Pub Date : 2022-06-30DOI: 10.15407/biotech15.03.035
G. Andriiash
The aim of the work was to obtain a producer strain with increased lysine accumulation using the chemical mutagenesis method. Methods. To achieve the goal, we used the method of treating the lysine-producing strain with the chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine, cultivating the resulting clone and determining the accumulation of lysine in flasks and a bioreactor. Results. The optimal concentrations and duration of mutagen action for the production of mutant microorganisms were found. Сlones with the maximum lysine accumulation were selected. Mutagenesis was carried out consecutively three times. As a result, lysine-producing strain Brevibacterium sp. IMV B-7796 auxotrophic regarding leucine and threonine with maximum accumulation of the target amino acid was obtained. Conclusions. The lysine producer Brevibacterium sp. IMV B-7796 was obtained, which produced 65.0 g/dm3 of lysine in a bioreactor under conditions of periodic cultivation with feeding. The Brevibacterium sp. IMV B-7796 strain was proposed as a basis for the creation of a genetically modified strain with increased accumulation of lysine for further use in industrial lysine technology.
{"title":"CHEMICAL MUTAGENESIS OF THE LYSINE-PRODUCING STRAIN Brevibacterium sp. IMV B-7447","authors":"G. Andriiash","doi":"10.15407/biotech15.03.035","DOIUrl":"https://doi.org/10.15407/biotech15.03.035","url":null,"abstract":"The aim of the work was to obtain a producer strain with increased lysine accumulation using the chemical mutagenesis method. Methods. To achieve the goal, we used the method of treating the lysine-producing strain with the chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine, cultivating the resulting clone and determining the accumulation of lysine in flasks and a bioreactor. Results. The optimal concentrations and duration of mutagen action for the production of mutant microorganisms were found. Сlones with the maximum lysine accumulation were selected. Mutagenesis was carried out consecutively three times. As a result, lysine-producing strain Brevibacterium sp. IMV B-7796 auxotrophic regarding leucine and threonine with maximum accumulation of the target amino acid was obtained. Conclusions. The lysine producer Brevibacterium sp. IMV B-7796 was obtained, which produced 65.0 g/dm3 of lysine in a bioreactor under conditions of periodic cultivation with feeding. The Brevibacterium sp. IMV B-7796 strain was proposed as a basis for the creation of a genetically modified strain with increased accumulation of lysine for further use in industrial lysine technology.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47225134","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 : 2022-06-30DOI: 10.15407/biotech15.03.043
V. Toptikov
Aim. The purpose of the work was to determine the effect of trichloroacetic (TCA) and perchloric (HClO4) acids on the result of ninhydrin reaction with various amino acids. Methods. A standard method of amino acid detection using a ninhydrin reagent was applied. Optical spectra and density of reaction products were determined spectrophotometrically. Results and conclusions. As a result, it was found that the studied acids change the spectral characteristics of the products of the ninhydrin reaction with amino acids. TCA significantly reduced the optical density of chromophores, and HClO4 also led to a significant shift of the spectra of the reaction products into the short-wavelength region. An exception was the reaction with proline, as a result of which a well-defined maximum appeared in the product spectrum: λ= 620 nm in the presence of TCA and λ=515 nm with HClO4. At the same time, in the presence of HClO4, the reaction became highly specific for proline. Conditions. The ninhydrin reaction with proline upon addition of HClO4 were analyzed in detail. As a result, a new method of highly specific determination of proline in the presence of other amino acids was proposed.
