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Purification and characterization of platelet aggregation inhibitor from the venom of Bitis arietans 白斑双翅虫毒液中血小板聚集抑制剂的纯化及特性研究
Pub Date : 2022-12-08 DOI: 10.15407/ubj94.05.007
O. Platonov, V. Nikulina, Y. Kucheryavyi, V. Gryshchuk, Y. Stohniy, V. Chernyshenko, O. Slominskyi, A. Rebriev, K. Savchenko, L. Garmanchuk
Disintegrins are the antagonists of integrin receptors that can be found mostly in snakes’ venom. They can inhibit platelet aggregation, thus preventing the formation of blood clots. By blocking the integrin receptors of cancer cells, disintegrins can inhibit proliferation and metastasis. Thus, the search for new sources of disintegrins and development of methods of their purification is an important task of modern biotechnolo­gy. This work was dedicated to the purification and characterization of inhibiting polypeptides from Bitis arietans­ venom. Crude venom of B. arietans was fractionated using ion-exchange chromatography on Q Sepharose followed by size-exclusion chromatography on Superdex 75 using FPLC method. Analysis of molecular weight of protein components was performed using SDS-PAGE and MALDI-TOF analysis on Voyager-DE. Aggregation of platelet-rich plasma (PRP) in the presence of platelet aggregation inhibitor was investigated using aggregometry on the AR2110. MTT test was used for measuring HeLa cells proliferation and survival in vitro. Two-step chromatography allowed us to obtain fraction that contained polypeptides possessing the dose-dependent inhibitory action on adenosine diphosphate (ADP)-induced platelet aggregation in PRP. SDS-PAGE showed that obtained fraction contained two polypeptides with molecular weight 9.0 and 13.67 kDa according­ to MALDI-TOF analysis. Purified polypeptides inhibited ADP-induced platelet aggregation with IC50 0.09 mg/ml. However, 0.005 mg/ml of fraction suppressed viability of HeLa cells according to MTT test on 20%. Discovered biological effects of fractions allowed us to conclude the possible use of these polypeptides as anti-aggregatory or anti-proliferative agents. Keywords: antithrombotic action, disintegrins, glycoprotein IIb/IIIa, platelets, snake venom
崩解素是整合素受体的拮抗剂,主要存在于蛇毒中。它们可以抑制血小板聚集,从而防止血栓的形成。崩解素通过阻断癌细胞的整合素受体,抑制癌细胞的增殖和转移。因此,寻找崩解素的新来源和发展其纯化方法是现代生物技术的重要任务。本文研究了从黄斑双翅虫毒液中提取的抑制多肽的纯化和特性。采用Q Sepharose离子交换层析,Superdex 75高效液相色谱法进行分离纯化。在Voyager-DE上使用SDS-PAGE和MALDI-TOF分析蛋白质组分的分子量。在AR2110上应用聚集法研究富血小板血浆(PRP)在血小板聚集抑制剂存在下的聚集。采用MTT法检测HeLa细胞体外增殖和存活情况。两步色谱法使我们能够获得含有多肽的部分,这些多肽对PRP中二磷酸腺苷(ADP)诱导的血小板聚集具有剂量依赖性抑制作用。SDS-PAGE经MALDI-TOF分析,得到的部分含有分子量分别为9.0和13.67 kDa的多肽。纯化多肽抑制adp诱导的血小板聚集,IC50为0.09 mg/ml。MTT试验显示,0.005 mg/ml对20%的HeLa细胞有抑制作用。发现的生物效应分数使我们得出结论,这些多肽作为抗聚集或抗增殖剂的可能用途。关键词:抗血栓作用,崩解素,糖蛋白IIb/IIIa,血小板,蛇毒
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引用次数: 0
Distribution and polymorphism of enzymes involved in antioxidant protection and xenobiotics biotransformation in the mediterranean mussel Mytilus galloprovincialis 地中海贻贝(Mytilus galloprovincialis)抗氧化保护和外源生物转化酶的分布与多态性
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.067
V. Toptikov
