutes. Aliquots of plasma were stored at -20 °C. Results. To determine the concentrations of thrombin and plasmin, TDCs of the formation and dissolution of blood plasma clots, initiated by the APTT reagent, were used. Based on the values of τ obtained, a calibration curve was constructed in the coordinates 1/τ – [Thr] (the rate of protofibrils formation in s-1 vs thrombin concentration in NIH units in 1 ml). Conclusion. The proposed methods to determine the activity of thrombin and plasmin made it possible to quantitatively calculate the rate of prothrombin activation in the lag period, the concentration and activity of thrombin based on the rate of fibrin and protofibrils formation as well as the activity and concentration of plasmin at the point of the complete clot dissolution,","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42844302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.011
K. Baidakova
Aim. To search fibrinogenolytic enzymes among protein components of Vipera renardi snake venom. Methods. Venom of V. renardi as the lyophilized powder was supplied by Trypillia serpentarium. It was dissolved in 0.05 M Tris-HCl buffer pH 8.3 and fractionated on Superdex G-75 using FPLC system Acta Prime. Peaks were tested for their ability to directly cleave fibrinogen. Hydrolytic products were analyzed by SDS-PAGE. Enzyme-electrophoresis with fibrinogen co-polymerized in 12% polyacrylamide gel was used for the identification of protein that can cleave fibrinogen.. Results. Venom of V. renardi was fractionated on 4 fractions using size-exclusion chromatography. SDS-PAGE of fibrinogen hydrolysis products showed the presence of fibrinogen-specific protease in the 1st and 2nd fractions of venom. 2nd fraction was much more active and according to the data of enzyme electrophoresis contained protease with molecular mass 25 kDa. Conclusions. Fractionation of V. renardi snake venom allowed to detect a protease with apparent molecular mass 25 kDa that can cleave fibrinogen molecule.
的目标。目的:在雷氏蝮蛇毒液的蛋白质成分中寻找纤维蛋白原分解酶。方法。冻干粉毒液由蛇锥虫(Trypillia serpentarium)提供。溶解于0.05 M pH 8.3的Tris-HCl缓冲液中，在Superdex G-75上用FPLC系统进行分离。测试了它们直接切割纤维蛋白原的能力。水解产物采用SDS-PAGE分析。采用纤维蛋白原在12%聚丙烯酰胺凝胶中共聚合的酶电泳法鉴定可切割纤维蛋白原的蛋白。结果。采用排色色谱法对毒进行了4个部分的分离。纤维蛋白原水解产物的SDS-PAGE显示在毒液的第一和第二部分存在纤维蛋白原特异性蛋白酶。酶电泳结果显示，第2部分活性更强，含有分子量为25 kDa的蛋白酶。结论。通过对renardi蛇毒的分离，检测到一种表观分子质量为25 kDa的蛋白酶，该蛋白酶可裂解纤维蛋白原分子。
{"title":"FIBRINOGEN-SPECIFIC PROTEASE IN THE Vipera renardi SNAKE VENOM","authors":"K. Baidakova","doi":"10.15407/biotech16.02.011","DOIUrl":"https://doi.org/10.15407/biotech16.02.011","url":null,"abstract":"Aim. To search fibrinogenolytic enzymes among protein components of Vipera renardi snake venom. Methods. Venom of V. renardi as the lyophilized powder was supplied by Trypillia serpentarium. It was dissolved in 0.05 M Tris-HCl buffer pH 8.3 and fractionated on Superdex G-75 using FPLC system Acta Prime. Peaks were tested for their ability to directly cleave fibrinogen. Hydrolytic products were analyzed by SDS-PAGE. Enzyme-electrophoresis with fibrinogen co-polymerized in 12% polyacrylamide gel was used for the identification of protein that can cleave fibrinogen.. Results. Venom of V. renardi was fractionated on 4 fractions using size-exclusion chromatography. SDS-PAGE of fibrinogen hydrolysis products showed the presence of fibrinogen-specific protease in the 1st and 2nd fractions of venom. 2nd fraction was much more active and according to the data of enzyme electrophoresis contained protease with molecular mass 25 kDa. Conclusions. Fractionation of V. renardi snake venom allowed to detect a protease with apparent molecular mass 25 kDa that can cleave fibrinogen molecule.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45440004","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.042
Y. Raynich
