首页 > 最新文献

Biotechnologia Acta最新文献

英文 中文
ACTIVITY OF AMP DEAMINASE AND 5′-NUCLEOTIDASE IN THE CYTOSOLIC KIDNEY FRACTION OF RATS UNDER THE CONDITIONS OF DIFFERENT PROTEIN AND SUCROSE CONTENT IN A DIET 不同蛋白质和蔗糖含量条件下大鼠肾细胞质部分amp脱氨酶和5′-核苷酸酶活性的变化
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.049
A. S. Boichuk
ACTIVITY OF AMP DEAMINASE AND 5′-NUCLEOTIDASE IN THE CYTOSOLIC KIDNEY FRACTION OF RATS UNDER THE CONDITIONS OF DIFFERENT PROTEIN AND SUCROSE CONTENT IN A DIET
不同蛋白质和蔗糖含量条件下大鼠肾细胞质部分amp脱氨酶和5′-核苷酸酶活性的变化
{"title":"ACTIVITY OF AMP DEAMINASE AND 5′-NUCLEOTIDASE IN THE CYTOSOLIC KIDNEY FRACTION OF RATS UNDER THE CONDITIONS OF DIFFERENT PROTEIN AND SUCROSE CONTENT IN A DIET","authors":"A. S. Boichuk","doi":"10.15407/biotech15.02.049","DOIUrl":"https://doi.org/10.15407/biotech15.02.049","url":null,"abstract":"ACTIVITY OF AMP DEAMINASE AND 5′-NUCLEOTIDASE IN THE CYTOSOLIC KIDNEY FRACTION OF RATS UNDER THE CONDITIONS OF DIFFERENT PROTEIN AND SUCROSE CONTENT IN A DIET","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":"45394257","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}
引用次数: 0
INFLUENCE OF SYSTEMIC INFLAMMATORY RESPONSE SYNDROME ON THE DEVELOPMENT OF OXIDATIVE STRESS DURING SIMULATION OF CHRONIC ALCOHOL INTOXICATION IN RATS 慢性酒精中毒大鼠模拟过程中全身炎症反应综合征对氧化应激发展的影响
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.062
A. Mykytenko
The aim of our study was to analyze changes in the development of oxidative stress in the liver of rats with chronic alcohol intoxication against the background of systemic inflammatory response syndrome based on the study of catalase and superoxide dismutase activity, concentration of malonic dialdehyde, oxidatively modified proteins and sulfide anion and superoxide anion production. Methods. Experimental studies were performed on 12 male Wistar rats weighing 180‒220 g. Animals were divided into two groups: 1 ‒ control and 2 ‒ animals, on which we simulated alcoholic hepatitis and SIRS. The activity of catalase and superoxide dismutase (SOD), the concentration of malonic dialdehyde (MDA) , oxidatively modified proteins (OMP) sulfide anion and superoxide anion production were studied in the rat liver homogenate. The obtained results were subjected to statistical processing using the Mann-Whitney test. Results. Analyzing the development of oxidative stress in the liver of rats, on which we simulated the combined effects of SIRS and prolonged alcohol intoxication, we found that the activity of SOD increased by 1.72 times (P<0.05), and catalase decreased by 1.18 times (P<0.05) compared with the control group. The production of superoxide anion radical in the liver of rats increased 2.21 times (P<0.05) in the group of animals with combined exposure to bacterial LPS and alcohol intoxication compared to control. The concentration of MDA increased 2.25 times (P<0.05), and OMP by 9.5 times (P<0.05) compared with control group. The concentration of sulfide anion in the liver of rats under the conditions of modeling the combined effects of SIRS and alcohol intoxication decreased by 1.44 times (P <0.05) compared with the control. Conclusions. Modeling of alcohol intoxication against the background of systemic inflammatory response syndrome leads to oxidative damage to lipid and protein structures of the liver due to increased production of superoxide anion radical and imbalance of antiradical protection.
本研究旨在通过对过氧化氢酶和超氧化物歧化酶活性、丙二醛浓度、氧化修饰蛋白以及硫阴离子和超氧化物阴离子产生的研究,分析在全身炎症反应综合征背景下,慢性酒精中毒大鼠肝脏氧化应激发展的变化。方法。对12只体重180-220 g的雄性Wistar大鼠进行了实验研究。将动物分为两组:1组为对照组,2组为动物,在其上模拟酒精性肝炎和SIRS。研究了大鼠肝匀浆中过氧化氢酶和超氧化物歧化酶(SOD)的活性、丙二醛(MDA)的浓度、氧化修饰蛋白(OMP)的硫化物阴离子和超氧化物阴离子的产生。使用Mann-Whitney检验对所获得的结果进行统计处理。后果通过分析大鼠肝脏氧化应激的发展,模拟SIRS和长期酒精中毒的联合作用,发现与对照组相比,SOD活性增加了1.72倍(P<0.05),过氧化氢酶活性降低了1.18倍(P<0.01)。与对照组相比,细菌LPS和酒精中毒联合暴露组大鼠肝脏中超氧阴离子自由基的产生增加了2.21倍(P<0.05)。MDA浓度比对照组增加2.25倍(P<0.05),OMP增加9.5倍(P>0.05)。在模拟SIRS和酒精中毒联合作用的条件下,大鼠肝脏中硫阴离子浓度比对照组降低了1.44倍(P<0.05)。结论。在全身炎症反应综合征背景下对酒精中毒进行建模,由于超氧化物阴离子自由基的产生增加和抗自由基保护的失衡,导致肝脏脂质和蛋白质结构的氧化损伤。
{"title":"INFLUENCE OF SYSTEMIC INFLAMMATORY RESPONSE SYNDROME ON THE DEVELOPMENT OF OXIDATIVE STRESS DURING SIMULATION OF CHRONIC ALCOHOL INTOXICATION IN RATS","authors":"A. Mykytenko","doi":"10.15407/biotech15.02.062","DOIUrl":"https://doi.org/10.15407/biotech15.02.062","url":null,"abstract":"The aim of our study was to analyze changes in the development of oxidative stress in the liver of rats with chronic alcohol intoxication against the background of systemic inflammatory response syndrome based on the study of catalase and superoxide dismutase activity, concentration of malonic dialdehyde, oxidatively modified proteins and sulfide anion and superoxide anion production. Methods. Experimental studies were performed on 12 male Wistar rats weighing 180‒220 g. Animals were divided into two groups: 1 ‒ control and 2 ‒ animals, on which we simulated alcoholic hepatitis and SIRS. The activity of catalase and superoxide dismutase (SOD), the concentration of malonic dialdehyde (MDA) , oxidatively modified proteins (OMP) sulfide anion and superoxide anion production were studied in the rat liver homogenate. The obtained results were subjected to statistical processing using the Mann-Whitney test. Results. Analyzing the development of oxidative stress in the liver of rats, on which we simulated the combined effects of SIRS and prolonged alcohol intoxication, we found that the activity of SOD increased by 1.72 times (P<0.05), and catalase decreased by 1.18 times (P<0.05) compared with the control group. The production of superoxide anion radical in the liver of rats increased 2.21 times (P<0.05) in the group of animals with combined exposure to bacterial LPS and alcohol intoxication compared to control. The concentration of MDA increased 2.25 times (P<0.05), and OMP by 9.5 times (P<0.05) compared with control group. The concentration of sulfide anion in the liver of rats under the conditions of modeling the combined effects of SIRS and alcohol intoxication decreased by 1.44 times (P <0.05) compared with the control. Conclusions. Modeling of alcohol intoxication against the background of systemic inflammatory response syndrome leads to oxidative damage to lipid and protein structures of the liver due to increased production of superoxide anion radical and imbalance of antiradical protection.","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":"43663444","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}
引用次数: 0