The aim of the work was to determine the tissue distribution, activity and polymorphism of 13 enzymes involved in antioxidative protection and xenobiotics biotransformation in a five bivalve mussel organs (hepatopancreas, ctenidia, mantle, leg, adductor muscles). Iso-allozyme analysis was performed by electrophoresis, correlation relationships between the studied enzymes in the whole mussel body were carried out using Spearman’s correlation analysis. It was found that all studied enzymes are polymorphic, each organ differed in the level of enzyme activity and a set of multiple forms. The coordinated functioning of protective enzymes in various mussel organs was demonstrated. Keywords: antioxidant enzymes, coordination of protective enzymes, enzymes of biotransformation, iso-allozyme analysis, Mytilus galloprovincialis
研究了双壳贝类5个器官(肝胰腺、锥体、套膜、腿、内收肌)中参与抗氧化保护和外源生物转化的13种酶的组织分布、活性和多态性。采用电泳法进行同工酶分析,采用Spearman相关分析法对各酶在整个贻贝体内的相关关系进行分析。结果表明,所研究的酶均具有多态性,各器官酶活性水平不同,存在一组多种形式。证明了保护酶在贻贝各器官中的协同作用。关键词:抗氧化酶,协同保护酶,生物转化酶,同工酶分析,蘑菇
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引用次数: 0
Fatty acid profile of the liver lipids under acute fungicide action and intake of a biologically active preparation in rats 急性杀菌剂作用下大鼠肝脏脂质脂肪酸谱及生物活性制剂的摄入
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.047
S. Khyzhnyak, S. Midyk, A. O. Velinska, O. Arnauta, L. Н. Kalachniuk
The widespread use of fungicides in agriculture leads to a negative consequences for both humans and the environment. The acute effect of fungicides containing tebuconazole alone or in combination with triadimefon on fatty acid (FAs) composition of the total lipids in the liver of rats was studied. FAs content was analyzed by gas chromatography with the use of flame ionization detector. The results obtained indicate that the acute effect of triazole fungicides one-time oral administration to rats is characterized by toxic reactions and is accompanied by the total liver lipids FAs profile redistribution, similar for the studied fungicides. In particular, the reduction in the total content of monounsaturated FAs (by 26% – 37%) and in the ω3/ω6 ratio was observed. It was demonstrated that the subsequent two-week introduction of the preparation based on Milk thistle seeds oil extract had a positive effect and improved the FAs profile of lipids in dynamics after fungicide intake. Keywords: fatty acids, fungicides, Milk thistle seeds extract, rat liver lipids, triazoles
杀菌剂在农业中的广泛使用对人类和环境都造成了负面影响。研究了含戊康唑的杀菌剂单用或联用三二美酮对大鼠肝脏总脂质脂肪酸组成的急性影响。采用气相色谱法和火焰电离检测器分析FAs含量。结果表明,三唑类杀菌剂一次性口服对大鼠的急性作用表现为毒性反应,并伴有肝总脂质FAs谱的重分布,与所研究的杀菌剂相似。特别是,单不饱和脂肪酸的总含量降低了26% - 37%,ω3/ω6比值也降低了。结果表明,随后两周引入乳蓟籽油提取物的制剂具有积极作用,并在摄入杀菌剂后动态改善了脂质FAs谱。关键词:脂肪酸,杀菌剂,水飞蓟籽提取物,大鼠肝脂,三唑
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引用次数: 0
Structural proteins in the mechanisms of betacoronavirus virion assembly 冠状病毒病毒粒子组装机制中的结构蛋白
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.005
I. Zaloilo, О. Zaloilo, Y. Rud, L. Buchatskyi
The emergence of SARS-CoV-2 caused an urgent need to investigate the molecular mechanisms of its reproduction. However, the detailed step-by-step mechanism of SARS-CoV-2 virion assembly has not been described yet. In the presented review the data on the role of structural proteins in the efficient assembly of betacoronavirus particles are analyzed.
