Физико-химические свойства мутантных форм L-аспарагиназы из Rhodospirillum rubrum, обладающих антителомеразной активностью

Rru_A3730 protein is a bacterial Rhodospirillum rubrum L-asparaginase (RrA), which is known by its anticancer activity. RrA variants with point amino acid substitutions in the region of 150 amino acids residues: RrA17N, K149E, RrAE149R, V150P, F151T, RrА17N, E149R, V150P, RrAE149R, V150P, showed antiproliferative properties, and also by their ability to suppress telomerase activity. This work is devoted to comparison of physical-chemical and catalytic properties of these mutant forms of RrA. It is shown that pH optimum is in the alkaline zone (8.5 – 9.3); L-glutaminase and D-asparaginase activity is respectively not more than 0.1% and 1.6% of L-asparaginase for all studied variants of RrA. The presence of the N17-terminal amino acid sequence MASMTGGQMGRGSSRQ of the capsid protein of bacteriophage T7 in the RrA structure leads to an increase in the thermal stability of mutant RrA analogues (from 50°C to 56°C) and their resistance to denaturation in the presence of 3 – 4 M urea. It is of Metal ions exhibit multidirectional effects on L-asparaginase activity of RrA. K+, Ca2+, Zn2+, Cs+, Co2+ in significantly affect the activity of L-asparaginase, while Mn2+, Cu2+, Fe3+ ions inhibit it. There was no correlation between antitelomerase (antiproliferative) activity and kinetic properties of mutant forms of L-asparaginase RrA.Белок Rru_A3730, известный как бактериальная L-аспарагиназа Rhodospirillum rubrum, представляет интерес в качестве потенциального противоопухолевого средства, особенно её варианты с точечными аминокислотными заменами в районе 150 аминокислотного остатка (а.к.о.): RrA17N, K149E, RrAE149R, V150P, F151T, RrА17N, E149R, V150P, RrAE149R, V150P, обладающие не только антипролиферативными свойствами, но и способностью подавлять активность теломеразы. Данная работа посвящена сравнению физико-химических и каталитических свойств этих мутантных форм RrA. Показано, что для всех изученных вариантов RrA рН оптимум находится в щелочной зоне (8.5 – 9.3); L-глутаминазная и D-аспарагиназная активность составляют, соответственно, не более 0.1% и 1.6% от L-аспарагиназной. Присутствие 17N-концевой аминокислотной последовательности MASMTGGQQMGRGSSRQ капсидного белка бактериофага Т7 в структуре RrA приводит к повышению термостабильности мутантных аналогов RrA (от 50°С до 56°С) и их устойчивости к денатурации в присутствии 3 – 4 М мочевины. Выявлен разнонаправленный эффект ионов металлов на L-аспарагиназную активность вариантов RrA: ионы K+, Ca2+, Zn2+, Cs+, Co2+ существенно не влияют на активность L-аспарагиназы, добавление ионов Mn2+, Cu2+, Fe3+ приводит к снижению активности. Не обнаружено корреляции между антителомеразной (антипролиферативной) активностью и кинетическими свойствами мутантных форм L-аспарагиназы RrA

О неустойчивости решений динамических уравнений на временной шкале

В роботi наведено результати аналiзу нестiйкостi динамiчних рiвнянь на часовiй шкалi. Застосовнiсть отриманого результату iлюструється на прикладi системи другого порядку.We present new results on the instability for dynamic equations on time scales. To demonstrate the applicability, we use some examples of dynamic equations of the second order

Multifaceted ammonia transporters

Ammonia homeostasis is essential for the normal functioning of macro-and microorganisms. The change in concentration of ammonia derivatives in intracellular and extracellular environments is a marker of nitrogen metabolism imbalance. Transport of these molecules is now known to occur by both simple diffusion and special membrane-associated transporters belonging to the Amt/MEP/Rh family. This protein family is subdivided into two subfamilies: the ammonium transporter (AMT)-methylammonium/ammonium permeases (MEP) and the rhesus (Rh) proteins. In this review, we systemize and generalize the long-established and some recent findings on the role of these proteins in nitrogen metabolism in general, and in the ammonia balance in particular. The similarities and differences of these systems in various living beings are discussed. The paper also focuses on the characteristics of several aspects of the classification and on the physiological importance of these proteins and their relationships to certain pathological processes. We also enumerate the prospects and unique challenges of research in this field of science. Deeper theoretical and practical research will provide a better understanding of the mechanisms underlying the structural-functional evolution of ammonia transport proteins, as well as the level of their involvement in the signaling pathways associated with the pathophysiology of nitrogen metabolism. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

