Hybrid multicyclophanes based on thiacalix[4]arene and pillar[5]arene: Synthesis and influence on the formation of polyaniline

© 2018 the Partner Organisations. For the first time, fragments of a pillar[5]arene were spatially preorganized with a thiacalix[4]arene core in a single multimacrocyclic structure. It was shown that the synthesized hybrid multicyclophanes bind aniline and do not interact with p-toluenesulfonic acid. Supramolecular assistance of the synthesized multicyclophanes in oxidative polymerization of aniline in aqueous p-toluenesulfonic acid solutions was studied. It was found that the use of the multicyclophane template in the reaction of the oxidative polymerization of aniline led to the formation of emeraldine with a higher molecular weight and a similar conductivity (1-2 mSm cm-1), which formed more stable emeraldine dispersions in acetone in comparison with traditionally obtained polyaniline

Kinetics of casein hydrolysis by peptidase from Bacillus thuringiensis var. israelensis

The kinetics of enzyme reaction is generally studied using the Michaelis-Menten equation and various methods of its linearization. Each method has its advantages and drawbacks, so their comparison for determining the kinetics of new enzymes action is topical. The aim of this work was to study the kinetics of casein hydrolysis catalyzed by new peptidase from Bacillus thuringiensis var. israelensis IMB B-7465 using several methods of enzyme activity assessment and Michaelis-Menten equation linearization. The satisfactory agreement between kinetic constants values obtained by the methods of Lineweaver-Burk, Hanes, Eadie-Hofstee, Cornish-Bowden-Eisenthal was established. The Lineweaver-Burk method was shown to be optimal for determining Km and Vmax of casein hydrolysis. Estimation of caseinolytic activity with the use of ortho-phthalic dialdehyde allowed more accurate Vmax determination compared to the use of Anson and Kunitz methods

Экспериментальные подходы к созданию тканеспецифического матрикса для биоискусственной печени

Shortage of donor organs for liver transplantation in the treatment of end-stage liver disease dictates the need to develop alternative methods that include technologies on tissue engineering and regenerative medicine. Objective: to study the ability of a tissue-specific matrix from decellularized human liver fragments (DHLF) to maintain adhesion and proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and HepG2 under static conditions and in a flow-through bioreactor. Materials and methods. Treatment with surfactants (SAS) – sodium dodecyl sulfate, Triton X-100 – followed by exposure to DNase was used for decellularization of human liver fragments (no more than 8 mm3). Biochemical screening included the determination of DNA quantity in the test samples. Efficiency of surfactant washing was assessed by the cytotoxicity of the matrix in the NIH 3T3 fibroblast culture. Viability and metabolic activity of cells were assessed via vital staining with a complex of fluorescent dyes LIVE/DEAD ® and PrestoBlue™ (Invitrogen, USA). Morphological examination of the liver cell-engineered constructs was carried out through histological staining and scanning electron microscopy with lanthanide contrast. Results. It was shown that the liver decellularization method used allows to obtain a biocompatible matrix with a residual DNA quantity <1%, which is capable of maintaining adhesion and proliferation of hAT-MSCs and HepG2. On day 7 of cultivation in the bioreactor, there was formation of a single conglomerate of the DHLF matrix with numerous groups of viable cells with a high nuclear-cytoplasmic ratio. The urea content in the culture medium is 1.5 ± 0.1 mmol/L, exceeding that of samples obtained under static conditions. This indicates the metabolic activity of HepG2 in the composition of the obtained culture systems. It was shown that constant flow of the culture medium in the perfusion bioreactor increased the proliferative activity of HepG2 and allowed to provide a more uniform colonization by matrix cells in comparison with static cultivation conditions. Conclusion. The conditions for uniform colonization of DHLFs in a flow-through bioreactor with cell cultures were established. The ability of the matrix to maintain adhesion and proliferation of hADSCs and HepG2 for 11 days indicates that it could be used in liver tissue engineering.Дефицит донорских органов для трансплантации печени при лечении терминальных стадий печеночной недостаточности диктует необходимость разработки альтернативных методов, к которым относятся технологии тканевой инженерии и регенеративной медицины. Целью работы было исследование способности тканеспецифического матрикса из децеллюляризованных фрагментов печени человека (ДФПч) поддерживать адгезию и пролиферацию мезенхимальных стромальных клеток жировой ткани человека (МСК ЖТч) и HepG2 в статических условиях и в проточном биореакторе. Материалы и методы. Для децеллюляризации фрагментов (не более 8 мм3) печени человека использовали обработку поверхностноактивными веществами (ПАВ) – додецилсульфатом натрия, Тритоном Х-100 с последующей экспозицией в ДНКазе. Биохимические исследования включали определение количества ДНК в исследуемых образцах. Эффективность отмывки от ПАВ оценивали по цитотоксичности матрикса на культуре фибробластов NIH 3T3. Оценку жизнеспособности и метаболической активности клеток проводили методом прижизненного окрашивания комплексом флюоресцентных красителей LIVE/DEAD ® и PrestoBlue™ (Invitrogen, США). Морфологическое исследование клеточно-инженерных конструкций печени проводили с использованием методов гистологического окрашивания и сканирующей электронной микроскопии с лантаноидным контрастированием. Результаты. Показано, что использованная методика децеллюляризации печени позволяет получать биосовместимый матрикс с остаточным количеством ДНК менее 1%, способный поддерживать адгезию и пролиферацию МСК ЖТч и HepG2. В биореакторе на 7-е сутки культивирования наблюдали образование единого конгломерата матрикса ДФПч с многочисленными группами жизнеспособных клеток с высоким ядерно-цитоплазматическим отношением. Содержание мочевины в культуральной среде превышает значение для образцов, полученных в статических условиях, и составляет 1,5 ± 0,1 ммоль/л, что свидетельствует о метаболической активности HepG2 в составе полученных культуральных систем. Показано, что постоянный поток культуральной среды перфузионного биореактора способствовал росту пролиферативой активности HepG2 и позволил обеспечить более равномерную колонизацию клетками матрикса по сравнению со статическими условиями культивирования. Заключение. Найдены условия равномерного заселения ДФПч в проточном биореакторе клеточными культурами. Способность матрикса поддерживать адгезию и пролиферацию МСК ЖТч и HepG2 в течение 11 суток свидетельствует о возможности его использования в тканевой инженерии печени