SARS-CoV-2的出现引起了对其繁殖分子机制研究的迫切需要。然而,SARS-CoV-2病毒粒子组装的详细分步机制尚未描述。本文对结构蛋白在冠状病毒粒子高效组装中的作用进行了分析。
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引用次数: 1
Studies of the molecular basis of eukaryotic transcription. Roger Kornberg the Nobel prize in Chemistry 2006 真核生物转录的分子基础研究。罗杰·科恩伯格获2006年诺贝尔化学奖
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.093
O. P. Matyshvska
The Nobel Prize in Chemistry 2006 was awarded to an American biochemist and professor of structural biology at Stanford University Roger Kornberg for his fundamental research on the molecular mechanisms of copying genetic information in eukaryotic cells. What are these molecular mechanisms? How is transcription complex formed and what is its structure? R. Kornberg devoted tirelessly 20 years of his work to answer these questions. The article is focused on his research and also describes Roger Kornberg’s life and scientific career. Keywords: DNA, protein crystals, RNA, RNA polymerase II, Roger Kornberg, transcription, transcription complex
2006年诺贝尔化学奖被授予美国生物化学家、斯坦福大学结构生物学教授罗杰·科恩伯格(Roger Kornberg),以表彰他在真核细胞中复制遗传信息的分子机制方面的基础研究。这些分子机制是什么?转录复合体是如何形成的,它的结构是什么?R. Kornberg不知疲倦地投入了20年的工作来回答这些问题。这篇文章的重点是他的研究,也描述了罗杰·科恩伯格的生活和科学生涯。关键词:DNA,蛋白质晶体,RNA, RNA聚合酶II, Roger Kornberg,转录,转录复合物
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引用次数: 0
Assessing the in vitro anti-glycation efficacy of vitamins A, C, D, E 评价维生素A、C、D、E的体外抗糖化作用
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.061
N. P. Dalbanjan, A. J. Kadapure, P. Huded, V. Chachadi, S. Nayaka, Praveen Kumar S.K.
Accreted sugars in the blood react with protein’s amino group via Schiff base to form Amadori compounds. Further, these compounds execute irreversible chemical modifications generating advanced glycation end products (AGEs). The current study investigated the effects of vitamins in a glycation-prone in vitro environment. Glycation model was reached by incubating BSA with 0.1 M glucose/fructose in 0.1 M phosphate-buffered saline. Intrinsic (tyrosine/tryptophan) and AGEs fluorescence was monitored with fluorescence spectrophotometer. Ellman’s test depicted that native BSA contains more free thiol groups than glycated BSA. It was shown that BSA is more susceptible to glycation in the presence of fructose than glucose, and vitamin D followed by vitamin E and A can significantly rescue the BSA from glycation progression. Keywords: advanced glycation end products, anti-glycation, BSA, Ellman’s test, fluorescence, vitamins
血液中积聚的糖通过希夫碱与蛋白质的氨基反应形成阿玛多里化合物。此外,这些化合物进行不可逆的化学修饰,产生晚期糖基化终产物(AGEs)。本研究探讨了维生素在体外易糖基化环境中的作用。在0.1 M磷酸盐缓冲盐水中,用0.1 M葡萄糖/果糖孵育牛血清白蛋白,形成糖基化模型。用荧光分光光度计监测本征(酪氨酸/色氨酸)和AGEs荧光。埃尔曼的试验表明,天然牛血清白蛋白比糖化牛血清白蛋白含有更多的游离巯基。结果表明,在果糖的存在下,牛血清白蛋白比葡萄糖更容易发生糖基化,维生素D、维生素E和维生素A可以显著地挽救牛血清白蛋白的糖基化进程。关键词:晚期糖基化终产物,抗糖基化,牛血清白蛋白,Ellman试验,荧光,维生素
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引用次数: 0
Localization and level of proapoptotic protein regulators in a rat lung tissue during development of acute experimental bronchopneumonia 急性实验性支气管肺炎大鼠肺组织中促凋亡蛋白调节因子的定位和水平
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.036
D. Ziablitsev, A. Tykhomyrov, O. Dyadyk, S. Kolesnikova, S. Ziablitsev
