Pub Date : 2023-06-30DOI: 10.15407/biotech16.03.065
Bhatia Sudhir
Aim: The Isolation of nucleic acid is an important step for conducting different molecular assays in many laboratories around the world. It is also a common practice that user is isolating the ribonucleic acid (RNA) from the samples with mini column once and throwing away the supernatant. This makes isolated RNA as limiting factor in many studies as this issue has not been addressed in literature. Therefore, we decided to conduct whether it is a loss of ribonucleic acid during the mini column isolation method. Method: In this research, the mini column isolations were done with different samples of human tissues from placenta and umbilical cords and subsequent isolations of supernatants. Yields and successful isolations of RNA were assessed with spectrometric instrument and real time PCR machine. Results: It was found that there is loss of abundant quantity of RNA during the subsequent isolations. The amount measured with UV spectrometer indicates that some times 2nd and 3rd isolation have more RNA than the first isolation. Realtime PCR for house keeping gene beta actin shows that presence of RNA can be seen up to 6 isolation cycles from supernatants. Conclusion: There is loss of RNA in subsequent isolations with mini column method, therefore it is possible to isolate more RNA from subsequent supernatant isolations. User should do the multiple isolations to get higher yield of RNA.
目的:在世界各地的许多实验室中，核酸的分离是进行不同分子分析的重要步骤。通常的做法是用户用迷你柱从样品中分离核糖核酸(RNA)一次，并将上清扔掉。这使得分离的RNA在许多研究中成为限制因素，因为这个问题在文献中没有得到解决。因此，我们决定在迷你柱分离法中进行是否为核糖核酸的丢失。方法:采用胎盘和脐带不同的人体组织样品进行微柱分离，然后分离上清。用光谱仪和实时PCR仪对RNA的产率和成功分离进行评估。结果:在随后的分离过程中发现大量的RNA丢失。紫外分光光度计测定的数量表明，有时第2和第3分离株比第1分离株含有更多的RNA。实时聚合酶链反应(real - time PCR)显示，从上清液中可以看到多达6个分离周期的RNA的存在。结论:小柱法在后续分离中存在RNA损失，因此可以从后续的上清分离中分离出更多的RNA。用户应进行多次分离以获得更高的RNA产率。
{"title":"LOSS OF AN ABUNDANT QUANTITY OF RIBONUCLEIC ACID DURING MINI COLUMN ISOLATION METHOD","authors":"Bhatia Sudhir","doi":"10.15407/biotech16.03.065","DOIUrl":"https://doi.org/10.15407/biotech16.03.065","url":null,"abstract":"Aim: The Isolation of nucleic acid is an important step for conducting different molecular assays in many laboratories around the world. It is also a common practice that user is isolating the ribonucleic acid (RNA) from the samples with mini column once and throwing away the supernatant. This makes isolated RNA as limiting factor in many studies as this issue has not been addressed in literature. Therefore, we decided to conduct whether it is a loss of ribonucleic acid during the mini column isolation method. Method: In this research, the mini column isolations were done with different samples of human tissues from placenta and umbilical cords and subsequent isolations of supernatants. Yields and successful isolations of RNA were assessed with spectrometric instrument and real time PCR machine. Results: It was found that there is loss of abundant quantity of RNA during the subsequent isolations. The amount measured with UV spectrometer indicates that some times 2nd and 3rd isolation have more RNA than the first isolation. Realtime PCR for house keeping gene beta actin shows that presence of RNA can be seen up to 6 isolation cycles from supernatants. Conclusion: There is loss of RNA in subsequent isolations with mini column method, therefore it is possible to isolate more RNA from subsequent supernatant isolations. User should do the multiple isolations to get higher yield of RNA.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67094928","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 : 2023-06-30DOI: 10.15407/biotech16.03.045
N. Matvieieva
Wild plant species are of great interest as a source of pharmacologically valuable compounds but a great number of them are endemic and/or endangered ones. Modern plant biotechnology can provide reliable methods for their utilization without disturbing natural populations. In vitro culture methods for Rhodiola species are being intensively developed to include them into various biotechnological programmes. Aim. Development of a protocol for direct Rhodiola rosea L. plant regeneration from leaf explants. Methods. The leaves of R. rosea aseptically growing plants were used as the explants. Several variants of Murashige and Skoog (1962) agar-solidified culture medium supplemented with different combinations of auxins (1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)) and cytokinins (kinetin and 6-benzylaminopurine (BAP)) were estimated as potential regeneration-inducing media. Regeneration frequency was calculated as the percentage of leaves that produced shoots. Results. The use of MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot formation with 100% frequency. An increase in the 2,4-D content up to 2.5 mg/l and decrease in BAP content to 1.0 mg/l resulted in decreasing of the regeneration frequency to 62.5%. Regeneration frequency was 25% and 62%, respectively, on the media containing 1.0 mg/l kinetin + 2.5 mg/l 2,4-D and 2.5 mg/l kinetin + 1.0 mg/l 2,4-D. Conclusions. R. rosea leaf explants have demonstrated high regeneration capacity with using the studied combinations of plant growth regulators. MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot regeneration in leaf explants with the frequency of 100%. The frequency of regeneration was lower in the case of substitution of BAP for kinetin. The other types of morphogenesis (formation of adventitious roots and/or callus) were also observed.