GLUTAMINE DEPRIVATION AFFECTS THE EXPRESSION OF GENES WHICH CONTROL PYRUVATE DEHYDROGENASE ACTIVITY: THE IMPACT OF ERN1 KNOCKDOWN 谷氨酰胺剥夺影响控制丙酮酸脱氢酶活性的基因表达:ern1敲低的影响
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.070
M. Sliusar
The aim of the current investigation was to study the expression of genes encoded pyruvate dehydrogenase subunits (PDHA1, PDHB, PDHX, DLAT, and DLD) in U87 glioma cells in response to glutamine deprivation in U87 glioma cells in relation to knockdown of ERN1 for evaluation of a possible dependence of the expression of these important regulatory genes from glutamine supply and ERN1 signaling. Methods. The expression of PDHA1, PDHB, PDHX, DLAT, and DLD genes was studied by real-time qPCR in control U87 glioma cells (transfected by vector) and cells with knockdown of ERN1 (transfected by dnERN1) after exposure to glutamine deprivation condition. Total RNA was extracted from glioma cells using TRIZOL reagent. An RNA quantity as well as spectral characteristics was measured using NanoDrop One. 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 %). Results. It was shown that the expression level of PDH1, PDHB, DLAT, and DLD genes was down-regulated in control glioma cells treated by glutamine deprivation. At the same time, ERN1 knockdown is suppressed the effect of glutamine deprivation on PDHB and DLD gene expressions in glioma cells, but did not change significantly the impact of glutamine deprivation on the expression of PDHA1, DLAT, and PDHX genes. Conclusions. The results of this investigation demonstrated that the expression of PDH1, PDHB, PDHX, DLAT, and DLD genes was significantly affected by exposure of U87 glioma cells under glutamine deprivation condition and that the effect of glutamine deprivation on the expression of most these genes was modified in cells with knockdown of ERN1, a major signaling pathway of the endoplasmic reticulum stress.
当前研究的目的是研究U87胶质瘤细胞中编码丙酮酸脱氢酶亚基(PDHA1、PDHB、PDHX、DLAT和DLD)基因的表达,以响应谷氨酰胺剥夺U87胶质瘤细胞中ERN1的敲低,以评估这些重要调控基因的表达可能依赖于谷氨酰胺供应和ERN1信号传导。方法。采用实时荧光定量pcr技术研究了谷氨酰胺剥夺条件下U87胶质瘤细胞(载体转染)和ERN1敲低细胞(dnERN1转染)中PDHA1、PDHB、PDHX、DLAT和DLD基因的表达。用TRIZOL试剂从胶质瘤细胞中提取总RNA。使用NanoDrop One测量RNA数量和光谱特性。对于mrna的逆转录,我们使用Thermo Scientific Verso cDNA合成试剂盒(德国)。将mRNA表达值归一化至ACTB mRNA水平,并以对照(100%)的百分比表示。结果。结果表明,在谷氨酰胺剥夺的对照胶质瘤细胞中,PDH1、PDHB、DLAT和DLD基因的表达水平下调。同时,ERN1敲低抑制了谷氨酰胺剥夺对胶质瘤细胞PDHB和DLD基因表达的影响,但没有显著改变谷氨酰胺剥夺对PDHA1、DLAT和PDHX基因表达的影响。结论。本研究结果表明,U87胶质瘤细胞暴露在谷氨酰胺剥夺条件下,PDH1、PDHB、PDHX、DLAT和DLD基因的表达受到显著影响,并且在内质网应激的主要信号通路ERN1被敲低的细胞中,谷氨酰胺剥夺对这些基因表达的影响被修饰。
{"title":"GLUTAMINE DEPRIVATION AFFECTS THE EXPRESSION OF GENES WHICH CONTROL PYRUVATE DEHYDROGENASE ACTIVITY: THE IMPACT OF ERN1 KNOCKDOWN","authors":"M. Sliusar","doi":"10.15407/biotech15.02.070","DOIUrl":"https://doi.org/10.15407/biotech15.02.070","url":null,"abstract":"The aim of the current investigation was to study the expression of genes encoded pyruvate dehydrogenase subunits (PDHA1, PDHB, PDHX, DLAT, and DLD) in U87 glioma cells in response to glutamine deprivation in U87 glioma cells in relation to knockdown of ERN1 for evaluation of a possible dependence of the expression of these important regulatory genes from glutamine supply and ERN1 signaling. Methods. The expression of PDHA1, PDHB, PDHX, DLAT, and DLD genes was studied by real-time qPCR in control U87 glioma cells (transfected by vector) and cells with knockdown of ERN1 (transfected by dnERN1) after exposure to glutamine deprivation condition. Total RNA was extracted from glioma cells using TRIZOL reagent. An RNA quantity as well as spectral characteristics was measured using NanoDrop One. 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 %). Results. It was shown that the expression level of PDH1, PDHB, DLAT, and DLD genes was down-regulated in control glioma cells treated by glutamine deprivation. At the same time, ERN1 knockdown is suppressed the effect of glutamine deprivation on PDHB and DLD gene expressions in glioma cells, but did not change significantly the impact of glutamine deprivation on the expression of PDHA1, DLAT, and PDHX genes. Conclusions. The results of this investigation demonstrated that the expression of PDH1, PDHB, PDHX, DLAT, and DLD genes was significantly affected by exposure of U87 glioma cells under glutamine deprivation condition and that the effect of glutamine deprivation on the expression of most these genes was modified in cells with knockdown of ERN1, a major signaling pathway of the endoplasmic reticulum stress.","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":"49033634","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}
引用次数: 0
THE ACQUISITION OF RESISTANCE IN HUMAN NON-SMALL LUNG ADENOCARCINOMA MOR CELLS IS ASSOCIATED WITH UP-REGULATION OF ADAPTOR PROTEIN RUK/CIN85 AND EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) 人非小肺腺癌细胞耐药的获得与受体蛋白ruk / cin85的上调和上皮-间质转化(emt)有关。
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.064
Y. Raynich
The aim of this study was to elucidate the regulatory role of Ruk/CIN85 in chemoresistance and EMT using human NSCLC MOR cells as a model. Methods. MOR (ECACC 84112312) cell line and drug-resistant cell line MOR/0.2R (ECACC 96042335) were cultured under standard conditions in DMEM medium. Knockdown of Ruk/CIN85 in MOR/0.2R cells was performed using shRNA lentiviral technology. Expression levels of Ruk/CIN85, vimentin and E-cadherin were estimated by RT-PCR. Results and Discussion. According to the results of qPCR, MOR/0.R cells showed an extremely higher level of Ruk/CIN85 mRNA expression, more than 10 times higher than the parental MOR cells. Preliminary data revealed that knockdown of Ruk/CIN85 in the MOR/0.2R cells led to significant decrease of their resistance to doxorubicin and development of epithelial phenotype. High content of RukCIN85 in doxorubicin-resistant (MOR/R) cells strongly correlate with their mesenchymal phenotype (high expression level of vimentin and low – E-cadherin), while its down-regulation is followed by restoration of expression values characteristic of parental MOR cells. Conclusions. In summary, high expression level of Ruk/CIN85 in doxorubicin-resistant MOR cells and the reversion of EMT-related transcriptome parameters and sensitivity to drug due to knockdown of adaptor protein in this subline suggests its involvement in regulation of EMT as well as cancer cells chemoresistance. Thus, the adaptor protein Ruk/CIN85 can be considered as a tissue-specific marker of carcinogenesis and perspective target for drug development.