The aim of the present study was to find out the role of Ruk/CIN85 in modulation of activities/content of key enzymes/components of glycolysis and hydrogen peroxide using as a model human NSCLC MOR wild type and resistant to drugs MOR/0.2R cells. Materials and methods. MOR (ECACC 84112312) and MOR/0.2R (ECACC 96042335), drug-resistant cell line, were cultured in the complete RPMI medium under standard conditions. Enzymes activity, content of metabolites and protein in cell extracts and the conditioned cell culture medium were estimated by spectrophotometric and fluorometric assays. Results. Using RT2-PCR it was revealed that the level of Ruk/CIN85 mRNA in drug-resistant MOR cells was 10 times higher than in parental MOR cells. The activities of lysyl oxidase (LOX) and diamine oxidase (DAO) were significantly higher in resistant cells It has been shown that these enzymes are associated with aggressiveness of tumor cells. Based on the obtained results, we draw a conclusion that observed changes in the intensity of glycolysis, amine oxidases activities and content of hydrogen peroxide in doxorubicin-resistant MOR/0.2R cells positively correlate with the expression level of the adaptor protein studied. Conclusions. In conclusion, it can be assumed that the adaptor protein Ruk/CIN85 is involved in metabolome reprogramming and may function as an important component of regulatory networks required for the acquisition of drug resistant phenotype by NSCLC cells.
{"title":"INCREASED EXPRESSION LEVEL OF ADAPTOR PROTEIN Ruk/CIN85 IN DOXORUBICIN-RESISTANT HUMAN NON-SMALL LUNG ADENOCARCINOMA MOR CELLS IS ASSOCIATED WITH THEIR METABOLIC REPROGRAMMING","authors":"Y. Raynich","doi":"10.15407/biotech16.02.042","DOIUrl":"https://doi.org/10.15407/biotech16.02.042","url":null,"abstract":"The aim of the present study was to find out the role of Ruk/CIN85 in modulation of activities/content of key enzymes/components of glycolysis and hydrogen peroxide using as a model human NSCLC MOR wild type and resistant to drugs MOR/0.2R cells. Materials and methods. MOR (ECACC 84112312) and MOR/0.2R (ECACC 96042335), drug-resistant cell line, were cultured in the complete RPMI medium under standard conditions. Enzymes activity, content of metabolites and protein in cell extracts and the conditioned cell culture medium were estimated by spectrophotometric and fluorometric assays. Results. Using RT2-PCR it was revealed that the level of Ruk/CIN85 mRNA in drug-resistant MOR cells was 10 times higher than in parental MOR cells. The activities of lysyl oxidase (LOX) and diamine oxidase (DAO) were significantly higher in resistant cells It has been shown that these enzymes are associated with aggressiveness of tumor cells. Based on the obtained results, we draw a conclusion that observed changes in the intensity of glycolysis, amine oxidases activities and content of hydrogen peroxide in doxorubicin-resistant MOR/0.2R cells positively correlate with the expression level of the adaptor protein studied. Conclusions. In conclusion, it can be assumed that the adaptor protein Ruk/CIN85 is involved in metabolome reprogramming and may function as an important component of regulatory networks required for the acquisition of drug resistant phenotype by NSCLC cells.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45507792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.018
V. Derkachov
The aim of our study was to determine the ability of broccoli sprouts to influence the intensity of lipid peroxidation in mice fed a high-calorie cafeteria diet. Materials and methods. In this study, C57BL/6J mice were used. For determinatiom of lipid peroxides (LOOH), we used a method based on the ability of lipid peroxides to convert Fe2+to Fe3+. Then Fe3+ forms a complex with xylenol orange that absorbs light at 580 nm at low pH. The reaction mixture contained coumene hydroperoxide (1 mM), FeSO4*7H2O(1 M), xylenol (4 mM), water and supernatant. Differences between groups were analyzed by Duncan’s test for multiple comparision. Results. During the experiment, we monitored the changes in body mass of mice fed with different diets. There were no differences in LOOH levels in cortexes of mice from all experimental groups, but there was a tendency to the lower content of LOOH in the brain of mice fed with cafeteria diet and broccoli. No statistical differences in levels of LOOH were found between groups, but LOOH levels tended to the highest in the groups fed with broccoli sprouts alone and cafeteria diet. A significant difference was observed in the muscles (C) between the broccoli sprout group and the cafeteria diet + broccoli group. We also found a significant difference between the group fed with the cafeteria diet and the cafeteria diet + broccoli, which may