{"title":"In vitro DIRECT SHOOT REGENERATION FROM Rhodiola rosea L. LEAF EXPLANTS","authors":"N. Matvieieva","doi":"10.15407/biotech16.03.045","DOIUrl":"https://doi.org/10.15407/biotech16.03.045","url":null,"abstract":"Wild plant species are of great interest as a source of pharmacologically valuable compounds but a great number of them are endemic and/or endangered ones. Modern plant biotechnology can provide reliable methods for their utilization without disturbing natural populations. In vitro culture methods for Rhodiola species are being intensively developed to include them into various biotechnological programmes. Aim. Development of a protocol for direct Rhodiola rosea L. plant regeneration from leaf explants. Methods. The leaves of R. rosea aseptically growing plants were used as the explants. Several variants of Murashige and Skoog (1962) agar-solidified culture medium supplemented with different combinations of auxins (1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)) and cytokinins (kinetin and 6-benzylaminopurine (BAP)) were estimated as potential regeneration-inducing media. Regeneration frequency was calculated as the percentage of leaves that produced shoots. Results. The use of MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot formation with 100% frequency. An increase in the 2,4-D content up to 2.5 mg/l and decrease in BAP content to 1.0 mg/l resulted in decreasing of the regeneration frequency to 62.5%. Regeneration frequency was 25% and 62%, respectively, on the media containing 1.0 mg/l kinetin + 2.5 mg/l 2,4-D and 2.5 mg/l kinetin + 1.0 mg/l 2,4-D. Conclusions. R. rosea leaf explants have demonstrated high regeneration capacity with using the studied combinations of plant growth regulators. MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot regeneration in leaf explants with the frequency of 100%. The frequency of regeneration was lower in the case of substitution of BAP for kinetin. The other types of morphogenesis (formation of adventitious roots and/or callus) were also observed.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43711911","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 : 2023-06-30DOI: 10.15407/biotech16.03.059
I.M. Malinovska
The purpose was to study the patterns of dissolution (solubilization) of phosphorus-containing minerals in aqueous and polysaccharide solutions of organic acids in order to model the mechanism of mineral destruction by soil bacteria synthesizing organic acids and exopolysaccharides. Methods. Model, laboratory-analytical, microbiological, statistical. Results. The destructive effect of organic acids on minerals is manifested both in aqueous and polysaccharide solutions. The introduction of bacterial polysaccharide into an aqueous solution of acids increases the decomposition of phosphorus-containing minerals by 1.34̶ 4.96 times. The influence of the chemical structure of acid molecules on the intensity of mineral decomposition is mainly manifested in the presence of bacterial polysaccharide, while in an aqueous solution the effectiveness of acid action depends on the nature of the mineral being destroyed. To the maximum degree, polysaccharide increases the destruction of minerals in a solution of citric acid: molten magnesium phosphate ̶ 2.55 times, thermophosphate ̶ 2.11 times, phosphate flour ̶ 4.96 times. Decomposition of phosphorus compounds in solutions of ascorbic and oxalic acids enhances bacterial polysaccharide to a lesser extent than in citric acid solution. Modeling the destruction of phosphorus-containing minerals under non-sterile conditions (soil conditions) made it possible to establish that organic acids under non-sterile conditions are subject to consumption by soil microbiota, especially ascorbic and citric acids, and to a lesser extent - succinic. Aqueous solutions of organic acids after 18 hours of incubation in non-sterile conditions lose their leaching activity by 1.06 ̶12.1 times. The introduction of a polysaccharide into aqueous solutions of acids makes it possible to avoid their rapid consumption by microorganisms, because of which the efficiency of mineral leaching under non-sterile conditions decreasшes by only 5–20% compared to sterile ones. Conclusions. The introduction of a bacterial polysaccharide into a solution of organic acids enables the latter to be transferred to a sorbed state, as a result of which their susceptibility to consumption by microorganisms is sharply reduced. Thus, polysaccharide-forming bacteria not only destroy minerals more intensively than microorganisms synthesizing only low-molecular-weight metabolites, but also synthesize a more stable and long-term functioning leaching complex in the soil.
{"title":"DECOMPOSITION OF PHOSPHORUS-CONTAINING COMPOUNDS IN AQUEOUS AND POLYSACCHARIDE SOLUTIONS OF ORGANIC ACIDS","authors":"I.M. Malinovska","doi":"10.15407/biotech16.03.059","DOIUrl":"https://doi.org/10.15407/biotech16.03.059","url":null,"abstract":"The purpose was to study the patterns of dissolution (solubilization) of phosphorus-containing minerals in aqueous and polysaccharide solutions of organic acids in order to model the mechanism of mineral destruction by soil bacteria synthesizing organic acids and exopolysaccharides. Methods. Model, laboratory-analytical, microbiological, statistical. Results. The destructive effect of organic acids on minerals is manifested both in aqueous and polysaccharide solutions. The introduction of bacterial polysaccharide into an aqueous solution of acids increases the decomposition of phosphorus-containing minerals by 1.34̶ 4.96 times. The influence of the chemical structure of acid molecules on the intensity of mineral decomposition is mainly manifested in the presence of bacterial polysaccharide, while in an aqueous solution the effectiveness of acid action depends on the nature of the mineral being destroyed. To the maximum degree, polysaccharide increases the destruction of minerals in a solution of citric acid: molten magnesium phosphate ̶ 2.55 times, thermophosphate ̶ 2.11 times, phosphate flour ̶ 4.96 times. Decomposition of phosphorus compounds in solutions of ascorbic and oxalic acids enhances bacterial polysaccharide to a lesser extent than in citric acid solution. Modeling the destruction of phosphorus-containing minerals under non-sterile conditions (soil conditions) made it possible to establish that organic acids under non-sterile conditions are subject to consumption by soil microbiota, especially ascorbic and citric acids, and to a lesser extent - succinic. Aqueous solutions of organic acids after 18 hours of incubation in non-sterile conditions lose their leaching activity by 1.06 ̶12.1 times. The introduction of a polysaccharide into aqueous solutions of acids makes it possible to avoid their rapid consumption by microorganisms, because of which the efficiency of mineral leaching under non-sterile conditions decreasшes by only 5–20% compared to sterile ones. Conclusions. The introduction of a bacterial polysaccharide into a solution of organic acids enables the latter to be transferred to a sorbed state, as a result of which their susceptibility to consumption by microorganisms is sharply reduced. Thus, polysaccharide-forming bacteria not only destroy minerals more intensively than microorganisms synthesizing only low-molecular-weight metabolites, but also synthesize a more stable and long-term functioning leaching complex in the soil.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41362050","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 : 2023-06-30DOI: 10.15407/biotech16.03.024
O. M. Klyuchko