Using DFT to calculate the parameters of the crystal field in Mn²⁺ doped hydroxyapatite crystals

Crystal field parameters for two nonequivalent positions Ca (I) and Ca (II) for hydroxy-apatite (HAp) crystals from the density functional theory (DFT) are calculated. Calculations are compared with the experimental electron paramagnetic resonance (EPR) spectra (registered at two microwave frequencies) for the synthesized Mn-HAp powders Ca9.995Mn0.005(PO4)6(OH)2. It is found that in the investigated species, the manganese is redistributed between both calcium sites with prevalence in Ca (I). Agreement between the calculated and experimental data proves that crystal field parameters in HAp can be calculated in the classical DFT model using the distributed electron density

D-amino acids in nature, agriculture and biomedicine

Information accumulated over the past decades on the physiological functions and metabolic pathways of biosynthesis and degradation of D-amino acids has led to a renewed interest in their study. These isomers are known to form both in nature and during the chemical synthesis of L-amino acids for feeding and pharmacological purposes, as well as in the industrial processing of some raw materials. This article discusses the positive and negative effects of D-amino acids on the human body, animals and the environment. In addition, the scientific data concerning the mechanisms of cytotoxic action of D-amino acids and their industrial and biomedical potential are summarized. © 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

Finding key points in a hand image using heatmaps

© Published under licence by IOP Publishing Ltd. This paper presents an approach to finding key points in the image of a hand using heat maps. For this, a convolutional model of a neural network and a training method on heatmaps were combined. An open bank of palm photos, supplemented by a set of own images and synthetic data, was used as a dataset. Direct and inverse transformations of two-dimensional coordinates on the plane into heat maps with a 2D Gaussian function centered at coordinate points were applied, data was prepared, the model was trained and tested. As a result of this approach, a neural network model able to recognise real world images was obtained

Induction of Telomerase Catalytic Subunit Alternative Splicing by Apoptotic Endonuclease G in Mouse and Rat Lymphocytes

It is known that apoptotic endonuclease G (EndoG) induces alternative splicing (AS) of telomerase catalytic subunit TERT (telomerase reverse transcriptase) mRNA and inhibits telomerase activity in tumor cells and activated human T cells. The aim of this study was to investigate the possibility of TERT mRNA AS induction and inhibition of telomerase activity by EndoG in activated mouse and rat lymphocytes. To induce EndoG expression, mouse and rat CD4+, CD8+ T cells, B cells, and NK cells were transfected with the pEndoG-GFP plasmid or incubated with the DNA-damaging agent cisplatin in vitro. The increase in the EndoG expression resulted in decreased expression of full-length active TERT variant, enhanced synthesis of the truncated splicing variant, and decreased telomerase activity. An increase in the EndoG expression, a change in the mRNA pool of TERT splicing variants, and inhibition of telomerase activity were observed in mouse and rat lymphocytes after cisplatin administration in vivo. Thus, EndoG is capable of inducing TERT mRNA AS and regulating telomerase activity in mouse and rat lymphocytes. © 2018, Pleiades Publishing, Ltd

Индукция альтернативного сплайсинга каталитической субъединицы теломеразы апоптотической эндонуклеазой EndoG в лимфоцитах мыши и крысы