indicate protective effects of broccoli on lipid peroxidation on cafeteria diet. Conclusions. Mice fed with cafeteria diet and broccoli spouts had higher body mass than control mice fed with standard group. Hypothalamus of mice fed with standard diet with broccoli spouts or with cafeteria diet showed a tendency to higher LOOH levels, whereas no effects of the diets were found on cortexes LOOH levels. The cafeteria diet + broccoli group had the lowest muscle LOOH content compared to all other groups. Also, LOOH levels tended to be lower in the cortexes in the hypothalamus of mice fed with cafeteria diet + broccoli as compared with the cafeteria diet group. This suggests the potential protective effects of broccoli spouts.
{"title":"THE EFFECT OF BROCCOLI SPROUTS ON OXIDATIVE STRESS MARKERS IN MICE FED WITH CAFETERIA DIET","authors":"V. Derkachov","doi":"10.15407/biotech16.02.018","DOIUrl":"https://doi.org/10.15407/biotech16.02.018","url":null,"abstract":"The aim of our study was to determine the ability of broccoli sprouts to influence the intensity of lipid peroxidation in mice fed a high-calorie cafeteria diet. Materials and methods. In this study, C57BL/6J mice were used. For determinatiom of lipid peroxides (LOOH), we used a method based on the ability of lipid peroxides to convert Fe2+to Fe3+. Then Fe3+ forms a complex with xylenol orange that absorbs light at 580 nm at low pH. The reaction mixture contained coumene hydroperoxide (1 mM), FeSO4*7H2O(1 M), xylenol (4 mM), water and supernatant. Differences between groups were analyzed by Duncan’s test for multiple comparision. Results. During the experiment, we monitored the changes in body mass of mice fed with different diets. There were no differences in LOOH levels in cortexes of mice from all experimental groups, but there was a tendency to the lower content of LOOH in the brain of mice fed with cafeteria diet and broccoli. No statistical differences in levels of LOOH were found between groups, but LOOH levels tended to the highest in the groups fed with broccoli sprouts alone and cafeteria diet. A significant difference was observed in the muscles (C) between the broccoli sprout group and the cafeteria diet + broccoli group. We also found a significant difference between the group fed with the cafeteria diet and the cafeteria diet + broccoli, which may indicate protective effects of broccoli on lipid peroxidation on cafeteria diet. Conclusions. Mice fed with cafeteria diet and broccoli spouts had higher body mass than control mice fed with standard group. Hypothalamus of mice fed with standard diet with broccoli spouts or with cafeteria diet showed a tendency to higher LOOH levels, whereas no effects of the diets were found on cortexes LOOH levels. The cafeteria diet + broccoli group had the lowest muscle LOOH content compared to all other groups. Also, LOOH levels tended to be lower in the cortexes in the hypothalamus of mice fed with cafeteria diet + broccoli as compared with the cafeteria diet group. This suggests the potential protective effects of broccoli spouts.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44119469","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.013
M. I. Bekala
Aim. To investigate the changes in MMPs expression and activity as well as invasiveness of human lung adenocarcinoma A549 cells with up-/down-regulation of Ruk/CIN85. Methods. There were used A549 cells with stable overexpression (subline RukUp) and knockdown of Ruk/CIN85 (subline RukDown), as well as corresponding vector control sublines Mock and Scr. Cells were cultured in the complete DMEM medium under standard conditions. mRNA expression levels were estimated by RT2-PCR, enzymatic activity was assessed using gelatin zymography. Invasiveness of cancer cells was studied using Boyden chambers coated with Matrigel. Results. Analysis of mRNA expression of MMPs in RukUp and RukDown cells revealed that MMP-2 and MMP-9 were preferentially overexpressed in RukDown cells, while RukUp subline did not exhibit significant difference compared with corresponding control. These findings were confirmed and complemented by study of enzyme activities. The gelatinolytic activities of both MMP-2 and MMP-9 were dramatically increased in RukDown subline, compared to respective control. we