Radiation is an important and dangerous factor in contemporary reality in some regions of industrial countries, after technological accidents at nuclear objects, chemical enterprises, etc. This is also the reality of some contemporary military activities and armed conflicts. Radiation damages of organisms can arise also due to the natural reasons — aviation or space flights at high altitudes or even long stay on mountain heights. Natural reasons of such effects have been studied insufficiently for today. Purpose. To outline briefly some results of studies of the characteristics of ionizing radiation at different heights above the Earth. To describe briefly the influence of radiation factors on biological organisms and main mechanisms of these effects. To describe effects that cause pathological changes in organisms of people exposed to the low doses of radiation for a long time and methods of post-radiation rehabilitation of affected people in highlands conditions. Methods. Space satellite exploring of the Earth atmosphere at different altitudes above sea level with measurements of different characteristics of solar and galactic radiation (mainly X-ray, gamma radiation, as well as other types of ionizing radiation in some other ranges). Comparative analysis of the results of longterm observation of patients in hospital conditions using many standard laboratory methods of their states examinations. The conducted scientific research consisted of a complex of methodological techniques and approaches: clinical and physiological studies of respiratory and cardiovascular systems, hematological and immunological states, and functional state of higher nervous activity, mental and neurotic state; administration of antihypoxants, histochemical, biophysical and other methods were used to evaluate oxybiotic processes. Mathematical processing of the results, as well as methods of mathematical modeling was applied. Results. The results of the measurements of ionizing radiation levels during the satellite exploring of the Earth atmosphere at different altitudes were analyzed and presented in schemes. The mechanisms of damaging radiation effects in organisms at nano level were described: water radiolysis, “oxygen effect” as radio sensitizer, formation of various types of free radicals and peroxides with future consequences for organic compounds, cells, tissues, organs, and organisms. The results of medical treatment and rehabilitation at the EMBS of the persons irradiated by the low doses of radiation were presented, observed and discussed. Many of represented results were obtained thanks to the collective work of the great commands of our predecessors in science who searched for the possibilities of medical treatment and rehabilitation of patients who obtained low doses of radiation during long time. The contemporary results of possibilities of some developed pathological states pharmacological corrections were discussed; practical recommendations were done. Co
{"title":"RADIATION PHENOMENA: SOME NATURAL SOURCES, MECHANISMS OF EFFECTS, WAYS OF BIOLOGICAL ORGANISMS PROTECTION AND REHABILITATION","authors":"O. M. Klyuchko","doi":"10.15407/biotech16.03.024","DOIUrl":"https://doi.org/10.15407/biotech16.03.024","url":null,"abstract":"Radiation is an important and dangerous factor in contemporary reality in some regions of industrial countries, after technological accidents at nuclear objects, chemical enterprises, etc. This is also the reality of some contemporary military activities and armed conflicts. Radiation damages of organisms can arise also due to the natural reasons — aviation or space flights at high altitudes or even long stay on mountain heights. Natural reasons of such effects have been studied insufficiently for today. Purpose. To outline briefly some results of studies of the characteristics of ionizing radiation at different heights above the Earth. To describe briefly the influence of radiation factors on biological organisms and main mechanisms of these effects. To describe effects that cause pathological changes in organisms of people exposed to the low doses of radiation for a long time and methods of post-radiation rehabilitation of affected people in highlands conditions. Methods. Space satellite exploring of the Earth atmosphere at different altitudes above sea level with measurements of different characteristics of solar and galactic radiation (mainly X-ray, gamma radiation, as well as other types of ionizing radiation in some other ranges). Comparative analysis of the results of longterm observation of patients in hospital conditions using many standard laboratory methods of their states examinations. The conducted scientific research consisted of a complex of methodological techniques and approaches: clinical and physiological studies of respiratory and cardiovascular systems, hematological and immunological states, and functional state of higher nervous activity, mental and neurotic state; administration of antihypoxants, histochemical, biophysical and other methods were used to evaluate oxybiotic processes. Mathematical processing of the results, as well as methods of mathematical modeling was applied. Results. The results of the measurements of ionizing radiation levels during the satellite exploring of the Earth atmosphere at different altitudes were analyzed and presented in schemes. The mechanisms of damaging radiation effects in organisms at nano level were described: water radiolysis, “oxygen effect” as radio sensitizer, formation of various types of free radicals and peroxides with future consequences for organic compounds, cells, tissues, organs, and organisms. The results of medical treatment and rehabilitation at the EMBS of the persons irradiated by the low doses of radiation were presented, observed and discussed. Many of represented results were obtained thanks to the collective work of the great commands of our predecessors in science who searched for the possibilities of medical treatment and rehabilitation of patients who obtained low doses of radiation during long time. The contemporary results of possibilities of some developed pathological states pharmacological corrections were discussed; practical recommendations were done. Co","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44563865","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 : 2023-06-30DOI: 10.15407/biotech16.03.051
Yu. A. Shesterenko
L-DOPA (3,4-dihydroxyphenyl-L-alanine) is a drug of choice in Parkinson's disease treatment. However the chemical method of its synthesis has a number of drawbacks, so biotechnological approaches are being explored as an alternative. Aim. The goal is to develop a new affordable and effective method of biosynthesis of L-DOPA using mushroom tyrosinase, immobilized using an economical carrier, which ensures stability and enzyme multiple uses. Methods. Agaricus bisporus isolated tyrosinase was used in the work. L-DOPA biosynthesis was carried out in aqueous and organic medium. The obtained product was analyzed using mass spectrometry, specific rotation and melting point. The enzyme immobilization was carried out in poly-N-vinylpyrrolidone (PVP), the interaction with the carrier, pH-optimum and the application frequency were determined. Results. A partially purified preparation of tyrosinase was isolated from Agaricus bisporus. In aqueous solution in enzyme presence, only 5.1% of L-DOPA was obtained due to the subsequent formation of complex polycyclic compounds. The biosynthesis of L-DOPA derivative in methylene chloride with the addition of a buffer solution made it possible to obtain a product with a yield of 55%. Tyrosinase immobilized in PVP showed activity 30% higher than free in CH2Cl2 medium and carried out biocatalysis for 7 cycles. Conclusions. A method of L-DOPA synthesizing using an available biocatalyst based on immobilized tyrosinase was developed, which enabled to obtain L-DOPA during 7 cycles of use in a methylene chloride medium.