{"title":"THE INFLUENCE OF ACID PROTEIN PRECIPITANTS ON THE SPECIFICITY OF THE REACTION OF NINHYDRIN WITH AMINO ACIDS","authors":"V. Toptikov","doi":"10.15407/biotech15.03.043","DOIUrl":"https://doi.org/10.15407/biotech15.03.043","url":null,"abstract":"Aim. The purpose of the work was to determine the effect of trichloroacetic (TCA) and perchloric (HClO4) acids on the result of ninhydrin reaction with various amino acids. Methods. A standard method of amino acid detection using a ninhydrin reagent was applied. Optical spectra and density of reaction products were determined spectrophotometrically. Results and conclusions. As a result, it was found that the studied acids change the spectral characteristics of the products of the ninhydrin reaction with amino acids. TCA significantly reduced the optical density of chromophores, and HClO4 also led to a significant shift of the spectra of the reaction products into the short-wavelength region. An exception was the reaction with proline, as a result of which a well-defined maximum appeared in the product spectrum: λ= 620 nm in the presence of TCA and λ=515 nm with HClO4. At the same time, in the presence of HClO4, the reaction became highly specific for proline. Conditions. The ninhydrin reaction with proline upon addition of HClO4 were analyzed in detail. As a result, a new method of highly specific determination of proline in the presence of other amino acids was proposed.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45944077","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}
{"title":"Interprofessional Training and Commitment to Research increasingly useful for the Health Professions.","authors":"Leopoldo Sarli, Massimo Guasconi, Giovanna Artioli","doi":"10.23750/abm.v93iS2.13182","DOIUrl":"10.23750/abm.v93iS2.13182","url":null,"abstract":"<p><p>Editorial.</p>","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":"10 1","pages":"e2022241"},"PeriodicalIF":0.0,"publicationDate":"2022-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9534220/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82726354","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-04-01DOI: 10.15407/biotech15.02.068
O. Rudnytska
Aim. In this study we investigate the impact of low doses of graphene oxide on the expression of key regulatory genes which control cell proliferation as well as microRNAs in normal human astrocytes. Methods. The expression level of genes related to cell proliferation was studied by real-time qPCR in normal human astrocytes line NHA/TS (Cambrex Bio Science, Walkersville, MD, USA) using SYBRGreen Mix and specific for each mRNA forward and reverse primers. These astrocytes were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Graphene oxide (2 mg/ml, dispersion in water) was received from Sigma-Aldrich Chemie GmbH, Germany. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). For polyadenylation and reverse transcription of miRNAs we used Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The expression level of microRNAs was studied by real-time qPCR using SYBRGreen Mix and specific for each miRNA forward primers and universal reverse primer. For normalization of microRNA expressions the level of U6 RNA expression was used. Results. It was shown that the expression level of TOB1, HSPA5, EDEM1, MYBL1, and MYBL2 significantly increased in normal human astrocytes line NHA/TS, which were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Up-regulation of these genes expression was dose-dependent: bigger dose of graphene oxide (4 ng/ml of medium) introduced more significant changes in the expression of all these genes. Furthermore, bioinformatics analysis of 3′-untranslated regions of mRNA allowed identifying binding sites of microRNA: miR-19a for MYBL1, miR-143 for MYBL2 and miR-182 for TOB1. It was also shown that the expression of all these microRNA significantly down-regulated by graphene oxide, supporting the idea of both post-transcriptional and transcriptional regulation of MYBL1, MYBL2 and TOB1 gene expressions. Conclusions. Graphene oxide significantly disturbs genome stability by up-regulation of the expression of key regulatory genes and down-regulation of microRNA.