本研究的目的是以人NSCLC MOR细胞为模型,阐明Ruk/CIN85在化疗耐药性和EMT中的调节作用。方法。MOR(ECACC 84112312)细胞系和耐药细胞系MOR/0.2R(ECACC96042335)在DMEM培养基中在标准条件下培养。使用shRNA慢病毒技术在MOR/0.2R细胞中敲除Ruk/CIN85。RT-PCR检测Ruk/CIN85、波形蛋白和E-钙粘蛋白的表达水平。结果和讨论。根据qPCR的结果,MOR/0.R细胞显示出非常高的Ruk/CIN85mRNA表达水平,比亲代MOR细胞高出10倍以上。初步数据显示,MOR/0.2R细胞中Ruk/CIN85的敲除导致其对阿霉素的耐药性显著降低和上皮表型的发展。在阿霉素抗性(MOR/R)细胞中,RukCIN85的高含量与其间充质表型(波形蛋白和低E-钙粘蛋白的高表达水平)密切相关,而其下调后,亲代MOR细胞的表达值恢复。结论。总之,Ruk/CIN85在阿霉素耐药MOR细胞中的高表达水平以及由于该亚系中衔接蛋白的敲低而导致的EMT相关转录组参数的逆转和对药物的敏感性表明其参与了EMT和癌症细胞化疗耐药性的调节。因此,衔接蛋白Ruk/CIN85可以被认为是致癌作用的组织特异性标志物和药物开发的前景靶点。
{"title":"THE ACQUISITION OF RESISTANCE IN HUMAN NON-SMALL LUNG ADENOCARCINOMA MOR CELLS IS ASSOCIATED WITH UP-REGULATION OF ADAPTOR PROTEIN RUK/CIN85 AND EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT)","authors":"Y. Raynich","doi":"10.15407/biotech15.02.064","DOIUrl":"https://doi.org/10.15407/biotech15.02.064","url":null,"abstract":"The aim of this study was to elucidate the regulatory role of Ruk/CIN85 in chemoresistance and EMT using human NSCLC MOR cells as a model. Methods. MOR (ECACC 84112312) cell line and drug-resistant cell line MOR/0.2R (ECACC 96042335) were cultured under standard conditions in DMEM medium. Knockdown of Ruk/CIN85 in MOR/0.2R cells was performed using shRNA lentiviral technology. Expression levels of Ruk/CIN85, vimentin and E-cadherin were estimated by RT-PCR. Results and Discussion. According to the results of qPCR, MOR/0.R cells showed an extremely higher level of Ruk/CIN85 mRNA expression, more than 10 times higher than the parental MOR cells. Preliminary data revealed that knockdown of Ruk/CIN85 in the MOR/0.2R cells led to significant decrease of their resistance to doxorubicin and development of epithelial phenotype. High content of RukCIN85 in doxorubicin-resistant (MOR/R) cells strongly correlate with their mesenchymal phenotype (high expression level of vimentin and low – E-cadherin), while its down-regulation is followed by restoration of expression values characteristic of parental MOR cells. Conclusions. In summary, high expression level of Ruk/CIN85 in doxorubicin-resistant MOR cells and the reversion of EMT-related transcriptome parameters and sensitivity to drug due to knockdown of adaptor protein in this subline suggests its involvement in regulation of EMT as well as cancer cells chemoresistance. Thus, the adaptor protein Ruk/CIN85 can be considered as a tissue-specific marker of carcinogenesis and perspective target for drug development.","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":"49196865","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}
引用次数: 0
HPLC DETECTION OF ANTITHROMBITIC CALIX[4]ARENE IN BLOOD PLASMA OF ANIMALS HPLC法测定动物血浆中抗血栓杯[4]芳烃的含量
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.051
V. A. Didkivskyi
Previously sodium salt of 5,11,17,23-bis (dihydroxyphosphoryl) methylcalix[4]arene (C-145) was shown to be promising antithrombotic agent. Aim. This work was focused on the development of the method for the direct detection of this substance in blood plasma and estimation of pharmacokinetics of this compound. Methods. C-145 was injected into the Wistar rat’s lateral tail vein and into rabbit’s marginal vein of the ear (12 mg/kg) or was administrated per-oral. The anticoagulant effects of C-145 in blood plasma were confirmed by activated partial thromboplastin time (APTT) test. HPLC was performed using Agilent 1100 series (Agilent, USA) on the phase cyano ZorbaxCN Column which parameters were L×I.D. 25 cm×4.6 mm. Results. The maximal antithrombotic effect after the intravenous or per-oral administration of C-145 was observed after 4-6 hours. In particular clotting time in APTT-test in these blood plasma samples was prolonged trice and more (120 s against 46 s in control). Normalization of blood clotting was achieved after 24 hours after the injection. To develop a method for direct C-145 detection in blood plasma we selected samples with maximal prolongation of clotting time. For accurate analysis of blood plasma samples proteins were saturated by 10 % trichloroacetic acid. After neutralization by NaHCO3 samples were prepared using 12-port vacuum unit for solid-phase extraction (Agilent, USA) with a Bond-Elut C18 cartridge. Samples that contained C-145 were eluted by 100% methanol for the HPLC analysis performed on the phase cyano ZorbaxCN Column equilibrated with an acetonitrile solution (ddH2O:AcCN 99:1). Elution was performed using a combined gradient of acetonitrile (100 %) and citrate buffer (0.1 M, pH 6.0). The elution zone of C-145 was detected on the 128th minute at 280 nm. Conclusion. Application of the developed methods allowed us to confirm the direct antithrombotic effect of calix[4]arene C-145 on blood of experimental animals during intravenous administration. Also HPLC technique enabled to detect this substance in blood plasma and most likely could be applied for other biological solutions and could be modified for the quantitative analysis in the pharmacokinetic studies as well.