{"title":"L-DOPA BIOSYNTHESIS WITH Agaricus bisporus TYROSINASES ASSISTANCE","authors":"Yu. A. Shesterenko","doi":"10.15407/biotech16.03.051","DOIUrl":"https://doi.org/10.15407/biotech16.03.051","url":null,"abstract":"L-DOPA (3,4-dihydroxyphenyl-L-alanine) is a drug of choice in Parkinson's disease treatment. However the chemical method of its synthesis has a number of drawbacks, so biotechnological approaches are being explored as an alternative. Aim. The goal is to develop a new affordable and effective method of biosynthesis of L-DOPA using mushroom tyrosinase, immobilized using an economical carrier, which ensures stability and enzyme multiple uses. Methods. Agaricus bisporus isolated tyrosinase was used in the work. L-DOPA biosynthesis was carried out in aqueous and organic medium. The obtained product was analyzed using mass spectrometry, specific rotation and melting point. The enzyme immobilization was carried out in poly-N-vinylpyrrolidone (PVP), the interaction with the carrier, pH-optimum and the application frequency were determined. Results. A partially purified preparation of tyrosinase was isolated from Agaricus bisporus. In aqueous solution in enzyme presence, only 5.1% of L-DOPA was obtained due to the subsequent formation of complex polycyclic compounds. The biosynthesis of L-DOPA derivative in methylene chloride with the addition of a buffer solution made it possible to obtain a product with a yield of 55%. Tyrosinase immobilized in PVP showed activity 30% higher than free in CH2Cl2 medium and carried out biocatalysis for 7 cycles. Conclusions. A method of L-DOPA synthesizing using an available biocatalyst based on immobilized tyrosinase was developed, which enabled to obtain L-DOPA during 7 cycles of use in a methylene chloride medium.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41313849","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/biotech16.02.030
Y. Kucheriavyi
The aim of our study was to compare the structure of clots formed as a result of thrombin-induced fibrin polymerization in the presence or absence of monoclonal fibrin-specific antibodies fragments as factors that change the clot structure. We concentrated on the final stage of fibrin clot formation at maximal turbidity point for every sample. Methods. Fibrin polymerization was studied by transmission electron microscopy (TEM) of negatively contrasted samples on H-600 Transmission Electron Microscope (“Hitachi”,Japan); 1% water solution of uranyl acetate (“Merck”, Germany) was used as a negative contrast. For sample preparation, in sterile glass tubes were sequentially added 0.32 mg/mL human fibrinogen, 0.025 M CaCl2 in 0.05 M ammonium formiate buffer (pH 7.9), and a total sample volume was 0.22 mL. The polymerization of fibrin was initiated by the introduction of thrombin at a final concentration of 0.25 NIH/mL. After 180 s, aliquots were taken from the polymerization medium. Each aliquot was diluted to a final fibrinogen concentration of 0.07 mg/mL; 0.01 mL probes of fibrinogen solution were transferred to a carbon lattice, which was treated with a 1% uranyl acetate solution after 2 minutes. Investigations were per-formed using an H-600 electron microscope at 75 kV. Electron microscopic images were obtained at magnification of 20,000 -50,000. Results. Two monoclonal antibodies fragments were obtained towards the mixture of separated Aα-, Bβ- and γ-chains of fibrinogen. Antibodies fragments that were marked as III-1D and I-4A, had different epitopes within fragment Аα105-206 of D-region of fibrinogen. It was shown that addition of antibody fragment I-4A lead to formation of abnormal fibrils that were thinner than in the control sample and were organized in the dense network (Figure). Control sample exhibited the thick fibrils with well-structured classically organized network. The difference between control and I-4A samples demonstrated that antibody I-4A disrupted the structure of polymerized fibrin. In the same time the fibrils obtained in the presence of antibody fragment III-1D were closer to the control ones. Conclusions. TEM is an informative method for the study of the fibrin network formation. Its application allows to estimate the disruption in fi brin formation directly. In a combination with turbidity study and other functional tests TEM can provide important information about molecular mechanisms of clot formation.