revealed that MMPs regulation was inversely correlated with invasion potential of Ruk/CIN85 up/down A549 cells. In particular, it was established that invasiveness of RukUp cells was 2 times higher in comparison with respective control subline. Alternatively, invasion ratio was significantly decreased in RukDown cells (0,5 times) in comparison with control. Conclusions. According to the data received, it is possible to suggest that up-regulation of adaptor protein Ruk/CIN85 in A549 cells can lead to the very aggressive MMP-independent mode of migration that rely on cycles of expansion and contraction of the cell body mediated by the cortically localized actin and myosin.
{"title":"https://biotechnology.kiev.ua/index.php/en/journal-archive-en/2023-en/2023-no2-en/fibrinogen-specific-protease-in-the-vipera-renardi-snake-venom-k-v-baidakova-y-m-stohnii-o-m-platonov","authors":"M. I. Bekala","doi":"10.15407/biotech16.02.013","DOIUrl":"https://doi.org/10.15407/biotech16.02.013","url":null,"abstract":"Aim. To investigate the changes in MMPs expression and activity as well as invasiveness of human lung adenocarcinoma A549 cells with up-/down-regulation of Ruk/CIN85. Methods. There were used A549 cells with stable overexpression (subline RukUp) and knockdown of Ruk/CIN85 (subline RukDown), as well as corresponding vector control sublines Mock and Scr. Cells were cultured in the complete DMEM medium under standard conditions. mRNA expression levels were estimated by RT2-PCR, enzymatic activity was assessed using gelatin zymography. Invasiveness of cancer cells was studied using Boyden chambers coated with Matrigel. Results. Analysis of mRNA expression of MMPs in RukUp and RukDown cells revealed that MMP-2 and MMP-9 were preferentially overexpressed in RukDown cells, while RukUp subline did not exhibit significant difference compared with corresponding control. These findings were confirmed and complemented by study of enzyme activities. The gelatinolytic activities of both MMP-2 and MMP-9 were dramatically increased in RukDown subline, compared to respective control. we revealed that MMPs regulation was inversely correlated with invasion potential of Ruk/CIN85 up/down A549 cells. In particular, it was established that invasiveness of RukUp cells was 2 times higher in comparison with respective control subline. Alternatively, invasion ratio was significantly decreased in RukDown cells (0,5 times) in comparison with control. Conclusions. According to the data received, it is possible to suggest that up-regulation of adaptor protein Ruk/CIN85 in A549 cells can lead to the very aggressive MMP-independent mode of migration that rely on cycles of expansion and contraction of the cell body mediated by the cortically localized actin and myosin.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46338645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.044
N. Stefanyshyn
Aim. To investigate how starvation during early stage of fly development affects carbohydrate metabolism in imago flies and their progeny of F1 generation. Methods. Wild-type Canton-S strain Drosophila melanogaster flies were used in all experiments. Flies of parental and offspring generations were used for the determination of glycogen and glucose content using the diagnostic kit Glucose-Mono-400-P according to the manufacturer's instructions. Results represent as the mean ± SEM of 3-4 replicates per group. According Student's t-test significant difference between groups was P<0.05. Graphing and statistical analysis were performed by using GraphPad Prism. Results. Starvation during development significantly influenced the level of hemolymph and body glucose in imago flies of parental generation. Hemolymph glucose concentration was lower by 34% (P=0.008) and 32% (P=0.033) in experimental females and males, respectively, as compared to control groups. Starvation during development led to lower level of body glucose in adult parental flies of both sexes. Adult males F1, generated by parents that were starved during development, showed 3-fold lower glycogen content, as compared to control. Conclusions. Starvation at early stage of development led to lower hemolymph glucose and body glucose level in imago flies. Moreover, parental starvation decreased glycogen pool in F1 males.