目标在这项研究中，我们研究了低剂量氧化石墨烯对正常人星形胶质细胞中控制细胞增殖的关键调控基因以及微小RNA表达的影响。方法。通过实时qPCR在正常人星形胶质细胞系NHA/TS（Cambrex Bio Science，Walkersville，MD，USA）中研究与细胞增殖相关的基因的表达水平，使用SYBRGreen Mix并对每种信使核糖核酸正向和反向引物特异。用氧化石墨烯（1和4ng/ml培养基）处理这些星形胶质细胞24小时。从德国Sigma-Aldrich Chemie GmbH获得氧化石墨烯。使用TRIZOL试剂提取总RNA。对于mRNA的逆转录，我们使用Thermo Scientific Verso cDNA合成试剂盒（德国）。将mRNA表达值标准化为ACTB mRNA水平，并表示为对照的百分比（100%）。对于miRNA的多腺苷酸化和逆转录，我们使用Mir-X miRNA第一链合成试剂盒（Takara，日本）。使用SYBRGreen Mix通过实时qPCR研究微小RNA的表达水平，并对每种miRNA正向引物和通用反向引物具有特异性。为了使微小RNA表达正常化，使用了U6 RNA表达水平。后果研究表明，在用氧化石墨烯（1和4ng/ml培养基）处理24小时的正常人星形胶质细胞系NHA/TS中，TOB1、HSPA5、EDEM1、MYBL1和MYBL2的表达水平显著增加。这些基因表达的上调是剂量依赖性的：更大剂量的氧化石墨烯（4 ng/ml培养基）会使所有这些基因的表达发生更显著的变化。此外，对信使核糖核酸3′-非翻译区的生物信息学分析允许鉴定微小核糖核酸的结合位点：MYBL1的miR-19a、MYBL2的miR-143和TOB1的miR-182。研究还表明，氧化石墨烯显著下调了所有这些微小RNA的表达，支持了MYBL1、MYBL2和TOB1基因表达的转录后和转录调控的观点。结论。氧化石墨烯通过上调关键调控基因的表达和下调微小RNA显著干扰基因组稳定性。
{"title":"GRAPHENE OXIDE AFFECT THE EXPRESSION OF PROLIFERATION RELATED GENES AND microRNA IN NORMAL HUMAN ASTROCYTES","authors":"O. Rudnytska","doi":"10.15407/biotech15.02.068","DOIUrl":"https://doi.org/10.15407/biotech15.02.068","url":null,"abstract":"Aim. In this study we investigate the impact of low doses of graphene oxide on the expression of key regulatory genes which control cell proliferation as well as microRNAs in normal human astrocytes. Methods. The expression level of genes related to cell proliferation was studied by real-time qPCR in normal human astrocytes line NHA/TS (Cambrex Bio Science, Walkersville, MD, USA) using SYBRGreen Mix and specific for each mRNA forward and reverse primers. These astrocytes were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Graphene oxide (2 mg/ml, dispersion in water) was received from Sigma-Aldrich Chemie GmbH, Germany. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). For polyadenylation and reverse transcription of miRNAs we used Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The expression level of microRNAs was studied by real-time qPCR using SYBRGreen Mix and specific for each miRNA forward primers and universal reverse primer. For normalization of microRNA expressions the level of U6 RNA expression was used. Results. It was shown that the expression level of TOB1, HSPA5, EDEM1, MYBL1, and MYBL2 significantly increased in normal human astrocytes line NHA/TS, which were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Up-regulation of these genes expression was dose-dependent: bigger dose of graphene oxide (4 ng/ml of medium) introduced more significant changes in the expression of all these genes. Furthermore, bioinformatics analysis of 3′-untranslated regions of mRNA allowed identifying binding sites of microRNA: miR-19a for MYBL1, miR-143 for MYBL2 and miR-182 for TOB1. It was also shown that the expression of all these microRNA significantly down-regulated by graphene oxide, supporting the idea of both post-transcriptional and transcriptional regulation of MYBL1, MYBL2 and TOB1 gene expressions. Conclusions. Graphene oxide significantly disturbs genome stability by up-regulation of the expression of key regulatory genes and down-regulation of microRNA.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46973198","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 : 2022-04-01DOI: 10.15407/biotech15.02.015
R. Kocatürk
With the development of molecular techniques over time more than %60 of epilepsy has associated with mitochondrial (mt) dysfunction. Ketogenic diet (KD) has been used in the treatment of epilepsy since the 1920s. Aim. To evaluate the evidence behind KD in mt dysfunction in epilepsy. Methods. Databases PubMed, Google Scholar and MEDLINE were searched in an umbrella approach to 12 March 2021 in English. To identify relevant studies specific search strategies were devised for the following topics: (1) mitochondrial dysfunction (2) epilepsy (3) KD treatment. Results. From 1794 papers, 36 articles were included in analysis: 16 (%44.44) preclinical studies, 11 (%30.55) case reports, 9 (%25) clinical studies. In all the preclinic studies, KD regulated the number of mt profiles, transcripts of metabolic enzymes and encoding mt proteins, protected the mice against to seizures and had an anticonvulsant mechanism. Case reports and clinical trials have reported patients with good results in seizure control and mt functions, although not all of them give good results as well as preclinical. Conclusion. Healthcare institutions, researchers, neurologists, health promotion organizations, and dietitians should consider these results to improve KD programs and disease outcomes for mt dysfunction in epilepsy.