{"title":"TRANSMISSION ELECTRON MICROSCOPY FOR THE DIRECT ANALYSIS OF FIBRIN CLOT STRUCTURE","authors":"Y. Kucheriavyi","doi":"10.15407/biotech16.02.030","DOIUrl":"https://doi.org/10.15407/biotech16.02.030","url":null,"abstract":"The aim of our study was to compare the structure of clots formed as a result of thrombin-induced fibrin polymerization in the presence or absence of monoclonal fibrin-specific antibodies fragments as factors that change the clot structure. We concentrated on the final stage of fibrin clot formation at maximal turbidity point for every sample. Methods. Fibrin polymerization was studied by transmission electron microscopy (TEM) of negatively contrasted samples on H-600 Transmission Electron Microscope (“Hitachi”,Japan); 1% water solution of uranyl acetate (“Merck”, Germany) was used as a negative contrast. For sample preparation, in sterile glass tubes were sequentially added 0.32 mg/mL human fibrinogen, 0.025 M CaCl2 in 0.05 M ammonium formiate buffer (pH 7.9), and a total sample volume was 0.22 mL. The polymerization of fibrin was initiated by the introduction of thrombin at a final concentration of 0.25 NIH/mL. After 180 s, aliquots were taken from the polymerization medium. Each aliquot was diluted to a final fibrinogen concentration of 0.07 mg/mL; 0.01 mL probes of fibrinogen solution were transferred to a carbon lattice, which was treated with a 1% uranyl acetate solution after 2 minutes. Investigations were per-formed using an H-600 electron microscope at 75 kV. Electron microscopic images were obtained at magnification of 20,000 -50,000. Results. Two monoclonal antibodies fragments were obtained towards the mixture of separated Aα-, Bβ- and γ-chains of fibrinogen. Antibodies fragments that were marked as III-1D and I-4A, had different epitopes within fragment Аα105-206 of D-region of fibrinogen. It was shown that addition of antibody fragment I-4A lead to formation of abnormal fibrils that were thinner than in the control sample and were organized in the dense network (Figure). Control sample exhibited the thick fibrils with well-structured classically organized network. The difference between control and I-4A samples demonstrated that antibody I-4A disrupted the structure of polymerized fibrin. In the same time the fibrils obtained in the presence of antibody fragment III-1D were closer to the control ones. Conclusions. TEM is an informative method for the study of the fibrin network formation. Its application allows to estimate the disruption in fi brin formation directly. In a combination with turbidity study and other functional tests TEM can provide important information about molecular mechanisms of clot formation.","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":"43026265","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/biotech16.02.021
O. Hrabovskyi
The aim of this work is to study the intermolecular interactions of fibrin with D-domain-containing fragments of fibrin(ogen): D-dimer and D-fragment. Materials and methods. Human fibrinogen was obtained from the human blood plasma by salt extraction using 16 % Na2SO4. The content of protein coagulated by thrombin – 96-98%. Analytical size-exclusion chromatography for the detection of molecular complexes was performed on the Sepharose 6B column (30 x 0.5 cm). Components of the analyzed mixture (0.8 ml) were separated by standard chromatography protocol: speed of elution – 0.5 ml/min; collected samples volume – 0.5 ml. Optical density of collected samples was measured by spectrophotometer POP (Optizen, Daejeon, Korea). Composition of each sample was analyzed by SDS-PAGE. Relative amounts of studied compounds in samples were analyzed using densitometry of scanned electropherograms with Totallab TL100 software. Molecular modeling of complexes formed by fibrin desAB and its fragments were performed using UCSF Chimera 1.16 on the basis on earlier developed protofibril structure. The structure of the D-region (PDB ID:1LTJ) was prepared in the same in-protein molecular docking was performed using HDOCK web server. Results. To investigate the complex formation between fibrin desAB. The appearance of D- and DD-fragments in the elution zone of 5.5 mL, which does not overlap with the elution zone of individual fragments (7.5-9.5 mL), was detected, indicating the formation of a ternary complex. Densitometry of electropherograms using TotalLab TL-100 demonstrated that the average densities of pixels in bands of fibrin desAB, D-dimer and D-fragment were equal. It means that the ternary complex of fibrin desAB with D-dimer and D-fragment was composed in the approximate ratio of fibrin desAB, D-dimer and D-fragment 1:1:1. Molecular docking in the HDOCK software was used to establish the spatial arrangement of the D-fragment in relation to the fibrin desAB molecule bound to the D-dimer. Conclusions. We obtained and characterized the ternary complex of fibrin desAB, D-dimer and D-fragment by size-exclusion chromatography followed by SDS-PAGE. Further study of the structure and properties of this complex may clarify certain issues related to fibrin polymerization, namely the process of protofibril formation and their spatial branching.
{"title":"DETECTION OF TERNARY COMPLEX OF FIBRIN DESAB WITH D-DIMER AND D-FRAGMENT OF FIBRIN","authors":"O. Hrabovskyi","doi":"10.15407/biotech16.02.021","DOIUrl":"https://doi.org/10.15407/biotech16.02.021","url":null,"abstract":"The aim of this work is to study the intermolecular interactions of fibrin with D-domain-containing fragments of fibrin(ogen): D-dimer and D-fragment. Materials and methods. Human fibrinogen was obtained from the human blood plasma by salt extraction using 16 % Na2SO4. The content of protein coagulated by thrombin – 96-98%. Analytical size-exclusion chromatography for the detection of molecular complexes was performed on the Sepharose 6B column (30 x 0.5 cm). Components of the analyzed mixture (0.8 ml) were separated by standard chromatography protocol: speed of elution – 0.5 ml/min; collected samples volume – 0.5 ml. Optical density of collected samples was measured by spectrophotometer POP (Optizen, Daejeon, Korea). Composition of each sample was analyzed by SDS-PAGE. Relative amounts of studied compounds in samples were analyzed using densitometry of scanned electropherograms with Totallab TL100 software. Molecular modeling of complexes formed by fibrin desAB and its fragments were performed using UCSF Chimera 1.16 on the basis on earlier developed protofibril structure. The structure of the D-region (PDB ID:1LTJ) was prepared in the same in-protein molecular docking was performed using HDOCK web server. Results. To investigate the complex formation between fibrin desAB. The appearance of D- and DD-fragments in the elution zone of 5.5 mL, which does not overlap with the elution zone of individual fragments (7.5-9.5 mL), was detected, indicating the formation of a ternary complex. Densitometry of electropherograms using TotalLab TL-100 demonstrated that the average densities of pixels in bands of fibrin desAB, D-dimer and D-fragment were equal. It means that the ternary complex of fibrin desAB with D-dimer and D-fragment was composed in the approximate ratio of fibrin desAB, D-dimer and D-fragment 1:1:1. Molecular docking in the HDOCK software was used to establish the spatial arrangement of the D-fragment in relation to the fibrin desAB molecule bound to the D-dimer. Conclusions. We obtained and characterized the ternary complex of fibrin desAB, D-dimer and D-fragment by size-exclusion chromatography followed by SDS-PAGE. Further study of the structure and properties of this complex may clarify certain issues related to fibrin polymerization, namely the process of protofibril formation and their spatial branching.","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":"43122081","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/biotech16.02.035
A.Y. NEVIDNYK-PRAVDA
Aim. To investigate the development and treatment with imidopyran and prednisolone of hemolytic anemia in dogs caused by the protozoan parasite Babesia canis. Methods. 17 domestic dogs weighing 5-10 kg aged 2-5 years were used for the study. The parameters of the general blood analysis were determined using the MicroCC-20 Plus automated hematology analyzer (HTI, USA). Microscopy with a Leica DM4 electric microscope (Germany) was carried out to study the condition of erythrocytes, counting the number of leukocytes and platelets. Results. The main indicator of the development of anemia in animals is the number of erythrocytes, hemoglobin, and hematocrit. Development of babesiosis lead to the hemolytic anemia investigated in dogs before treatment: the number of erythrocytes lower than normal by 20-30%, the level of hemoglobin 40-55%, the average concentration of hemoglobin in erythrocytes 10- 18%, hematocrit 20-30% and the number of platelets 40-50%. Conclusions. The results of the study showed that treatment with imidopyran and prednisolone is effective in cases of babesiosis for dogs caused by the protozoan parasite Babesia canis. Moreover, such treatment decreases the risks of the anemic state development for these animals.