{"title":"STARVATION DURING DEVELOPMENT AFFECTS METABOLISM IN DROSOPHILA","authors":"N. Stefanyshyn","doi":"10.15407/biotech16.02.044","DOIUrl":"https://doi.org/10.15407/biotech16.02.044","url":null,"abstract":"Aim. To investigate how starvation during early stage of fly development affects carbohydrate metabolism in imago flies and their progeny of F1 generation. Methods. Wild-type Canton-S strain Drosophila melanogaster flies were used in all experiments. Flies of parental and offspring generations were used for the determination of glycogen and glucose content using the diagnostic kit Glucose-Mono-400-P according to the manufacturer's instructions. Results represent as the mean ± SEM of 3-4 replicates per group. According Student's t-test significant difference between groups was P<0.05. Graphing and statistical analysis were performed by using GraphPad Prism. Results. Starvation during development significantly influenced the level of hemolymph and body glucose in imago flies of parental generation. Hemolymph glucose concentration was lower by 34% (P=0.008) and 32% (P=0.033) in experimental females and males, respectively, as compared to control groups. Starvation during development led to lower level of body glucose in adult parental flies of both sexes. Adult males F1, generated by parents that were starved during development, showed 3-fold lower glycogen content, as compared to control. Conclusions. Starvation at early stage of development led to lower hemolymph glucose and body glucose level in imago flies. Moreover, parental starvation decreased glycogen pool in F1 males.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47478318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.032
Y. O. Kustovskiy
The aim of this research was to determine the structural patterns of IVM allosteric interaction with residues of its binding site located in the transmembrane domain of α-homopentameric glutamate-gated chloride channel (GluClα) of Caenorhabditis elegans. Methods. To consider different conformational states of IVM binding site two complexes of IVM bound to C. elegans GluClα (each with five site conformations) with identifiers 3RHW and 3RIF were obtained from PDB. The structures were examined in Analyzer Mode of SeeSAR v.12.1.0, in which contributions of IVM atoms into the complex affinity and their interactions with site structural patterns were determined for each site conformation using the HYDE scoring function. The residues belonging to identified structural patterns were classified by their properties using the Taylor’s classification of amino acids. Results. According to the results, the benzofuran group is critical for IVM recognition and binding: it interacts with the T-A-S-N-D-I-L-Q-I-P pattern, which is formed by T257, A258, S260, and N264 of M2, D277 and I280 of M3 of (+) subunit and L218, Q219, I222, P223 of M1 of (–) subunit. Due to the size and hydrophobicity of macrocycle, its different parts interact with residues of all site-forming structural elements mentioned above resulting in the V-I-G-A-M and I-V-D-L patterns. While the V-I-G-A-M pattern is formed by the residues of (+) subunit (V278, I280, G281, A282, and M284 of M3), the I-V-D-L pattern contains residues of both subunits: I273 of M2-M3, D277 and V278 of M3 of (+) subunit and L218 of M1 of (–) subunit. Finally, the spiroketal group interacts with M-T-F-C-M-I of (+) subunit (M284, T285, and F288 of M3) and (–) subunit (С225, M226, and I229 of M1). As opposed to other functional groups, the disaccharide is located outside of the binding site pocket. It interacts with I273 of M2-M3 of (+) subunit and L217, L218, and I222 of M1 of (–) subunit; however, considering that these residues are not united spatially, no pattern for the disaccharide can be determined based on the structural information which was analyzed. The determined structural patterns of IVM allosteric interaction with GluClα can be used in search of IVM binding site on its potential targets, in the development of hypotheses of IVM binding to identified sites, and to rationalize the drug design of new GluCl ligands. Conclusions. The structural patterns with high affinity for IVM functional groups have been determined based on the results of HYDE assessment and visual analysis of IVM-GluClα complexes and the possible implementations of patterns knowledge have been described. The identified patterns can be further corrected and extended using the structural information of other IVM targets deposited in PDB.