先前,5,11,17,23-双(二羟基磷酰)甲基杯[4]芳烃(C-145)钠盐被证明是有前途的抗血栓剂。的目标。本工作的重点是建立血浆中该物质的直接检测方法和药代动力学的估计。方法。C-145经Wistar大鼠尾侧静脉和家兔耳缘静脉注射(12 mg/kg)或经口给药。采用活化部分凝血活素时间(APTT)试验证实C-145在血浆中的抗凝作用。采用Agilent 1100系列(Agilent, USA)高效液相色谱法,色谱柱为相氰ZorbaxCN,参数为L×I.D。25 cm×4.6 mm。结果。静脉或口服C-145后4-6小时观察到最大的抗血栓作用。特别是在aptt试验中,这些血浆样品的凝血时间延长了三倍以上(120秒,而对照组为46秒)。注射后24小时血凝恢复正常。为了建立一种直接检测血浆中C-145的方法,我们选择了凝血时间最长的样品。为了准确分析血浆样品,蛋白质被10%的三氯乙酸饱和。样品经NaHCO3中和后,采用Bond-Elut C18滤筒,采用12孔真空固相萃取装置(Agilent, USA)制备。含有C-145的样品用100%甲醇洗脱,用乙腈溶液(ddH2O:AcCN 99:1)平衡相ZorbaxCN柱进行HPLC分析。使用乙腈(100%)和柠檬酸缓冲液(0.1 M, pH 6.0)的联合梯度进行洗脱。第128分钟在280 nm处检测到C-145的洗脱区。结论。应用所开发的方法,我们证实了杯盏芳烃C-145在静脉给药时对实验动物血液的直接抗血栓作用。此外,高效液相色谱技术可以检测血浆中的这种物质,很可能应用于其他生物溶液,也可以用于药代动力学研究中的定量分析。
{"title":"HPLC DETECTION OF ANTITHROMBITIC CALIX[4]ARENE IN BLOOD PLASMA OF ANIMALS","authors":"V. A. Didkivskyi","doi":"10.15407/biotech15.02.051","DOIUrl":"https://doi.org/10.15407/biotech15.02.051","url":null,"abstract":"Previously sodium salt of 5,11,17,23-bis (dihydroxyphosphoryl) methylcalix[4]arene (C-145) was shown to be promising antithrombotic agent. Aim. This work was focused on the development of the method for the direct detection of this substance in blood plasma and estimation of pharmacokinetics of this compound. Methods. C-145 was injected into the Wistar rat’s lateral tail vein and into rabbit’s marginal vein of the ear (12 mg/kg) or was administrated per-oral. The anticoagulant effects of C-145 in blood plasma were confirmed by activated partial thromboplastin time (APTT) test. HPLC was performed using Agilent 1100 series (Agilent, USA) on the phase cyano ZorbaxCN Column which parameters were L×I.D. 25 cm×4.6 mm. Results. The maximal antithrombotic effect after the intravenous or per-oral administration of C-145 was observed after 4-6 hours. In particular clotting time in APTT-test in these blood plasma samples was prolonged trice and more (120 s against 46 s in control). Normalization of blood clotting was achieved after 24 hours after the injection. To develop a method for direct C-145 detection in blood plasma we selected samples with maximal prolongation of clotting time. For accurate analysis of blood plasma samples proteins were saturated by 10 % trichloroacetic acid. After neutralization by NaHCO3 samples were prepared using 12-port vacuum unit for solid-phase extraction (Agilent, USA) with a Bond-Elut C18 cartridge. Samples that contained C-145 were eluted by 100% methanol for the HPLC analysis performed on the phase cyano ZorbaxCN Column equilibrated with an acetonitrile solution (ddH2O:AcCN 99:1). Elution was performed using a combined gradient of acetonitrile (100 %) and citrate buffer (0.1 M, pH 6.0). The elution zone of C-145 was detected on the 128th minute at 280 nm. Conclusion. Application of the developed methods allowed us to confirm the direct antithrombotic effect of calix[4]arene C-145 on blood of experimental animals during intravenous administration. Also HPLC technique enabled to detect this substance in blood plasma and most likely could be applied for other biological solutions and could be modified for the quantitative analysis in the pharmacokinetic studies as well.","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":"43657804","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}
引用次数: 1
CHARACTERISTICS OF WOUND INFECTIONS AND METHODS OF THEIR TREATMENT USING PREPARATIONS OF BIOLOGICAL ORIGIN 伤口感染的特点及生物源性制剂的治疗方法
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.007
A. Oliinyk
Surgical wound infections are the most common patients’ complications in the postoperative period. In the modern clinic, they worsen the disease prognosis and remain the most important and acute health problem in all countries of the world. The aim of the work was to analyze current scientific data on the peculiarities of the pathogenesis of wound infections and types of their pathogens, as well as drugs of biological origin in the treatment of wound infections. The paper discusses in detail the problem of infection of wound injuries during surgery and domestic injuries of various kinds. The main pathogens of wound infections are considered. Specific pathogenicity factors for bacteria of the genera Staphylococcus, Pseudomonas, Enterobacteriaceae were analyzed. Based on the analysis of literature sources, a list of drugs of biotechnological origin that can be effectively used in combination therapy for the treatment and prevention of wound infections was determined. Conclusions. The result is the identification of those mechanisms of pathogenesis of wound infections that determine the effectiveness of the use of drugs of biological origin in this pathology treatment.