{"title":"EFFECTS OF IMIDOPYRAN AND PREDNISONE IN THE TREATMENT OF BABESIOSIOSIS-ASSOCIATED ANEMIA IN DOGS","authors":"A.Y. NEVIDNYK-PRAVDA","doi":"10.15407/biotech16.02.035","DOIUrl":"https://doi.org/10.15407/biotech16.02.035","url":null,"abstract":"Aim. To investigate the development and treatment with imidopyran and prednisolone of hemolytic anemia in dogs caused by the protozoan parasite Babesia canis. Methods. 17 domestic dogs weighing 5-10 kg aged 2-5 years were used for the study. The parameters of the general blood analysis were determined using the MicroCC-20 Plus automated hematology analyzer (HTI, USA). Microscopy with a Leica DM4 electric microscope (Germany) was carried out to study the condition of erythrocytes, counting the number of leukocytes and platelets. Results. The main indicator of the development of anemia in animals is the number of erythrocytes, hemoglobin, and hematocrit. Development of babesiosis lead to the hemolytic anemia investigated in dogs before treatment: the number of erythrocytes lower than normal by 20-30%, the level of hemoglobin 40-55%, the average concentration of hemoglobin in erythrocytes 10- 18%, hematocrit 20-30% and the number of platelets 40-50%. Conclusions. The results of the study showed that treatment with imidopyran and prednisolone is effective in cases of babesiosis for dogs caused by the protozoan parasite Babesia canis. Moreover, such treatment decreases the risks of the anemic state development for these animals.","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":"49521611","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/biotech16.02.026
M. V. Ivanochko
Aim. The purpose of this work was to investigate effects of the consumption of broccoli sprouts on the activity of antioxidant defense enzymes in the liver of. GPx isoforms. After photo fixation of gels, we determined activity of each isoform densitometrically using ImageJ software. Results were statistically analyzed by ANOVA followed by post-hoc Tukey's test. Data are presented as mean±SEM. Methods. Frozen tissue was homozeniged in lysis buffer, centrifuged and resulted supernatants were used for analysis. Activities of enzymes were determined by using electrophoresis in polyacrylamide gel. After electrophoresis we conducted dyeing of separate gels for detection of superoxide dismutase (SOD), glutathione-S-transferase (GST) or glutathione peroxidase (GPx) isoforms. After photo fixation of gels, we determined activity of each isoform densitometrically using ImageJ software. Results were statistically analyzed by ANOVA followed by post-hoc Tukey's test. Data are presented as mean±SEM. Results. In the hepatic tissue of all four groups of mice, two isoforms of SOD (SOD1 and SOD2) were detected in gels. The intensity of the bands of both isoforms was not significantly different between groups. Three isoforms of GST (GST1, GST2, GST3) were detected in the liver samples. The activity of GST1 did not significantly differ between the experimental groups. Activities of GST2 and GST3 forms were significantly higher in the group “Broccoli” compared to Control and Cafeteria Diet groups. We identified three isoforms of glutathione peroxidase (GPx1, GPx2, GPx3) in liver samples. The activity of GPx isoform 1 was not significantly different between the experimental groups. The activity of GPx2 was significantly higher in the group of mice that consumed Broccoli and Cafeteria Diet + Broccoli compared to Control. GPx2 activity was significantly higher in the Broccoli group compared to the Cafeteria Diet group. Activity of GPx3 was significantly higher in the Broccoli group compared to the Control and Cafeteria Diet group. Conclusions. Cafeteria diet did not significantly affect the activity of SOD isoforms, but led to redistribution of in the activity of GST and GPx isoforms in murine liver. Feeding with broccoli spouts significantly increased the activity of 2 and 3 isoforms of GST and GPx in murine liver compared to values in control mice and mice fed with cafeteria diet. Combination of Broccoli + Cafereria diet had small activating effects on antioxidant enzyme acivity, compared with cafeteria diet.