{"title":"USE OF KETOGENIC DIET THERAPY IN EPILEPSY WITH MITOCHONDRIAL DYSFUNCTION: A SYSTEMATIC AND CRITICAL REVIEW","authors":"R. Kocatürk","doi":"10.15407/biotech15.02.015","DOIUrl":"https://doi.org/10.15407/biotech15.02.015","url":null,"abstract":"With the development of molecular techniques over time more than %60 of epilepsy has associated with mitochondrial (mt) dysfunction. Ketogenic diet (KD) has been used in the treatment of epilepsy since the 1920s. Aim. To evaluate the evidence behind KD in mt dysfunction in epilepsy. Methods. Databases PubMed, Google Scholar and MEDLINE were searched in an umbrella approach to 12 March 2021 in English. To identify relevant studies specific search strategies were devised for the following topics: (1) mitochondrial dysfunction (2) epilepsy (3) KD treatment. Results. From 1794 papers, 36 articles were included in analysis: 16 (%44.44) preclinical studies, 11 (%30.55) case reports, 9 (%25) clinical studies. In all the preclinic studies, KD regulated the number of mt profiles, transcripts of metabolic enzymes and encoding mt proteins, protected the mice against to seizures and had an anticonvulsant mechanism. Case reports and clinical trials have reported patients with good results in seizure control and mt functions, although not all of them give good results as well as preclinical. Conclusion. Healthcare institutions, researchers, neurologists, health promotion organizations, and dietitians should consider these results to improve KD programs and disease outcomes for mt dysfunction in epilepsy.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44691003","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 : 2022-04-01DOI: 10.15407/biotech15.02.060
Y. Kucheriavyi
Aim. The purpose of our study was to compare hydrolytic action of proteases from Gloydius halys halys, Agkistrodon contortrix contortrix and Calloselasma rhodostoma rhodostoma snake venoms and from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium on αC-regions of fibrinogen molecule. Methods. Products of hydrolysis were characterized by SDS-PAGE under reducing conditions with following Western-Blot using the mouse monoclonal 1-5А (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibody. MALDI-TOF analysis of fibrinogen hydrolysis products was performed using a Voyager-DE. Results. Combination of SDS-PAGE, FPLC and MALDI-TOF analysis enabled to detect the peptide bonds cleaved by studied proteases. In particular proteases from Gloydius halys halys and Agkistrodon contortrix contortrix snake venoms cleaved peptide bond Aα413-414. Action of protease from Calloselasma rhodostoma rhodostoma on fibrinogen led to the formation of hydrolytic product generated from C-terminal portion of Aα-chain that corresponded to fragments generated by enzymes from two other snakes. On the other hand protease from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium cleaved peptide bond Aα504-505. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.
{"title":"LIMITED PROTEOLYSIS OF FIBRINOGEN αC-REGION REVEALS ITS STRUCTURE","authors":"Y. Kucheriavyi","doi":"10.15407/biotech15.02.060","DOIUrl":"https://doi.org/10.15407/biotech15.02.060","url":null,"abstract":"Aim. The purpose of our study was to compare hydrolytic action of proteases from Gloydius halys halys, Agkistrodon contortrix contortrix and Calloselasma rhodostoma rhodostoma snake venoms and from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium on αC-regions of fibrinogen molecule. Methods. Products of hydrolysis were characterized by SDS-PAGE under reducing conditions with following Western-Blot using the mouse monoclonal 1-5А (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibody. MALDI-TOF analysis of fibrinogen hydrolysis products was performed using a Voyager-DE. Results. Combination of SDS-PAGE, FPLC and MALDI-TOF analysis enabled to detect the peptide bonds cleaved by studied proteases. In particular proteases from Gloydius halys halys and Agkistrodon contortrix contortrix snake venoms cleaved peptide bond Aα413-414. Action of protease from Calloselasma rhodostoma rhodostoma on fibrinogen led to the formation of hydrolytic product generated from C-terminal portion of Aα-chain that corresponded to fragments generated by enzymes from two other snakes. On the other hand protease from Bacillus thuringiensis вар. israelensis IMV B-7465 culture medium cleaved peptide bond Aα504-505. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47464267","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 : 2022-04-01DOI: 10.15407/biotech15.02.053
I. V. Gavrylyak
The aim of our study was to evaluate tear levels of some protein endpoints that can reflect intensities of hypoxia, angiogenesis and tissue remodeling in wounded cornea. Methods. We examined 21 patients (21 eyes) with nonpenetrating corneal injuries. The patients underwent standard ophthalmological examination including previous history and ocular symptoms, visual acuity test, complete anterior and posterior eye segments examination using slit lamp biomicroscopy, evaluation of corneal staining with fluorescein, ophthalmoscopy. Healthy volunteers (n = 10) served as a control. Tear fluid was collected from patients and control volunteers with the use of a disposable tip micropipette. From the lower arch of the conjunctiva without instillation of anesthetic, tears were collected in a sterile plastic Eppendorf tube and frozen at -20 oC before laboratory examination. Proteins of tear fluids were separated by SDS-PAGE (loading 50 µg total protein per track). Then, levels of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and angiostatins were measured by western blot. Active MMP-9 levels were evaluated by gelatin zymography. The results of blot and zymography assays were processed by densitometric software and then analyzed statistically with the use of Mann-Whitney U-test. Results. Elevated HIF-1α (P<0.001) and angiostatins (P<0.05) levels were revealed by western blot in tear fluid samples collected from patients with injured cornea in comparison with the control group. It is noteworthy that extremely low amounts of VEGF were detected in tear fluid from injured eyes, in spite of abundance of its transcription inducer HIF-1α. Dramatically increased levels of active MMP-9 were found in the tear fluids of patients with corneal wounds, while no significant collagenolytic activity was observed in tears from healthy eyes. There is a strong correlation between extent of corneal lesions and changes in markers expression. Conclusions. Tear levels of HIF-1α and angiostatin as well as MMP-9 activity could represent valuable biomarkers of corneal injury severity in traumatic eye.