Исследование регенераторной и тканеспецифичной активности общ ей РНК клеток костного мозга

Aim. To establish the ability of the total RNA extracted from the body’s bone marrow cells (BMCs), in which liver tissue was damaged, to serve as a carrier of targeted regenerative signals to this organ.Materials and methods. By method of adoptive transfer in rats (n = 37) the  mitotic and proliferative activity of liver and kidney cells were studied in intact  recipients after intraperitoneal injection: the mononuclear BMCs – 2,5×106;  5,0×106; 3,5×107 cells – group 1 and the total RNA of the same BMCs  (30μg/100g of weight) – group 2 from donors in 12 hours after 70–75% of  hepatectomy; in group 3 (control), a saline solution was injected. RNA from  BMCs was extracted by the method developed by the «Evrogen» firm (Russia) with the reagent Extract RNA.Results. In group 2 in 48 and 72 h. there was the increasing of mitotic and  proliferative cell activity in the liver, but not in the kidneys (control of the  specificity of regenerative signals); in group 1 there was no transfer of  regenerative signals to these organs.Conclusion. The authors believe that the total RNA from BMCs, activated by hepatectomy, accumulates targeted (hepatospecific) regeneration signals, but  they are perceived only when RNA has been obtained by the damaged tissue.Цель. Установить способность суммарной РНК, выделенной из ККМ организма, в котором повреждена ткань печени, служить переносчиком адресных регенерационных сигналов именно в этот орган.Материалы и методы. Методом адоптивного переноса на крысах (n = 37) изучена митотическаая и пролиферативная активность клеток печени и почек у интактных реципиентов после внутрибрюшинного введения: мононуклеарных клеток костного мозга (ККМ) – 2,5×106;  5,0×106; 3,5×107 клеток – группа 1 и общей РНК из таких же ККМ (30 мкг/100 г веса) –  группа 2 от доноров через 12 часов после 70–75% гепатэктомии; в группе 3 (контроль)  вводили физраствор. РНК из ККМ выделяли методом, разработанным фирмой «Евроген» (Россия) с помощью реактива ExtractRNA.Результаты. В группе 2 на сроках 48 и 72 ч. отмечено увеличение митотической и  пролиферативной активности клеток в печени, но не в почках (контроль специфичности  регенерационных сигналов); в группе 1 не выявлен перенос регенерационных сигналов в эти  органы.Заключение. Авторы полагают, что общая РНК из активированных гепатэктомией ККМ аккумулирует адресные (гепатоспецифические) регенерационные сигналы, но  воспроизводятся они лишь при поступлении РНК в поврежденную ткань