Apoptotic endonuclease EndoG is known to induce alternative splicing (AS) of mRNA of telomerase catalytic subunit TERT (telomerase reverse transcriptase) in human cancer cells and activated T lymphocytes. The aim of this work was to study the possibility of induction AS of TERT mRNA and inhibition of telomerase activity by EndoG in activated lymphocytes of mice and rats. In order to overexpress EndoG, CD4+, CD8+ T-lymphocytes, B-lymphocytes and NK-cells from mice and rats were transfected with pEndoG-GFP plasmid or incubated with DNA-damaging agent cisplatin in vitro. EndoG overexpression was associated with down-regulation of the expression of full-length TERT and up-regulation of truncated spliced variant which resulted in inhibition of telomerase activity. Induction of EndoG and changes in the expression of TERT splice-variants accompanied by telomerase inhibition was observed in murine lymphocytes after in vivo cisplatin administration. Thus, EndoG was shown to induce AS of TERT mRNA and regulate telomerase activity in murine lymphocytes.Известно, что апоптотическая эндонуклеаза EndoG индуцирует альтернативный сплайсинг (АС) мРНК каталитической субъединицы теломеразы TERT (telomerase reverse transcriptase) и ингибирует активность теломеразы в опухолевых клетках и активированных Т-лимфоцитах человека. Цель работы - исследование возможности индукции АС мРНК TERT и ингибирования активности теломеразы эндонуклеазой EndoG в активированных лимфоцитах мыши и крысы. Для индукции экспрессии EndoG, CD4+ и CD8+ T-лимфоциты, В-лимфоциты и НК-клетки мышей и крыс трансфицировали плазмидой pEndoG-GFP или инкубировали их с ДНК-повреждающим агентом цисплатином in vitro. Увеличение экспрессии EndoG приводило к уменьшению экспрессии полноразмерного активного варианта TERT, повышению синтеза укороченного сплайс-варианта и понижению активности теломеразы. Увеличение экспрессии EndoG, изменение пула мРНК сплайс-вариантов TERT и ингибирование активности теломеразы наблюдали в лимфоцитах мышей и крыс после введения цисплатина in vivo. Таким образом, EndoG способна индуцировать АС мРНК TERT и регулировать активность теломеразы в лимфоцитах мышей и кры

Contact-independent suppressive activity of regulatory T cells is associated with telomerase inhibition, telomere shortening and target lymphocyte apoptosis

Regulatory T cells (Tregs) play a fundamental role in the maintenance of immunological tolerance by suppressing effector target T, B and NK lymphocytes. Contact-dependent suppression mechanisms have been well–studied, though contact-independent Treg activity is not fully understood. In the present study, we showed that human native Tregs, as well as induced ex vivo Tregs, can cause in vitro telomere-dependent senescence in target T, B and NK cells in a contact-independent manner. The co-cultivation of target cells with Tregs separated through porous membranes induced alternative splicing of the telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase), which suppressed telomerase activity. Induction of the hTERT splicing variant was associated with increased expression of the apoptotic endonuclease EndoG, a splicing regulator. Inhibited telomerase in target cells co-cultivated with Tregs for a long period of time led to a decrease in their telomere lengths, cell cycle arrest, conversion of the target cells to replicative senescence and apoptotic death. Induced Tregs showed the ability to up-regulate EndoG expression, TERT alternative splicing and telomerase inhibition in mouse T, B and NK cells after in vivo administration. The results of the present study describe a novel mechanism of contact-independent Treg cell suppression that induces telomerase inhibition through the EndoG-provoked alternative splicing of hTERT and converts cells to senescence and apoptosis phenotypes. © 2018 Elsevier Lt

Получение и характеристика нового мутантного гомолога хемотаксисного белка CheY из гипертермофильного анаэробного микроорганизма Thermotoga naphthophila

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.С использованием генно-инженерных методов сконструированы экспрессионные векторные конструкции, обеспечивающие эффективную продукцию рекомбинантных белков TnaCheY и TnaCheY-mut – гомологов хемотаксисного белка CheY гипертермофильного микроорганизма Thermotoga naphthophila – в клетках Escherichia coli BL21(DE3). Оптимизированы условия культивирования трансформированных штаммов. Изучено влияние эписомальной экспрессии гетерологичного хемотаксисного белка CheY на параметры кинетики роста культуры мезофильного микроорганизма E. coli. Предложена оптимальная схема очистки полученных белков с использованием термолизиса. На основе полученных данных в статье рассмотрены потенциальные области применения рекомбинантных вариантов термостабильного хемотаксисного белка CheY. На примере клеток лабораторного штамма E. coli BL21(DE3), экспериментально обоснована возможность использования эписомальной экспрессии подобных белков для управления временем культивирования и продукции фармацевтически и промышленно ценных метаболитов за счёт воздействия на отдельные этапы хемотаксиса бактерий