{"title":"PROTEIN MARKERS OF HYPOXIA AND ANGIOGENESIS IN TEAR FLUID OF PATIENTS WITH TRAUMATIC CORNEAL INJURY","authors":"I. V. Gavrylyak","doi":"10.15407/biotech15.02.053","DOIUrl":"https://doi.org/10.15407/biotech15.02.053","url":null,"abstract":"The aim of our study was to evaluate tear levels of some protein endpoints that can reflect intensities of hypoxia, angiogenesis and tissue remodeling in wounded cornea. Methods. We examined 21 patients (21 eyes) with nonpenetrating corneal injuries. The patients underwent standard ophthalmological examination including previous history and ocular symptoms, visual acuity test, complete anterior and posterior eye segments examination using slit lamp biomicroscopy, evaluation of corneal staining with fluorescein, ophthalmoscopy. Healthy volunteers (n = 10) served as a control. Tear fluid was collected from patients and control volunteers with the use of a disposable tip micropipette. From the lower arch of the conjunctiva without instillation of anesthetic, tears were collected in a sterile plastic Eppendorf tube and frozen at -20 oC before laboratory examination. Proteins of tear fluids were separated by SDS-PAGE (loading 50 µg total protein per track). Then, levels of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and angiostatins were measured by western blot. Active MMP-9 levels were evaluated by gelatin zymography. The results of blot and zymography assays were processed by densitometric software and then analyzed statistically with the use of Mann-Whitney U-test. Results. Elevated HIF-1α (P<0.001) and angiostatins (P<0.05) levels were revealed by western blot in tear fluid samples collected from patients with injured cornea in comparison with the control group. It is noteworthy that extremely low amounts of VEGF were detected in tear fluid from injured eyes, in spite of abundance of its transcription inducer HIF-1α. Dramatically increased levels of active MMP-9 were found in the tear fluids of patients with corneal wounds, while no significant collagenolytic activity was observed in tears from healthy eyes. There is a strong correlation between extent of corneal lesions and changes in markers expression. Conclusions. Tear levels of HIF-1α and angiostatin as well as MMP-9 activity could represent valuable biomarkers of corneal injury severity in traumatic eye.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46853264","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 : 2022-04-01DOI: 10.15407/biotech15.02.056
E. Iskandarov
Aim. In this study we focused on the search of fibrinogen-targeted proteases in the venom of Vipera renardi, Vipera nikolskii and Vipera berus. Venom of Vipera berus was also fractionated on Q-sepharose and action of separated fractions on human blood plasma, platelets and red cells was studied. Methods. Analysis of protein mixtures was performed using SDS-PAGE. Аction on the blood coagulation system was analyzed using the APTT assay. Identification of protein components with fibrinolytic activity was performed using enzyme-electrophoresis with fibrinogen as the substrate. Fractionation of V. berus venom was performed on Q-sepharose using FPLC system Acta Prime. Action of separated fractions on ADP-induced platelet aggregation in platelet rich blood plasma was analyzed using Aggregometer AP 2110. Hemolytic action of fractions was estimated using fresh human red cells. Amount of released hemoglobin was estimated by spectrophotometry on Optizen POP. Results. All studied venoms had different protein compositions with major protein fractions in the range from 25 kDa to 130 kDa. Both V. berus and V. nikolskii venoms taken in 1:200 dilutions reduced the time of clotting in APTT test from 25 to 13 s. In contrast, V. renardi venom in the same dilution prolonged the clotting time from 25 s to 180 s that we assumed as the result of fibrinogen-specific protease presence. According to enzyme-electrophoresis data all studied venoms contained fibrinogen-specific proteases with the apparent molecular weights for V. berus, V. nikolskii – 25-55 kDa. and V. renardi – 55-75 kDa. Fractionation of crude venom of V. berus allowed obtaining several fractions eluted at different concentrations of NaCl: 0.1; 0.2; 0.3; 0.5 М. Non-binded fraction was also collected. Conclusions. Thus, the components of Vipera venoms living in Ukraine can be used for basic biochemical research. At the same time, care should be taken in the case of envenomation, as the presence of fibrinogenolytic enzymes in the venom can lead to hemorrhage. Further characterization of fibrinogen-specific protease from V. berus venom is a promising task for biotechnology.