{"title":"THE SEARCH OF STRUCTURAL PATTERNS OF IVERMECTIN ALLOSTERIC INTERACTION WITH GLUTAMATE-GATED CHLORIDE CHANNEL OF Caenorhabditis elegans","authors":"Y. O. Kustovskiy","doi":"10.15407/biotech16.02.032","DOIUrl":"https://doi.org/10.15407/biotech16.02.032","url":null,"abstract":"The aim of this research was to determine the structural patterns of IVM allosteric interaction with residues of its binding site located in the transmembrane domain of α-homopentameric glutamate-gated chloride channel (GluClα) of Caenorhabditis elegans. Methods. To consider different conformational states of IVM binding site two complexes of IVM bound to C. elegans GluClα (each with five site conformations) with identifiers 3RHW and 3RIF were obtained from PDB. The structures were examined in Analyzer Mode of SeeSAR v.12.1.0, in which contributions of IVM atoms into the complex affinity and their interactions with site structural patterns were determined for each site conformation using the HYDE scoring function. The residues belonging to identified structural patterns were classified by their properties using the Taylor’s classification of amino acids. Results. According to the results, the benzofuran group is critical for IVM recognition and binding: it interacts with the T-A-S-N-D-I-L-Q-I-P pattern, which is formed by T257, A258, S260, and N264 of M2, D277 and I280 of M3 of (+) subunit and L218, Q219, I222, P223 of M1 of (–) subunit. Due to the size and hydrophobicity of macrocycle, its different parts interact with residues of all site-forming structural elements mentioned above resulting in the V-I-G-A-M and I-V-D-L patterns. While the V-I-G-A-M pattern is formed by the residues of (+) subunit (V278, I280, G281, A282, and M284 of M3), the I-V-D-L pattern contains residues of both subunits: I273 of M2-M3, D277 and V278 of M3 of (+) subunit and L218 of M1 of (–) subunit. Finally, the spiroketal group interacts with M-T-F-C-M-I of (+) subunit (M284, T285, and F288 of M3) and (–) subunit (С225, M226, and I229 of M1). As opposed to other functional groups, the disaccharide is located outside of the binding site pocket. It interacts with I273 of M2-M3 of (+) subunit and L217, L218, and I222 of M1 of (–) subunit; however, considering that these residues are not united spatially, no pattern for the disaccharide can be determined based on the structural information which was analyzed. The determined structural patterns of IVM allosteric interaction with GluClα can be used in search of IVM binding site on its potential targets, in the development of hypotheses of IVM binding to identified sites, and to rationalize the drug design of new GluCl ligands. Conclusions. The structural patterns with high affinity for IVM functional groups have been determined based on the results of HYDE assessment and visual analysis of IVM-GluClα complexes and the possible implementations of patterns knowledge have been described. The identified patterns can be further corrected and extended using the structural information of other IVM targets deposited in PDB.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46019124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.037