手术伤口感染是术后患者最常见的并发症。在现代临床中,它们恶化了疾病的预后,仍然是世界各国最重要和最紧迫的健康问题。这项工作的目的是分析目前的科学数据的特点,伤口感染的发病机制和类型的病原体,以及生物来源的药物在伤口感染的治疗。本文详细讨论了外科手术中创面损伤及各种家庭外伤的感染问题。讨论了伤口感染的主要病原体。分析了葡萄球菌、假单胞菌和肠杆菌科细菌的特异性致病性因素。在文献资料分析的基础上,确定了可有效用于联合治疗和预防创面感染的生物技术来源药物清单。结论。结果是确定了伤口感染的发病机制,这些机制决定了在这种病理治疗中使用生物来源药物的有效性。
{"title":"CHARACTERISTICS OF WOUND INFECTIONS AND METHODS OF THEIR TREATMENT USING PREPARATIONS OF BIOLOGICAL ORIGIN","authors":"A. Oliinyk","doi":"10.15407/biotech15.02.007","DOIUrl":"https://doi.org/10.15407/biotech15.02.007","url":null,"abstract":"Surgical wound infections are the most common patients’ complications in the postoperative period. In the modern clinic, they worsen the disease prognosis and remain the most important and acute health problem in all countries of the world. The aim of the work was to analyze current scientific data on the peculiarities of the pathogenesis of wound infections and types of their pathogens, as well as drugs of biological origin in the treatment of wound infections. The paper discusses in detail the problem of infection of wound injuries during surgery and domestic injuries of various kinds. The main pathogens of wound infections are considered. Specific pathogenicity factors for bacteria of the genera Staphylococcus, Pseudomonas, Enterobacteriaceae were analyzed. Based on the analysis of literature sources, a list of drugs of biotechnological origin that can be effectively used in combination therapy for the treatment and prevention of wound infections was determined. Conclusions. The result is the identification of those mechanisms of pathogenesis of wound infections that determine the effectiveness of the use of drugs of biological origin in this pathology treatment.","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":"48102567","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}
引用次数: 0
MOLECULAR BASIS OF THE DEVELOPMENT OF INSULIN RESISTANCE IN OBESE ADOLESCENT AND ADULT MEN 肥胖青少年和成年男性胰岛素抵抗发生的分子基础
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.072
Y. Viletska
The aim of this work was to study the association between the expression of glucose metabolism related genes and insulin resistance, which expression is changed in obese adolescents and adult men with and without insulin resistance, for better understanding the molecular basis of the development of obesity complications and evaluation of possible contribution of these genes in development of insulin resistance. Methods. The expression level of genes related to glucose metabolism and their regulations was studied by real-time qPCR in adipose tissue and blood cells using SYBRGreen Mix and specific for each mRNA forward and reverse primers. 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 %). Results. It was shown that in obese patients with insulin resistance the expression level of IRS1 (insulin receptor substrate 1), HK2 (hexokinase 2), PFKFB2 (6-phosphofructokinase/fructose-2,6-bisphosphatase 2) and PFKFB3 as well as circadian factors CLOCK and ARNTL genes in subcutaneous adipose tissue is significantly decreased as compared to obese men with normal sensitivity to insulin. At the same time, the development of insulin resistance in obese patients leads to up-regulation of PFKFB4, PER1, HSPA6, ALDH1A3, COL5A1, TIMP1, TIMP2, SPARC, and VCAN gene expressions in subcutaneous adipose tissue. The expression level of IGF1 (insulin-like growth factor 1) and IGFBP5 (IGF binding protein 5) as well as ENO1 (enolase 1) and ENO2 is down-regulated in the blood of obese adolescent with insulin resistance, but IGFBP2 and IGFBP7 gene expressions are significantly increased in these patients. Conclusions. The results of this investigation provide evidence that the development of insulin resistance in obese patients is associated with gene specific changes in the expression of many very important regulatory genes, which are endoplasmic reticulum stress responsible.
这项工作的目的是研究葡萄糖代谢相关基因的表达与胰岛素抵抗之间的关系,胰岛素抵抗的表达在患有和不患有胰岛素抵抗的肥胖青少年和成年男性中发生变化,以更好地了解肥胖并发症发展的分子基础,并评估这些基因在胰岛素抵抗发展中的可能贡献。方法。使用SYBRGreen Mix通过实时qPCR研究了与葡萄糖代谢相关的基因在脂肪组织和血细胞中的表达水平及其调控,并对每种信使核糖核酸正向和反向引物具有特异性。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%)。后果研究表明,在胰岛素抵抗的肥胖患者中,与对胰岛素敏感正常的肥胖男性相比,皮下脂肪组织中IRS1(胰岛素受体底物1)、HK2(己糖激酶2)、PFKFB2(6-磷酸果糖激酶/果糖-2,6-二磷酸酶2)和PFKFB3以及昼夜节律因子CLOCK和ARNTL基因的表达水平显著降低。同时,肥胖患者胰岛素抵抗的发展导致皮下脂肪组织中PFKFB4、PER1、HSPA6、ALDH1A3、COL5A1、TIMP1、TIMP2、SPARC和VCAN基因表达的上调。在患有胰岛素抵抗的肥胖青少年的血液中,IGF1(胰岛素样生长因子1)和IGFBP5(IGF结合蛋白5)以及ENO1(烯醇化酶1)和ENO2的表达水平下调,但这些患者的IGFBP2和IGFBP7基因表达显著增加。结论。这项研究的结果提供了证据,证明肥胖患者胰岛素抵抗的发展与许多非常重要的调节基因表达的基因特异性变化有关,这些基因是内质网应激的原因。
{"title":"MOLECULAR BASIS OF THE DEVELOPMENT OF INSULIN RESISTANCE IN OBESE ADOLESCENT AND ADULT MEN","authors":"Y. Viletska","doi":"10.15407/biotech15.02.072","DOIUrl":"https://doi.org/10.15407/biotech15.02.072","url":null,"abstract":"The aim of this work was to study the association between the expression of glucose metabolism related genes and insulin resistance, which expression is changed in obese adolescents and adult men with and without insulin resistance, for better understanding the molecular basis of the development of obesity complications and evaluation of possible contribution of these genes in development of insulin resistance. Methods. The expression level of genes related to glucose metabolism and their regulations was studied by real-time qPCR in adipose tissue and blood cells using SYBRGreen Mix and specific for each mRNA forward and reverse primers. 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 %). Results. It was shown that in obese patients with insulin resistance the expression level of IRS1 (insulin receptor substrate 1), HK2 (hexokinase 2), PFKFB2 (6-phosphofructokinase/fructose-2,6-bisphosphatase 2) and PFKFB3 as well as circadian factors CLOCK and ARNTL genes in subcutaneous adipose tissue is significantly decreased as compared to obese men with normal sensitivity to insulin. At the same time, the development of insulin resistance in obese patients leads to up-regulation of PFKFB4, PER1, HSPA6, ALDH1A3, COL5A1, TIMP1, TIMP2, SPARC, and VCAN gene expressions in subcutaneous adipose tissue. The expression level of IGF1 (insulin-like growth factor 1) and IGFBP5 (IGF binding protein 5) as well as ENO1 (enolase 1) and ENO2 is down-regulated in the blood of obese adolescent with insulin resistance, but IGFBP2 and IGFBP7 gene expressions are significantly increased in these patients. Conclusions. The results of this investigation provide evidence that the development of insulin resistance in obese patients is associated with gene specific changes in the expression of many very important regulatory genes, which are endoplasmic reticulum stress responsible.","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":"49480895","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}