{"title":"ACTION OF VENOM OF VIPERA SNAKE OF UKRAINE ON BLOOD COAGULATION in vitro","authors":"E. Iskandarov","doi":"10.15407/biotech15.02.056","DOIUrl":"https://doi.org/10.15407/biotech15.02.056","url":null,"abstract":"Aim. In this study we focused on the search of fibrinogen-targeted proteases in the venom of Vipera renardi, Vipera nikolskii and Vipera berus. Venom of Vipera berus was also fractionated on Q-sepharose and action of separated fractions on human blood plasma, platelets and red cells was studied. Methods. Analysis of protein mixtures was performed using SDS-PAGE. Аction on the blood coagulation system was analyzed using the APTT assay. Identification of protein components with fibrinolytic activity was performed using enzyme-electrophoresis with fibrinogen as the substrate. Fractionation of V. berus venom was performed on Q-sepharose using FPLC system Acta Prime. Action of separated fractions on ADP-induced platelet aggregation in platelet rich blood plasma was analyzed using Aggregometer AP 2110. Hemolytic action of fractions was estimated using fresh human red cells. Amount of released hemoglobin was estimated by spectrophotometry on Optizen POP. Results. All studied venoms had different protein compositions with major protein fractions in the range from 25 kDa to 130 kDa. Both V. berus and V. nikolskii venoms taken in 1:200 dilutions reduced the time of clotting in APTT test from 25 to 13 s. In contrast, V. renardi venom in the same dilution prolonged the clotting time from 25 s to 180 s that we assumed as the result of fibrinogen-specific protease presence. According to enzyme-electrophoresis data all studied venoms contained fibrinogen-specific proteases with the apparent molecular weights for V. berus, V. nikolskii – 25-55 kDa. and V. renardi – 55-75 kDa. Fractionation of crude venom of V. berus allowed obtaining several fractions eluted at different concentrations of NaCl: 0.1; 0.2; 0.3; 0.5 М. Non-binded fraction was also collected. Conclusions. Thus, the components of Vipera venoms living in Ukraine can be used for basic biochemical research. At the same time, care should be taken in the case of envenomation, as the presence of fibrinogenolytic enzymes in the venom can lead to hemorrhage. Further characterization of fibrinogen-specific protease from V. berus venom is a promising task for biotechnology.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67094919","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 : 2022-04-01DOI: 10.15407/biotech15.02.066
K. S. Romanenko
Aim. To study the possible protective effect of cannabimimetic lipid - N-stearoylethanolamine (NSE) on the lipid composition of the frontal cortex, hippocampus and on the state of episodic memory of old rats. Methods. Extraction of lipids from the tissues of the hippocampus and frontal cortex of rats was performed by the method of Bligh and Dyer. Phospholipids were separated by two-dimensional thin layer chromatography. Methyl esters of fatty acids from lipid extract were obtained by a modified method of Carreau and Dubaco. Quantitative analysis of fatty acid methyl esters was performed by gas-liquid chromatography on an Agilent GC7890 chromatograph with an Agilent 8987 mass detector. The fractions of free and esterified cholesterol were separated by one-dimensional thin layer chromatography. The dry cholesterol residue was analyzed on a Carlo Erba gas-liquid chromatograph. Results. The study of the diacyl (DF) and plasmalogen (PF) forms of phospholipids (PLs) content in the frontal cortex and hippocampus have shown a significant decrease in the plasmalogen form of PE (Phosphatidylethanolamine) (up to 15%) and an increase in its DF, compare to its content in young rats. Administration of NSE to old rats led to a significant increase in PF PE and did not cause significant changes in the content of PF in the composition of other PL of the frontal cortex of the brain and hippocampus. The decrease in the percentage of various phospholipids was found in frontal cortex and hippocampus of old rats: the content of phosphatidylcholine (PC) and phosphatidylinositol (PI) was significantly reduced in the frontal cortex and the decrease of diphosphatidylglycerol (DPG), PI and phosphatidylserine (PS) was found in the hippocampus, compare to the young animals. Administration of NSE to old rats had a different effects on the content of various phospholipids. The increase in the content of PC and PI in the frontal cortex and PS and DPG in the hippocampus is particularly pronounced due to NSE. An increase in the content of