O. Platonov, US I.V.
Aim. In our work, we studied platelet aggregation in blood plasma of pregnant women and estimated the possibility of ex vivo normalization of aggregation rate using a polypeptide from the Echis multisquamatus snake venom. Previous reports demonstrated that it directly interacts with glycoprotein IIb/IIIa receptors on the surface of platelets, preventing their adhesion, thereby affecting the degree of aggregation. Methods. chromatography followed by size-exclusion chromatography on Superdex 75 using the FPLC system (ӒKTA, GE Healthcare, USA). Analysis of molecular weight of protein components was performed using SDS-PAGE. The concentration of protein was measured using spectrophotometer Optizen POP (Korea) at 280 nm. The ability of obtained protein to inhibit platelet aggregation was measured directly by aggregometry. Blood samples of women with placental disfunction during pregnancy (n = 28) were kindly provided by “Perinatal Center of Kyiv”. This study was approved by the Ethics Commission of the Shupyk National Medical Academy of Postgraduate Education and the Ethics Commission of the Kyiv Perinatal Center (# 3 from 05/05/2020). Aggregation of platelet-rich plasma (PRP) induced by ADP was investigated using aggregometry on the AP 2110 (Solar, Belarus). We compared the rate of platelet aggregation in the presence vs absence of platelet aggregation inhibitor. Results. Two-step chromatography protocol allowed us to obtain the polypeptide from the venom of Echis multisquamatus that possessed the anti-aggregatory action. SDS-PAGE analysis confirmed the homogeneity of obtained polypeptide with apparent molecular weight 14 kDa that corresponds to the platelet aggregation inhibitor reported earlier. Initial studies of ADPinduced platelet aggregation allowed selecting active concentration for the effective inhibitory action as 0.02 mg/ml. Conclusions. Platelet aggregation inhibitor from Echis multisquamatis snake venom of can be assumed as the effective agent that reduce the rate of platelet aggregation. We demonstrated it efficacy in platelet rich plasma of pregnant women that had placenta dysfunction. The use of direct antagonist of platelet integrin receptors was assumed as the prospective approach for suppressing of platelet reactivity in particular during complicated pregnancy.
{"title":"Ex vivo STUDY OF THE ACTION OF INTEGRIN RECEPTORS ANTAGONIST FROM ECHIS MULTISQUAMATIS SNAKE VENOM ON PLATELETS OF PREGNANT WOMEN WITH COMPICATIONS DURING GESTATION","authors":"O. Platonov, US I.V.","doi":"10.15407/biotech16.02.037","DOIUrl":"https://doi.org/10.15407/biotech16.02.037","url":null,"abstract":"Aim. In our work, we studied platelet aggregation in blood plasma of pregnant women and estimated the possibility of ex vivo normalization of aggregation rate using a polypeptide from the Echis multisquamatus snake venom. Previous reports demonstrated that it directly interacts with glycoprotein IIb/IIIa receptors on the surface of platelets, preventing their adhesion, thereby affecting the degree of aggregation. Methods. chromatography followed by size-exclusion chromatography on Superdex 75 using the FPLC system (ӒKTA, GE Healthcare, USA). Analysis of molecular weight of protein components was performed using SDS-PAGE. The concentration of protein was measured using spectrophotometer Optizen POP (Korea) at 280 nm. The ability of obtained protein to inhibit platelet aggregation was measured directly by aggregometry. Blood samples of women with placental disfunction during pregnancy (n = 28) were kindly provided by “Perinatal Center of Kyiv”. This study was approved by the Ethics Commission of the Shupyk National Medical Academy of Postgraduate Education and the Ethics Commission of the Kyiv Perinatal Center (# 3 from 05/05/2020). Aggregation of platelet-rich plasma (PRP) induced by ADP was investigated using aggregometry on the AP 2110 (Solar, Belarus). We compared the rate of platelet aggregation in the presence vs absence of platelet aggregation inhibitor. Results. Two-step chromatography protocol allowed us to obtain the polypeptide from the venom of Echis multisquamatus that possessed the anti-aggregatory action. SDS-PAGE analysis confirmed the homogeneity of obtained polypeptide with apparent molecular weight 14 kDa that corresponds to the platelet aggregation inhibitor reported earlier. Initial studies of ADPinduced platelet aggregation allowed selecting active concentration for the effective inhibitory action as 0.02 mg/ml. Conclusions. Platelet aggregation inhibitor from Echis multisquamatis snake venom of can be assumed as the effective agent that reduce the rate of platelet aggregation. We demonstrated it efficacy in platelet rich plasma of pregnant women that had placenta dysfunction. The use of direct antagonist of platelet integrin receptors was assumed as the prospective approach for suppressing of platelet reactivity in particular during complicated pregnancy.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45713606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-04-28DOI: 10.15407/biotech16.02.047