引用次数: 0
EPITHELIAL-MESENCHYMAL TRANSITION IN MELANOMA PROGRESSION: THE CONTRIBUTION OF ADAPTOR PROTEIN RUK/CIN85 黑色素瘤进展中的上皮-间质转化:受体蛋白ruk / cin85的作用
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.074
B. V. Zhuravel
The purpose of this study was to test the hypothesis that Ruk/CIN85 overexpression/knockdown in melanoma cells may be involved in the regulation of EMT. Materials and methods. The mouse melanoma cell line B16-F10 and its sublines with up-/down-regulation of Ruk/CIN85 (generated early using lentiviral technology) were used as a model for research. Melanoma cells were cultured in the complete RPMI 1610 medium under standard conditions. Proliferative activity of the cells was estimated using the MTT-test, and cell migratory potential was studied by the wound-healing assay. The data obtained were analyzed with parametric Student`s t-test. Results were expressed as mean ± SEM and significance was set at P<0.05. Results and Discussion. Cutaneous melanoma genesis is a multi-step process initiated by the transformation of a normal melanocyte following an oncogenic insult. Due to the transcriptome and metabolome reprogramming in the course of EMT, transformed melanoma cells change their phenotype and acquire increased proliferative rate, cell motility, invasiveness, and metastatic potential. According to the data obtained, overexpression of Ruk/CIN85 in B16 mouse melanoma cells (subclones Up7 and Up21) led to an increase in their proliferative activity by 1,6 and 1.8 times, respectively, at 24th hour in comparison with control Mock cells . At the 48th hour, when the cells reached confluence, the cell viability of subclones did not differ from the control ones. No statistically significant changes in the proliferative activity of B16 cells with suppressed expression of the adaptor protein (subclone Down) were found. In accordance with previous data, B16 cells overexpressing Ruk/CIN85 were characterized by strongly increased motility rate (more than twofold for both Up7 and Up21 subclones compared to control Mock cells). At the same time, knockdown of Ruk/CIN85 in B16 cells resulted in a decrease in their migratory activity by about 30%. Conclusions. All findings obtained demonstrated that the malignancy traits of melanoma B16 cells are inversely modulated upon up- and down-changes in adaptor protein Ruk/CIN85 expression levels suggesting its possible role in the control of EMT.
本研究的目的是验证黑色素瘤细胞中Ruk/CIN85过表达/敲低可能参与EMT调控的假设。材料和方法。以早期利用慢病毒技术生成的Ruk/CIN85上调/下调的小鼠黑色素瘤细胞系B16-F10及其亚系为模型进行研究。在标准条件下,在完整的RPMI 1610培养基中培养黑色素瘤细胞。使用mtt试验估计细胞的增殖活性,并通过伤口愈合试验研究细胞的迁移潜力。所得数据采用参数Student 's t检验进行分析。结果以mean±SEM表示,P<0.05为显著性。结果和讨论。皮肤黑色素瘤的发生是一个多步骤的过程,由正常黑素细胞在癌性损伤后的转化所启动。由于EMT过程中的转录组和代谢组重编程,转化的黑色素瘤细胞改变其表型,获得更高的增殖率、细胞运动性、侵袭性和转移潜力。根据获得的数据,在B16小鼠黑色素瘤细胞(Up7和Up21亚克隆)中,Ruk/CIN85的过表达导致其增殖活性在24小时时比对照Mock细胞分别增加了1倍,6倍和1.8倍。48h,细胞到达汇合时,亚克隆的细胞活力与对照没有差异。抑制接头蛋白(Down亚克隆)表达后,B16细胞的增殖活性无显著变化。根据之前的数据,过表达Ruk/CIN85的B16细胞的特征是运动率显著增加(Up7和Up21亚克隆与对照Mock细胞相比,运动率都增加了两倍以上)。同时,在B16细胞中敲低Ruk/CIN85导致其迁移活性降低约30%。结论。所有研究结果表明,黑素瘤B16细胞的恶性性状随受体蛋白Ruk/CIN85表达水平的变化而呈负调节,提示其可能在EMT的控制中起作用。
{"title":"EPITHELIAL-MESENCHYMAL TRANSITION IN MELANOMA PROGRESSION: THE CONTRIBUTION OF ADAPTOR PROTEIN RUK/CIN85","authors":"B. V. Zhuravel","doi":"10.15407/biotech15.02.074","DOIUrl":"https://doi.org/10.15407/biotech15.02.074","url":null,"abstract":"The purpose of this study was to test the hypothesis that Ruk/CIN85 overexpression/knockdown in melanoma cells may be involved in the regulation of EMT. Materials and methods. The mouse melanoma cell line B16-F10 and its sublines with up-/down-regulation of Ruk/CIN85 (generated early using lentiviral technology) were used as a model for research. Melanoma cells were cultured in the complete RPMI 1610 medium under standard conditions. Proliferative activity of the cells was estimated using the MTT-test, and cell migratory potential was studied by the wound-healing assay. The data obtained were analyzed with parametric Student`s t-test. Results were expressed as mean ± SEM and significance was set at P<0.05. Results and Discussion. Cutaneous melanoma genesis is a multi-step process initiated by the transformation of a normal melanocyte following an oncogenic insult. Due to the transcriptome and metabolome reprogramming in the course of EMT, transformed melanoma cells change their phenotype and acquire increased proliferative rate, cell motility, invasiveness, and metastatic potential. According to the data obtained, overexpression of Ruk/CIN85 in B16 mouse melanoma cells (subclones Up7 and Up21) led to an increase in their proliferative activity by 1,6 and 1.8 times, respectively, at 24th hour in comparison with control Mock cells . At the 48th hour, when the cells reached confluence, the cell viability of subclones did not differ from the control ones. No statistically significant changes in the proliferative activity of B16 cells with suppressed expression of the adaptor protein (subclone Down) were found. In accordance with previous data, B16 cells overexpressing Ruk/CIN85 were characterized by strongly increased motility rate (more than twofold for both Up7 and Up21 subclones compared to control Mock cells). At the same time, knockdown of Ruk/CIN85 in B16 cells resulted in a decrease in their migratory activity by about 30%. Conclusions. All findings obtained demonstrated that the malignancy traits of melanoma B16 cells are inversely modulated upon up- and down-changes in adaptor protein Ruk/CIN85 expression levels suggesting its possible role in the control of EMT.","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":"48546376","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}