saturated fatty acids (FFAs ) and a decrease in the content of unsaturated FFAs in the frontal cortex and hippocampus of old rats also has been found. It has also been found that NSE administration to old rats promoted the growth of the free cholesterol level in the frontal cortex and hippocampus. The results of the New Object Recognition test in old rats have shown that a short-term memory has been improved by NSE. Conclusions. The administration of NSE to old rats causes an increase in PF of PLs in the frontal cortex and hippocampus of the brain, which can be considered as one of the mechanisms of neuroprotective action of NSE in aging. The changes in the phospholipids and fatty acids composition, and free cholesterol level of the frontal cortex and hippocampus of the brain of old rats caused by NSE administration have been shown to be adaptive and restorative. The New Object Recognition Behavioral Test has shown that NSE restores short-term m
{"title":"EFFECT OF N-STEAROYLETHANOLAMINE ON THE LIPID COMPOSITION OF THE FRONTAL CORTEX AND HIPPOCAMPUS OF THE RAT'S BRAIN AT THE AGING","authors":"K. S. Romanenko","doi":"10.15407/biotech15.02.066","DOIUrl":"https://doi.org/10.15407/biotech15.02.066","url":null,"abstract":"Aim. To study the possible protective effect of cannabimimetic lipid - N-stearoylethanolamine (NSE) on the lipid composition of the frontal cortex, hippocampus and on the state of episodic memory of old rats. Methods. Extraction of lipids from the tissues of the hippocampus and frontal cortex of rats was performed by the method of Bligh and Dyer. Phospholipids were separated by two-dimensional thin layer chromatography. Methyl esters of fatty acids from lipid extract were obtained by a modified method of Carreau and Dubaco. Quantitative analysis of fatty acid methyl esters was performed by gas-liquid chromatography on an Agilent GC7890 chromatograph with an Agilent 8987 mass detector. The fractions of free and esterified cholesterol were separated by one-dimensional thin layer chromatography. The dry cholesterol residue was analyzed on a Carlo Erba gas-liquid chromatograph. Results. The study of the diacyl (DF) and plasmalogen (PF) forms of phospholipids (PLs) content in the frontal cortex and hippocampus have shown a significant decrease in the plasmalogen form of PE (Phosphatidylethanolamine) (up to 15%) and an increase in its DF, compare to its content in young rats. Administration of NSE to old rats led to a significant increase in PF PE and did not cause significant changes in the content of PF in the composition of other PL of the frontal cortex of the brain and hippocampus. The decrease in the percentage of various phospholipids was found in frontal cortex and hippocampus of old rats: the content of phosphatidylcholine (PC) and phosphatidylinositol (PI) was significantly reduced in the frontal cortex and the decrease of diphosphatidylglycerol (DPG), PI and phosphatidylserine (PS) was found in the hippocampus, compare to the young animals. Administration of NSE to old rats had a different effects on the content of various phospholipids. The increase in the content of PC and PI in the frontal cortex and PS and DPG in the hippocampus is particularly pronounced due to NSE. An increase in the content of saturated fatty acids (FFAs ) and a decrease in the content of unsaturated FFAs in the frontal cortex and hippocampus of old rats also has been found. It has also been found that NSE administration to old rats promoted the growth of the free cholesterol level in the frontal cortex and hippocampus. The results of the New Object Recognition test in old rats have shown that a short-term memory has been improved by NSE. Conclusions. The administration of NSE to old rats causes an increase in PF of PLs in the frontal cortex and hippocampus of the brain, which can be considered as one of the mechanisms of neuroprotective action of NSE in aging. The changes in the phospholipids and fatty acids composition, and free cholesterol level of the frontal cortex and hippocampus of the brain of old rats caused by NSE administration have been shown to be adaptive and restorative. The New Object Recognition Behavioral Test has shown that NSE restores short-term m","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46592725","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 : 2022-04-01DOI: 10.15407/biotech15.02.058
R. S. Korshun