Apoptosis plays an important role in the development of acute inflammatory lung injury (AILI) and its consequences, which can be realized in different cells with distinct intensity and rate. The aim of this study was to determine the distribution and expression intensity of apoptosis markers in the lungs of rats in the AILI model with endotracheal introduction of capron thread and LPS. Immunoblotting and immunohistochemical studies were performed using monoclonal antibodies against Bax and caspase-3 proteins. It was shown that Bax level increased significantly with the peak on the 7th day. The second peak of Bax 40 dimeric form was noted on the 21st day. The level of both pro-caspase-3 and active caspase-3 was also dramatically increased with a maximum on the 5th day and the second peak of active caspase-3 content was observed on the 21st day. These changes reflected the activation of apoptosis in key trigger periods of AILI during the development of exudative hemorrhagic pneumonia and subsequent fibrotic remodeling of the lungs. Keywords: AILI, apoptosis, Bax, caspase-3, lipopolysaccharide
细胞凋亡在急性炎症性肺损伤(acute inflammatory lung injury, AILI)及其后果的发生发展中起着重要作用,其发生在不同的细胞中,且发生的强度和速率不同。本研究的目的是测定经气管内注入capron线和LPS的AILI模型大鼠肺中凋亡标志物的分布和表达强度。使用抗Bax和caspase-3蛋白的单克隆抗体进行免疫印迹和免疫组织化学研究。结果表明,Bax水平显著升高,在第7天达到峰值。bax40二聚体的第二峰出现在第21天。前caspase-3和活性caspase-3含量也显著增加,在第5天达到最大值,活性caspase-3含量在第21天出现第二个高峰。这些变化反映了在渗出性出血性肺炎发展和随后肺纤维化重塑过程中,aii关键触发期细胞凋亡的激活。关键词:AILI,凋亡,Bax, caspase-3,脂多糖
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引用次数: 2
Black tea and coffee impact on steroid hormones status in young men 红茶和咖啡对年轻男性类固醇激素水平的影响
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.083
M. F. Amirova, A. Dadashova, E. Huseynova, I. Kerimova, S. I. Hasanova, F. E. Guliyeva, S. R. Guliyeva, R. Rahimova, G. Vahabova
Tea and coffee alkaloids affect the hormonal status of the body. There are reports about the effect of caffeine on the body under stress, but nearly absent reports on the effect of tea and coffee alkaloids at rest. The aim of this work was to determine whether there is a significant difference in testosterone and cortisol concentrations in the blood of young men before and after drinking indicated beverages. The work was carried out on 21 healthy young males that were tested for blood cortisol levels before and after drinking tea or coffee on an empty stomach. The young men were divided into two groups: the first group comprised those whose cortisol levels increased after taking a single dose of tea, the second group – whose cortisol decreased. The third group comprised persons who took a single dose of strong-grain coffee. In addition, adrenaline and testosterone levels were determined as a hormonal panel. Our pioneer investigation found that coffee causes a significant decrease in cortisol levels at rest, but tea consumption by coffee drinkers leads to a more pronounced decrease in the cortisol levels than coffee. Keywords: black tea, coffee, cortisol, young man
茶和咖啡中的生物碱会影响身体的荷尔蒙状态。有关于咖啡因在压力下对身体的影响的报道,但几乎没有关于茶和咖啡生物碱在休息时的影响的报道。这项工作的目的是确定年轻男性在饮用指示饮料前后血液中的睾酮和皮质醇浓度是否有显著差异。研究人员对21名健康的年轻男性在空腹喝茶或喝咖啡前后的血液皮质醇水平进行了测试。这些年轻人被分为两组:第一组的人在喝了一剂茶后皮质醇水平上升,第二组的人皮质醇水平下降。第三组是只喝一剂浓咖啡的人。此外,肾上腺素和睾酮水平被确定为激素面板。我们的开创性研究发现,咖啡会导致休息时皮质醇水平显著下降,但喝咖啡的人喝茶会比喝咖啡更明显地降低皮质醇水平。关键词:红茶,咖啡,皮质醇,年轻人
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引用次数: 1
Lipid parameters in the blood of coronary artery diseases patients depending on age and dietary components: a case study 冠状动脉疾病患者血液中的脂质参数与年龄和饮食成分的关系:一个案例研究
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.054
S. Prasad