引用次数: 0
LONG-TERM EFFECTS OF SHAM SURGERY ON PHAGOCYTE FUNCTIONS IN RATS 假手术对大鼠吞噬细胞功能的长期影响
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.037
Zh. Oliynyk
Animal models of inflammatory disorders, including those of the nervous system are commonly used to explore the pathophysiological role of immune cell response in disease triggering and course and to develop biotechnology products for therapeutic use. Modeling some of these disorders, particularly neurodegenerative diseases, implies surgical manipulations for the intracerebral introduction of disease-initiating substances (toxins, amyloids etc.). Design of these experiments involves the use of sham-operated animals as a control of non-specific intrinsic side-effects elicited by surgical manipulations per se, including local and systemic inflammation, where phagocytic cells are key participants. Short-term post-surgical immunomodulatory effects are widely reported. However, no study thus far has examined the long term effects of sham-surgery on phagocyte functions. The purpose of this study was to evaluate the effect of sham-surgery, commonly used for modeling neurodegenerative diseases, on phagocyte functions in the far terms after the surgical manipulations. Materials and Methods. Adult male Wistar rats were used in the study. Sham surgery consisted of stereotactic unilateral injection of saline solution into the median forebrain bundle (sham-operated 1, SO1) or directly into the substantia nigra (sham-operated 2, SO2). Before the placebo surgery, animals were anaesthetized using nembutal and ketamine/xylazine correspondingly. Functional characteristics (phagocytic activity, oxidative metabolism, CD80/86 and CD206 expression) of phagocytes (microglia, peritoneal macrophages, circulating monocytes and granulocytes) were examined by flow cytometry. Differential leukocyte count was conducted using hematological analyzer. Results. Phagocytes from animals underwent of different protocols of placebo surgery, demonstrated various patterns of functional changes on day 29 after the manipulations. In animals from SO1 group, we observed signs of residual neuroinflammation (pro-inflammatory shift of microglia functional profile) along with ongoing resolution of systemic inflammation (anti-inflammatory metabolic shift of circulating phagocytes and peritoneal macrophages). In rats from SO2 group, pro-inflammatory polarized activation of peritoneal phagocytes was registered along with anti-inflammatory shift in microglia and circulating phagocytes. Conclusions. Sham surgery influences functions of phagocytic cells of different locations even in the far terms after the manipulations. These effects can be considered as combined long-term consequences of surgical brain injury and the use of anesthetics. Our observations evidences, that sham associated non-specific immunomodulatory effects should always be taken into consideration in animal models of inflammatory central nervous system diseases.
炎症性疾病(包括神经系统炎症性疾病)的动物模型通常用于探索免疫细胞反应在疾病触发和病程中的病理生理作用,并开发用于治疗的生物技术产品。对其中一些疾病,特别是神经退行性疾病进行建模,意味着在脑内引入疾病起始物质(毒素、淀粉样蛋白等)的手术操作。这些实验的设计包括使用假手术动物来控制手术操作本身引发的非特异性内在副作用,包括局部和全身炎症,其中吞噬细胞是关键参与者。术后短期免疫调节作用已被广泛报道。然而,到目前为止,还没有研究检验假手术对吞噬细胞功能的长期影响。本研究的目的是评估通常用于模拟神经退行性疾病的假手术在手术后对吞噬细胞功能的影响。材料和方法。本研究采用成年雄性Wistar大鼠。假手术包括将生理盐水单侧立体定向注射到正中前脑束(假手术1,SO1)或直接注射到黑质(假手术2,SO2)。在安慰剂手术之前,相应地使用戊巴比妥和氯胺酮/甲苯噻嗪对动物进行麻醉。通过流式细胞术检测吞噬细胞(小胶质细胞、腹膜巨噬细胞、循环单核细胞和粒细胞)的功能特征(吞噬活性、氧化代谢、CD80/86和CD206表达)。使用血液分析仪进行白细胞的差异计数。后果来自接受不同方案安慰剂手术的动物的吞噬细胞在操作后第29天表现出各种模式的功能变化。在SO1组的动物中,我们观察到残余神经炎症的迹象(小胶质细胞功能谱的促炎性转变),以及全身炎症的持续消退(循环吞噬细胞和腹膜巨噬细胞的抗炎代谢转变)。在SO2组的大鼠中,腹膜吞噬细胞的促炎极化激活以及小胶质细胞和循环吞噬细胞的抗炎转变被记录下来。结论。假手术影响不同位置的吞噬细胞的功能,甚至在手术后很长一段时间内也是如此。这些影响可以被认为是手术脑损伤和麻醉剂使用的综合长期后果。我们的观察结果证明,在炎症性中枢神经系统疾病的动物模型中,应始终考虑假手术相关的非特异性免疫调节作用。
{"title":"LONG-TERM EFFECTS OF SHAM SURGERY ON PHAGOCYTE FUNCTIONS IN RATS","authors":"Zh. Oliynyk","doi":"10.15407/biotech15.02.037","DOIUrl":"https://doi.org/10.15407/biotech15.02.037","url":null,"abstract":"Animal models of inflammatory disorders, including those of the nervous system are commonly used to explore the pathophysiological role of immune cell response in disease triggering and course and to develop biotechnology products for therapeutic use. Modeling some of these disorders, particularly neurodegenerative diseases, implies surgical manipulations for the intracerebral introduction of disease-initiating substances (toxins, amyloids etc.). Design of these experiments involves the use of sham-operated animals as a control of non-specific intrinsic side-effects elicited by surgical manipulations per se, including local and systemic inflammation, where phagocytic cells are key participants. Short-term post-surgical immunomodulatory effects are widely reported. However, no study thus far has examined the long term effects of sham-surgery on phagocyte functions. The purpose of this study was to evaluate the effect of sham-surgery, commonly used for modeling neurodegenerative diseases, on phagocyte functions in the far terms after the surgical manipulations. Materials and Methods. Adult male Wistar rats were used in the study. Sham surgery consisted of stereotactic unilateral injection of saline solution into the median forebrain bundle (sham-operated 1, SO1) or directly into the substantia nigra (sham-operated 2, SO2). Before the placebo surgery, animals were anaesthetized using nembutal and ketamine/xylazine correspondingly. Functional characteristics (phagocytic activity, oxidative metabolism, CD80/86 and CD206 expression) of phagocytes (microglia, peritoneal macrophages, circulating monocytes and granulocytes) were