Aim. To determine the role of Ruk/CIN85 in the control of breast adenocarcinoma cells metabolism, we performed systemic analysis of the activity levels/content of key enzymes/components of glycolysis and oxidative phosphorylation using as a model the weakly invasive human breast adenocarcinoma MCF-7 cell line (Mock); and its sublines with stable overexpression (G4 subline) and reverse down-regulation (G4vir subline) of the adaptor protein. Materials and methods. MCF-7 cells were cultured in the complete DMEM 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. First of all, biochemical indexes of aerobic glycolysis, activity levels of some key glycolytic enzymes and metabolites were evaluated. A significant increase in the activity of these enzymes, aldolase A (ALDOA) and lactate dehydrogenase A (LDHA), was found in G4 cells compared to Mock by 1.3 and 1.6 times, respectively. In addition, in the conditioned medium of G4 cells, an increase in lactate content by 1.5 times compared with the control was found, which corresponded to a change in LDHA activity. Knockdown of Ruk/CIN85 expression level in G4 subline resulted in a significant decrease of these parameters compared to G4 cells, ALDOA – 4 times, LDHA - 1.4 times, and lactate production - 2.5 times. It should be noted that in G4vir cells, LDHA activity returned to level of control cells, while ALDOA activity and lactate content additionally decreased by 3 times and 1.6 times, respectively. Therefore, the observed changes in the intensity of glycolysis in MCF-7 sublines positively correlate with the expression level of adaptor protein studied. To assess the metabolic status of mitochondria, the level of activity of the Krebs cycle enzyme, NAD-dependent malate dehydrogenase (MDH2), the catalyst of last stage of the cycle, was determined. A 2-fold decrease in MDH2 activity was found in the MCF-7 G4 subline relative to control Mock cells, as well as an increase in this index by 2.4 times in G4vir cells to control values. Unlike glycolysis, we observed the opposite pattern with respect to the intensity of Krebs cycle reactions depending on the expression level of Ruk/CIN85. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.
{"title":"ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS","authors":"R. S. Korshun","doi":"10.15407/biotech15.02.058","DOIUrl":"https://doi.org/10.15407/biotech15.02.058","url":null,"abstract":"Aim. To determine the role of Ruk/CIN85 in the control of breast adenocarcinoma cells metabolism, we performed systemic analysis of the activity levels/content of key enzymes/components of glycolysis and oxidative phosphorylation using as a model the weakly invasive human breast adenocarcinoma MCF-7 cell line (Mock); and its sublines with stable overexpression (G4 subline) and reverse down-regulation (G4vir subline) of the adaptor protein. Materials and methods. MCF-7 cells were cultured in the complete DMEM 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. First of all, biochemical indexes of aerobic glycolysis, activity levels of some key glycolytic enzymes and metabolites were evaluated. A significant increase in the activity of these enzymes, aldolase A (ALDOA) and lactate dehydrogenase A (LDHA), was found in G4 cells compared to Mock by 1.3 and 1.6 times, respectively. In addition, in the conditioned medium of G4 cells, an increase in lactate content by 1.5 times compared with the control was found, which corresponded to a change in LDHA activity. Knockdown of Ruk/CIN85 expression level in G4 subline resulted in a significant decrease of these parameters compared to G4 cells, ALDOA – 4 times, LDHA - 1.4 times, and lactate production - 2.5 times. It should be noted that in G4vir cells, LDHA activity returned to level of control cells, while ALDOA activity and lactate content additionally decreased by 3 times and 1.6 times, respectively. Therefore, the observed changes in the intensity of glycolysis in MCF-7 sublines positively correlate with the expression level of adaptor protein studied. To assess the metabolic status of mitochondria, the level of activity of the Krebs cycle enzyme, NAD-dependent malate dehydrogenase (MDH2), the catalyst of last stage of the cycle, was determined. A 2-fold decrease in MDH2 activity was found in the MCF-7 G4 subline relative to control Mock cells, as well as an increase in this index by 2.4 times in G4vir cells to control values. Unlike glycolysis, we observed the opposite pattern with respect to the intensity of Krebs cycle reactions depending on the expression level of Ruk/CIN85. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45108020","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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