КІНЕТИКА ГІДРОЛІЗУ КАЗЕЇНУ ЗА ДОПОМОГОЮ ВІЛЬНОЇ І ІММОБІЛІЗОВАНОЇ ПЕПТИДАЗИ BACILLUS THURINGIENSIS VAR. ISRAELENSIS ІМВ В-7465

In the process of proteases immobilization, their conformation, affinity of enzymes to the substrate, as well as other properties, may change. Therefore, their comprehensive study, including the reaction rates, affinity to the substrate and the determination of the mechanism of action using kinetic studies of free and immobilized peptidases, is an important task. This work aimed to determine the kinetic parameters of casein hydrolysis catalyzed by free and released from PVA/chitosan films peptidase from B. thuringiensis var. israelensis IMB B-7465. The kinetics of casein hydrolysis of free and released from PVA/chitosan films peptidase from Bacillus thuringiensis var. israelensis IMB B-7465 was studied. It is shown, that only the modified Anson method is applicable for determining the caseinolytic activity of peptidase, released from the matrix. It was revealed, that at relatively low substrate concentrations, the reaction rate of casein hydrolysis catalysed by free and released from PVA/chitosan films peptidase increased proportionally. When substrate concentration increased, the rate value approached its limit, and then began to decrease. That is, in the certain casein concentration range, the enzyme is inhibited by the substrate. The kinetic constants were measured within the ascending part of the curve relating the initial reaction rate and the substrate concentration. It was determined that the entrapment of the enzyme in the PVA/chitosan film does not significantly affect Vmax. of hydrolysis, while Km is increased 1.3-fold. It is associated with decreasing of the affinity of the enzyme to the substrate as a result of conformational changes in the protein globule, or due to the viscosity limitations, caused by polymers of the matrix. Substrate inhibition of free and released from films peptidase was studied. It was shown that the last Kis is 2.3-fold higher, than that of the free enzyme, which allows to hydrolyze higher concentrations of the substrate.Досліджено кінетику гідролізу казеїну вільною і вивільненою з плівок ПВС/хітозан пептидазою з Bacillus thuringiensis var. israelensis ІМВ В-7465. Показано, що тільки модифікований метод Ансона може бути застосований для визначення казеїнолітичної активності вивільненої з матриці пептидази. Визначено, що включення ензиму в плівку ПВС/хітозан істотно не впливає на Vмакс. гідролізу, тоді як Км збільшується в 1,3 рази. Виявлено інгібування вільної і вивільненої з плівок пептидази субстратом. Показано, що Кis останньої у 2,3 рази перевищує таку вільного ензиму, що дозволяє гідролізувати більші концентрації субстрату

Hybrid multicyclophanes based on thiacalix[4]arene and pillar[5]arene: Synthesis and influence on the formation of polyaniline

© 2018 the Partner Organisations. For the first time, fragments of a pillar[5]arene were spatially preorganized with a thiacalix[4]arene core in a single multimacrocyclic structure. It was shown that the synthesized hybrid multicyclophanes bind aniline and do not interact with p-toluenesulfonic acid. Supramolecular assistance of the synthesized multicyclophanes in oxidative polymerization of aniline in aqueous p-toluenesulfonic acid solutions was studied. It was found that the use of the multicyclophane template in the reaction of the oxidative polymerization of aniline led to the formation of emeraldine with a higher molecular weight and a similar conductivity (1-2 mSm cm-1), which formed more stable emeraldine dispersions in acetone in comparison with traditionally obtained polyaniline

Experimental implantation of tissue-engineering pancreatic construct

Aim: to study the effect of implantation of tissue-engineering pancreatic construct (TEPC) on the course of experimental diabetes mellitus.Materials and methods. The TEPC samples received as a result of joint incubation in vitro floating islet-like cultures (FILC), obtained from the pancreas of newborn rabbits, and the biopolymer microheterogeneous collagen hydrogel (BMCH). Stable diabetes mellitus was caused in Wistar rats by the method of fractional streptozotocin administration.Results. The formation of TEPC occurred at 7-10 days of incubation FILC with BMCH. At the same time, the presence of β-cells with insulin-producing activity was revealed in the TEPC. After intraperitoneal implantation of TEPC samples in rats with streptozotocin diabetes mellitus, there was a significant and persistent decrease in glycemia until the end of the 8-week period of the experiment. Morphological study of pancreas of recipient rats revealed signs of regeneration of own β-cells.Conclusion. The data obtained suggest the combined antidiabetic effect of intraperitoneal injection of TEPC, due to both the direct functioning of the implant and its stimulating effect on the regeneration of β-cells in their own islets of rats with streptozotocin diabetes mellitus