examined by flow cytometry. Differential leukocyte count was conducted using hematological analyzer. Results. Phagocytes from animals underwent of different protocols of placebo surgery, demonstrated various patterns of functional changes on day 29 after the manipulations. In animals from SO1 group, we observed signs of residual neuroinflammation (pro-inflammatory shift of microglia functional profile) along with ongoing resolution of systemic inflammation (anti-inflammatory metabolic shift of circulating phagocytes and peritoneal macrophages). In rats from SO2 group, pro-inflammatory polarized activation of peritoneal phagocytes was registered along with anti-inflammatory shift in microglia and circulating phagocytes. Conclusions. Sham surgery influences functions of phagocytic cells of different locations even in the far terms after the manipulations. These effects can be considered as combined long-term consequences of surgical brain injury and the use of anesthetics. Our observations evidences, that sham associated non-specific immunomodulatory effects should always be taken into consideration in animal models of inflammatory central nervous system diseases.","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":"45249506","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}
引用次数: 2
ADAPTOR PROTEIN RUK/CIN85 IS INVOLVED IN THE GLUCOSE METABOLISM REPROGRAMMING IN BREAST CANCER CELLS 衔接蛋白RUK/CIN85参与乳腺癌细胞葡萄糖代谢重编程
Pub Date : 2022-04-01 DOI: 10.15407/biotech15.02.047
M. I. Bekala
Aim. This study aimed to investigate the changes in glucose metabolism in mouse 4T1 breast adenocarcinoma cells with different levels of Ruk/CIN85 expression. Methods. We used 4T1 cells with stable overexpression (subline RukUp) or knockdown (subline RukDown) of Ruk/CIN85, as well as corresponding vector control sublines Mock and Scr. Cells were cultured in the complete RPMI-1640 medium under standard conditions. mRNA expression levels were estimated by RT2-PCR, enzymes activities were measured by spectrophotometric and/or fluorometric assays. Results. Analysis of mRNA expression of glucose metabolism-related genes in RukUp and RukDown cells revealed that glycolysis genes are preferentially overexpressed in RukUp cells, and downregulated in RukDown cells. Thus, RukUp cells were characterized by significantly overexpressed Slc2a1, Gck, Aldoa, and Ldha, while in RukDown cells these genes were either down regulated or not changed. However, the expression of TCA (tricarboxylic acid) cycle enzyme Mdh2 increased dramatically (by 7,8 times) in RukDown cells. In detail, we observed statistically significant changes in the activity of all studied enzymes in RukUp cells (increase by 1,5-1,9 times for glycolysis enzymes and G6PD, and decrease by 1,33-1,69 times for TCA enzymes). However, in RukDown cells we did not find any significant changes in glycolysis enzymes activities, but activities of mitochondrial IDH3 and MDH2 were elevated by 1,65 and 1,59 times, respectively. Conclusions. The results obtained indicate that adaptor protein Ruk/CIN85 is involved in the metabolic reprogramming during breast cancer progression. High level of Ruk/CIN85 expression is associated with potentiation of the Warburg effect.
目标本研究旨在研究不同Ruk/CIN85表达水平的小鼠4T1乳腺腺癌细胞葡萄糖代谢的变化。方法。我们使用具有Ruk/CIN85的稳定过表达(亚系RukUp)或敲除(亚系RukDown)的4T1细胞,以及相应的载体对照亚系Mock和Scr。细胞在标准条件下在完全RPMI-1640培养基中培养。通过RT2-PCR估计mRNA表达水平,通过分光光度法和/或荧光测定法测量酶活性。后果对RukUp和RukDown细胞中葡萄糖代谢相关基因的mRNA表达的分析显示,糖酵解基因在RukUp细胞中优先过表达,而在RukDown细胞中下调。因此,RukUp细胞的特征是显著过表达的Slc2a1、Gck、Aldoa和Ldha,而在RukDown细胞中,这些基因要么被下调,要么没有改变。然而,TCA(三羧酸)循环酶Mdh2在RukDown细胞中的表达显著增加(增加了7,8倍)。详细地说,我们观察到RukUp细胞中所有研究酶的活性发生了统计学上的显著变化(糖酵解酶和G6PD增加了1,5,1.9倍,TCA酶减少了1,33-1,69倍)。然而,在RukDown细胞中,我们没有发现糖酵解酶活性有任何显著变化,但线粒体IDH3和MDH2的活性分别升高了1.65和1.59倍。结论。所获得的结果表明,衔接蛋白Ruk/CIN85参与了乳腺癌症进展过程中的代谢重编程。高水平的Ruk/CIN85表达与Warburg效应的增强有关。
{"title":"ADAPTOR PROTEIN RUK/CIN85 IS INVOLVED IN THE GLUCOSE METABOLISM REPROGRAMMING IN BREAST CANCER CELLS","authors":"M. I. Bekala","doi":"10.15407/biotech15.02.047","DOIUrl":"https://doi.org/10.15407/biotech15.02.047","url":null,"abstract":"Aim. This study aimed to investigate the changes in glucose metabolism in mouse 4T1 breast adenocarcinoma cells with different levels of Ruk/CIN85 expression. Methods. We used 4T1 cells with stable overexpression (subline RukUp) or knockdown (subline RukDown) of Ruk/CIN85, as well as corresponding vector control sublines Mock and Scr. Cells were cultured in the complete RPMI-1640 medium under standard conditions. mRNA expression levels were estimated by RT2-PCR, enzymes activities were measured by spectrophotometric and/or fluorometric assays. Results. Analysis of mRNA expression of glucose metabolism-related genes in RukUp and RukDown cells revealed that glycolysis genes are preferentially overexpressed in RukUp cells, and downregulated in RukDown cells. Thus, RukUp cells were characterized by significantly overexpressed Slc2a1, Gck, Aldoa, and Ldha, while in RukDown cells these genes were either down regulated or not changed. However, the expression of TCA (tricarboxylic acid) cycle enzyme Mdh2 increased dramatically (by 7,8 times) in RukDown cells. In detail, we observed statistically significant changes in the activity of all studied enzymes in RukUp cells (increase by 1,5-1,9 times for glycolysis enzymes and G6PD, and decrease by 1,33-1,69 times for TCA enzymes). However, in RukDown cells we did not find any significant changes in glycolysis enzymes activities, but activities of mitochondrial IDH3 and MDH2 were elevated by 1,65 and 1,59 times, respectively. Conclusions. The results obtained indicate that adaptor protein Ruk/CIN85 is involved in the metabolic reprogramming during breast cancer progression. High level of Ruk/CIN85 expression is associated with potentiation of the Warburg effect.","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":"43790341","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}
引用次数: 0
期刊
Biotechnologia Acta
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1