的目标。本研究旨在探讨食用西兰花芽对大鼠肝脏抗氧化防御酶活性的影响。GPx亚型。凝胶光固定后，我们用ImageJ软件测定每个异构体的密度活性。结果采用方差分析和事后Tukey检验进行统计学分析。数据以平均值±SEM表示。方法。冷冻组织在裂解缓冲液中匀浆，离心，所得上清液用于分析。用聚丙烯酰胺凝胶电泳法测定了酶的活性。电泳后对分离凝胶进行染色，检测超氧化物歧化酶(SOD)、谷胱甘肽- s -转移酶(GST)和谷胱甘肽过氧化物酶(GPx)异构体。凝胶光固定后，我们用ImageJ软件测定每个异构体的密度活性。结果采用方差分析和事后Tukey检验进行统计学分析。数据以平均值±SEM表示。结果。在所有四组小鼠的肝组织中，凝胶中检测到SOD的两种异构体(SOD1和SOD2)。两种异构体的条带强度在组间无显著差异。肝脏样品中检测到GST的三种亚型(GST1、GST2、GST3)。各组间GST1活性差异无统计学意义。“西兰花”组的GST2和GST3活性显著高于对照组和自助饮食组。我们在肝脏样本中鉴定出谷胱甘肽过氧化物酶的三种亚型(GPx1, GPx2, GPx3)。GPx异构体1的活性在各试验组间无显著差异。与对照组相比，食用西兰花和自助饮食+西兰花组的GPx2活性明显更高。与自助饮食组相比，西兰花组的GPx2活性明显更高。与对照组和自助饮食组相比，西兰花组的GPx3活性明显更高。结论。自助饮食对小鼠肝脏SOD活性无显著影响，但导致GST和GPx活性的重新分布。与对照组和自助饮食小鼠相比，食用西兰花芽可显著提高小鼠肝脏中GST和GPx 2和3亚型的活性。与自助饲料相比，西兰花+自助饲料组合对抗氧化酶活性的激活作用较小。
{"title":"CONSUMPTION OF BROCCOLI SPROUTS INCREASED THE ACTIVITY OF GLUTATHIONE-DEPENDENT ANTIOXIDANT ENZYMES IN MURINE LIVER","authors":"M. V. Ivanochko","doi":"10.15407/biotech16.02.026","DOIUrl":"https://doi.org/10.15407/biotech16.02.026","url":null,"abstract":"Aim. The purpose of this work was to investigate effects of the consumption of broccoli sprouts on the activity of antioxidant defense enzymes in the liver of. GPx isoforms. After photo fixation of gels, we determined activity of each isoform densitometrically using ImageJ software. Results were statistically analyzed by ANOVA followed by post-hoc Tukey's test. Data are presented as mean±SEM. Methods. Frozen tissue was homozeniged in lysis buffer, centrifuged and resulted supernatants were used for analysis. Activities of enzymes were determined by using electrophoresis in polyacrylamide gel. After electrophoresis we conducted dyeing of separate gels for detection of superoxide dismutase (SOD), glutathione-S-transferase (GST) or glutathione peroxidase (GPx) isoforms. After photo fixation of gels, we determined activity of each isoform densitometrically using ImageJ software. Results were statistically analyzed by ANOVA followed by post-hoc Tukey's test. Data are presented as mean±SEM. Results. In the hepatic tissue of all four groups of mice, two isoforms of SOD (SOD1 and SOD2) were detected in gels. The intensity of the bands of both isoforms was not significantly different between groups. Three isoforms of GST (GST1, GST2, GST3) were detected in the liver samples. The activity of GST1 did not significantly differ between the experimental groups. Activities of GST2 and GST3 forms were significantly higher in the group “Broccoli” compared to Control and Cafeteria Diet groups. We identified three isoforms of glutathione peroxidase (GPx1, GPx2, GPx3) in liver samples. The activity of GPx isoform 1 was not significantly different between the experimental groups. The activity of GPx2 was significantly higher in the group of mice that consumed Broccoli and Cafeteria Diet + Broccoli compared to Control. GPx2 activity was significantly higher in the Broccoli group compared to the Cafeteria Diet group. Activity of GPx3 was significantly higher in the Broccoli group compared to the Control and Cafeteria Diet group. Conclusions. Cafeteria diet did not significantly affect the activity of SOD isoforms, but led to redistribution of in the activity of GST and GPx isoforms in murine liver. Feeding with broccoli spouts significantly increased the activity of 2 and 3 isoforms of GST and GPx in murine liver compared to values in control mice and mice fed with cafeteria diet. Combination of Broccoli + Cafereria diet had small activating effects on antioxidant enzyme acivity, compared with cafeteria diet.","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":"46334298","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/biotech16.02.024
E. Iskandarov
Aim. In this study we focused on the search of fibrinogen-targeted proteases in the venom of Vipera lebetina. Methods. Fractionation of the venom was performed using FPLC chromatographic system Acta Prime on Q Sepharose. Analysis of protein mixtures was performed using SDS-PAGE. Аction on blood coagulation system was analyzed using APTT assay [2]. Proteolytic action on fibrinogen and identification of protein components with fibrinolytic activity was performed using electrophoresis of mixture of fibrinogen solution (2 mg/ml) with venom`s fractions. For a comprehensive evaluation of the effect of the obtained fractions on hemostasis, an original approach with modified aggregatometry was used [3]. This approach made it possible to take into account the ability of fractions to activate platelets, initiate blood coagulation, or inhibit platelet aggregation. Hemolytic action of fractions was estimated using fresh human red cells. Amount of released hemoglobin was estimated by spectrophotometry on Optizen POP. Results. Crude venom of V. lebetina was fractionated using ion-exchange chromatography on Q Sepharose. Elution was performed using a stepwise gradient of NaCl (0.1, 0.2, 0.3, 0.5, and 0.7 M NaCl) in 0.05 M Tris-HCl buffer at pH 8.3. Fractions eluted at 0.1 and 0.2 M of NaCl contained several proteins with different molecular weights ranging from 75 kDa to low molecular weight fractions according to the SDS-PAGE. Proteins that cleave α- and β-chains of fibrinogen were found in fractions 0.1 and 0.2, indicating the presence of an enzyme with fibrinogenolytic activity in the venom of V. lebetina. The fractions 0.3, 0.5, and 0.7 did not show any significant fibrinogenolytic activity. After platelet aggregation study we concluded that fraction 0.1 contained a protein with fibrinogenolytic activity. An increase in platelet aggregation was observed for the fraction 0.2 after the addition of ADP. This may indicate the presence of an active compound that promotes platelet aggregation. Further research is necessary to determine its nature. Fractions 0.3, 0.5, and 0.7 had no effect on platelet aggregation. A decrease in blood plasma clotting time in APTT to 5 s and 7 s, compared to a control value of 70 s, was shown for fractions eluted at NaCl concentrations of 0.1 M and 0.2 M, respectively. The fractions 0.3, 0.5 had only a slight effect on reducing blood plasma clotting. A slightly increased level of hemolysis was shown in the presence of the unbound fraction and the whole venom. It can be suggested that proteins with phospholipase activity are present in the non-binded fraction. Conclusions. Thus, fibrinogen-specific proteases, hemolytic agents, activators of blood clotting were found in the venom of Vipera lebetina. Most of these compounds must to be purified and can be used for basic biochemical research.