Coronary artery disease (CAD) is the major cause of mortality worldwide and is defined as an elevated level of atherogenic cholesterol and low levels of HDL cholesterol. However, very few studies have been conducted in India on the association of blood lipid profile with age and diet. The study aims to estimate lipid parameters in the blood of CAD patients aged 40-70 years on different diets. Lipid parameters were analyzed by enzymatic method on EM-200 fully automated Biochemistry analyzer. The data obtained showed that the majority of patients who suffered from CAD were between the age group of 51-60 years. Patients with CAD had altered lipid profiles, with higher levels of serum TGs, total cholesterol and low levels of HDL. Vegetarians have been found to have reduced lipid risk factors for coronary heart disease compared to patients on a mixed diet. Keywords: age, cholesterol, coronary artery disease, dietary components, HDL, triglycerides
冠状动脉疾病(CAD)是世界范围内导致死亡的主要原因,其定义为致动脉粥样硬化胆固醇水平升高和高密度脂蛋白胆固醇水平降低。然而,在印度,很少有关于血脂与年龄和饮食之间关系的研究。本研究旨在评估40-70岁冠心病患者在不同饮食下的血脂参数。在EM-200全自动生化分析仪上用酶法分析脂质参数。获得的数据显示,患CAD的大多数患者年龄在51-60岁之间。冠心病患者血脂谱发生改变,血清总胆固醇、总胆固醇水平较高,HDL水平较低。研究发现,与混合饮食的患者相比,素食者患冠心病的脂质风险因素更低。关键词:年龄,胆固醇,冠状动脉疾病,膳食成分,HDL,甘油三酯
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引用次数: 0
Inhibition of plasma membrane Сa(2+),Mg(2+)-АТРase by сalixarene sulfonylamidines. Structure-activity relationship 链芳烃磺酰基胺对质膜Сa(2+),Mg(2+)-АТРase的抑制作用。结构与活性关系
Pub Date : 2022-11-10 DOI: 10.15407/ubj94.04.018
O. A. Shkrabak, T. O. Veklich, R. Rodik, V. Kalchenko, S. Kosterin
Previously we have already shown that tetrasulfonylamidinecalixarene C-90 inhibited plasma membrane Са2+,Mg2+-АТРаse of smooth muscle cells selectively to other ATPases of plasma membrane. To inhance the inhibitory effect of calixarenes several alkoxycalixarene sulfonylamidines structurally similar to calixa­rene C-90 were synthesized and their effects on the mentioned enzyme activity, the level of cytoplasmic Ca2+ concentration and hydrodynamic diameter of isolated smooth muscle cells were checked. It was shown that sulfonylamidino groups are crucial for Са2+,Mg2+-АТРаse inhibition, the efficiency of inhibition depends on their quantity and spatial orientation at the upper rim of calixarene macrocycle. Introduction of phenyl or tert-butyl groups into the upper rim and of long alkyl chains into the lower rim led to only slightl increase of inhibition efficiency. The inhibitory effect of studied calixarenes on Са2+,Mg2+-АТРаse correlated with effects on cytosolic Ca2+ concentration and hydrodynamic diameter of smooth muscle cells. The obtained results are important for creation of more effective and selective inhibitors of plasma membrane Са2+,Mg2+-АТРаse as regulators of smooth muscle contractility. Keywords: calixa­rene sulfonylamidines, intracellular Ca(2+) concentration, plasma membrane Са(2+);Mg(2+)-АТРаse, smooth muscle
之前我们已经证明,四磺酰酰胺二羧基arene C-90选择性地抑制平滑肌细胞的质膜Са2+,Mg2+-АТРаse对质膜其他atp酶的抑制作用。为了增强杯芳烃的抑制作用,合成了几种结构类似杯芳烃C-90的烷氧基杯芳烃磺酰基胺,并检测了它们对上述酶活性的影响,以及离体平滑肌细胞的细胞质Ca2+浓度水平和水动力直径。结果表明,磺胺基对Са2+、Mg2+-АТРаse的抑制作用至关重要,抑制效果取决于它们在杯芳烃大环上缘的数量和空间取向。上环引入苯基或叔丁基,下环引入长烷基链,抑制效率仅略有提高。杯芳烃对Са2+、Mg2+-АТРаse的抑制作用与细胞内Ca2+浓度和平滑肌细胞水动力直径的影响相关。所获得的结果对于创建更有效和选择性的质膜抑制剂Са2+,Mg2+-АТРаse作为平滑肌收缩性的调节剂具有重要意义。关键词:杯脲磺酰胺,胞内Ca(2+)浓度,质膜Са(2+), Mg(2+)-АТРаse,平滑肌
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引用次数: 1
期刊
The Ukrainian Biochemical Journal
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