Y. Tsaryk
Aim. The study of molecular mechanisms of hemostasis balance is one of the most vivid tasks for clinical biochemistry. In present communication we aimed to underline the constant connection between blood coagulation and fibrinolysis. Methods. Blood coagulation activation was estimated by the soluble fibrin accumulation. For it determination we used the sandwich ELISA method. As the catch-antibody we used fibrin-specific mAb FnI-3C. As the tag-antibody we used another mAb (II-4d) that has an epitope in the NH 2 -terminal fragment of the γ-chain of the D-region of the fibrin(ogen) molecule. The rate of activation of fibrinolysis was estimated by measuring of Fibrinolytic Potential (FP). It was measured by turbidimetric method with recording the scattering of light by a fibrin clot at 405 nm on a microplate reader Multiscan (Finland). The clot was formed in the microplate wells in blood plasma activated by APTT reagent in the presence or without t-PA. Results. SF was found in blood plasma of 12 pregnant women with placenta dysfunction. Six of studied patients had SF less than 4 µg/ml that were assumed as the control meanings. We divided patients on two groups according to this parameter. It was shown that patients of the 1st group (SF ≤ 4) exhibited FP as 24 ou/s. In the same time patients of the 2nd group (SF ≥ 4) had much higher FP – 62 ou/s. The level of statistical significance was P = 0,05. Conclusions. Blood coagulation activation (estimated by SF measurement) was shown to be accompanied by fibrinolysis activity increasing (measured by FP evaluation) in pregnant women with placental dysfunctions. These findings can be evidence of constant balance between blood coagulation and fibrinolysis that stabilize hemostasis in pathological conditions for avoiding thrombosis or hemorrhages.
{"title":"FIBRINOLYTIC POTENTIAL INCREASING DURING ACTIVATION OF BLOOD COAGULATION IN THE COURSE OF PREGNANCY WITH PLACENTAL DYSFUNCTION","authors":"Y. Tsaryk","doi":"10.15407/biotech16.02.047","DOIUrl":"https://doi.org/10.15407/biotech16.02.047","url":null,"abstract":"Aim. The study of molecular mechanisms of hemostasis balance is one of the most vivid tasks for clinical biochemistry. In present communication we aimed to underline the constant connection between blood coagulation and fibrinolysis. Methods. Blood coagulation activation was estimated by the soluble fibrin accumulation. For it determination we used the sandwich ELISA method. As the catch-antibody we used fibrin-specific mAb FnI-3C. As the tag-antibody we used another mAb (II-4d) that has an epitope in the NH 2 -terminal fragment of the γ-chain of the D-region of the fibrin(ogen) molecule. The rate of activation of fibrinolysis was estimated by measuring of Fibrinolytic Potential (FP). It was measured by turbidimetric method with recording the scattering of light by a fibrin clot at 405 nm on a microplate reader Multiscan (Finland). The clot was formed in the microplate wells in blood plasma activated by APTT reagent in the presence or without t-PA. Results. SF was found in blood plasma of 12 pregnant women with placenta dysfunction. Six of studied patients had SF less than 4 µg/ml that were assumed as the control meanings. We divided patients on two groups according to this parameter. It was shown that patients of the 1st group (SF ≤ 4) exhibited FP as 24 ou/s. In the same time patients of the 2nd group (SF ≥ 4) had much higher FP – 62 ou/s. The level of statistical significance was P = 0,05. Conclusions. Blood coagulation activation (estimated by SF measurement) was shown to be accompanied by fibrinolysis activity increasing (measured by FP evaluation) in pregnant women with placental dysfunctions. These findings can be evidence of constant balance between blood coagulation and fibrinolysis that stabilize hemostasis in pathological conditions for avoiding thrombosis or hemorrhages.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48478877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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