的目标。在本研究中，我们着重于在白蝮蛇毒液中寻找纤维蛋白原靶向蛋白酶。方法。采用FPLC色谱系统对Q Sepharose进行分离。用SDS-PAGE对蛋白混合物进行分析。应用APTT试验[2]分析Аction对凝血系统的影响。采用纤维蛋白原溶液(2mg /ml)与蛇毒组分混合电泳，对纤维蛋白原的蛋白水解作用和具有纤维蛋白溶解活性的蛋白组分进行鉴定。为了全面评估所获得的分数对止血的影响，我们采用了一种原始的方法，即改良的聚集法。这种方法使得考虑到组分激活血小板、启动血液凝固或抑制血小板聚集的能力成为可能。用新鲜的人红细胞估计其溶血作用。Optizen POP分光光度法测定血红蛋白释放量。结果。采用Q - Sepharose离子交换色谱法对芦笋粗毒液进行分离。在pH 8.3的0.05 M Tris-HCl缓冲液中，使用NaCl(0.1、0.2、0.3、0.5和0.7 M NaCl)逐步梯度洗脱。SDS-PAGE显示，在0.1和0.2 M NaCl洗脱的组分中含有不同分子量的蛋白质，分子量从75 kDa到低分子量不等。在0.1和0.2组分中发现了切割纤维蛋白原α-和β-链的蛋白质，这表明在紫叶弧菌毒液中存在一种具有纤维蛋白原裂解活性的酶。分数0.3、0.5和0.7未显示出任何显著的纤维蛋白原溶解活性。经过血小板聚集研究，我们得出结论，0.1馏分含有一种具有纤维蛋白原溶解活性的蛋白质。在加入ADP后，0.2分数的血小板聚集增加。这可能表明存在一种促进血小板聚集的活性化合物。需要进一步的研究来确定其性质。分数0.3、0.5和0.7对血小板聚集无影响。NaCl浓度分别为0.1 M和0.2 M时，APTT血浆凝固时间分别缩短至5 s和7 s，而对照值为70 s。分数0.3、0.5对降低血浆凝块只有轻微的作用。在未结合的部分和整个毒液存在时，溶血水平略有增加。可以认为，在非结合部分中存在具有磷脂酶活性的蛋白质。结论。因此，纤维蛋白原特异性蛋白酶，溶血剂，血液凝固活化剂被发现在蛇毒中。这些化合物大部分必须经过纯化，才能用于基础生化研究。
{"title":"ACTION OF VENOM OF VIPERA LEBETINA ON BLOOD COAGULATION in vitro","authors":"E. Iskandarov","doi":"10.15407/biotech16.02.024","DOIUrl":"https://doi.org/10.15407/biotech16.02.024","url":null,"abstract":"Aim. In this study we focused on the search of fibrinogen-targeted proteases in the venom of Vipera lebetina. Methods. Fractionation of the venom was performed using FPLC chromatographic system Acta Prime on Q Sepharose. Analysis of protein mixtures was performed using SDS-PAGE. Аction on blood coagulation system was analyzed using APTT assay [2]. Proteolytic action on fibrinogen and identification of protein components with fibrinolytic activity was performed using electrophoresis of mixture of fibrinogen solution (2 mg/ml) with venom`s fractions. For a comprehensive evaluation of the effect of the obtained fractions on hemostasis, an original approach with modified aggregatometry was used [3]. This approach made it possible to take into account the ability of fractions to activate platelets, initiate blood coagulation, or inhibit platelet aggregation. Hemolytic action of fractions was estimated using fresh human red cells. Amount of released hemoglobin was estimated by spectrophotometry on Optizen POP. Results. Crude venom of V. lebetina was fractionated using ion-exchange chromatography on Q Sepharose. Elution was performed using a stepwise gradient of NaCl (0.1, 0.2, 0.3, 0.5, and 0.7 M NaCl) in 0.05 M Tris-HCl buffer at pH 8.3. Fractions eluted at 0.1 and 0.2 M of NaCl contained several proteins with different molecular weights ranging from 75 kDa to low molecular weight fractions according to the SDS-PAGE. Proteins that cleave α- and β-chains of fibrinogen were found in fractions 0.1 and 0.2, indicating the presence of an enzyme with fibrinogenolytic activity in the venom of V. lebetina. The fractions 0.3, 0.5, and 0.7 did not show any significant fibrinogenolytic activity. After platelet aggregation study we concluded that fraction 0.1 contained a protein with fibrinogenolytic activity. An increase in platelet aggregation was observed for the fraction 0.2 after the addition of ADP. This may indicate the presence of an active compound that promotes platelet aggregation. Further research is necessary to determine its nature. Fractions 0.3, 0.5, and 0.7 had no effect on platelet aggregation. A decrease in blood plasma clotting time in APTT to 5 s and 7 s, compared to a control value of 70 s, was shown for fractions eluted at NaCl concentrations of 0.1 M and 0.2 M, respectively. The fractions 0.3, 0.5 had only a slight effect on reducing blood plasma clotting. A slightly increased level of hemolysis was shown in the presence of the unbound fraction and the whole venom. It can be suggested that proteins with phospholipase activity are present in the non-binded fraction. Conclusions. Thus, fibrinogen-specific proteases, hemolytic agents, activators of blood clotting were found in the venom of Vipera lebetina. Most of these compounds must to be purified and can be used for basic biochemical research.","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":"41797141","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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