90 research outputs found

    Усовершенствование экзонового метода для ускоренного получения кДНК гена реналазы крысы

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    We have improved our previously developed method of exon cloning of cDNA of eukaryotic genes to obtain the rat renalase gene cDNA. In contrast to the previously used step-by-step pairwise assembly of exons, in this work the procedure of full-length cDNA preparation was shortened due to simultaneous assembly of four neighboring exons at once (exons 1-4 and exons 6-9 of the rat renalase gene). The two obtained sequences (exons 1-4 and 6-9) were combined into a full-length cDNA of the rat renalase gene. The cDNA synthesized in this way was cloned into the prokaryotic vector pET-28a(+), which was then expressed in E. coli cells. The correctness of this approach was confirmed by sequencing resultant cDNA sequencing, which showed full (100%) identity with the nucleotide sequence available in the GenBank database (accession code: GenBankNM_001014167).Усовершенствован ранее разработанный нами метод экзонового клонирования кДНК эукариотических генов для получения кДНК гена реналазы крысы. В отличие от ранее использованного постадийного парного объединения экзонов, в данной работе процедура получения полноразмерной кДНК была сокращена за счет того, что мы использовали объединение сразу четырех соседних экзонов (1-4 и 6-9 гена крысы). Две полученные последовательности (экзонов 1-4 и 6-9) объединяли в полноразмерную кДНК гена реналазы крысы. Синтезированную таким образом кДНК клонировали в прокариотический вектор pET-28a(+) с последующей экспрессией в клетках E. coli. Корректность такого подхода подтверждена путем секвенирования полученной кДНК, которая показала полное (100%) совпадение с нуклеотидной последовательностью базы данных (код доступа GenBankNM_001014167)

    Особенности экспрессии и выделения укороченной рекомбинантной реналазы в прокариотических клетках

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    Renalase (RNLS) is a flavoproteinin which its N-terminal peptide (residues 1-17) has several important functions. In cells, it participates in the formation of the so-called Rossmanfold (residues 2-35), needed for «accommodation» of the FAD cofactor and for performing the catalytic functions of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). RNLS secretion into the extracellular space is accompanied by cleavage of this peptide. The resultant truncated extracellular RNLS cannot bind FAD and therefore performs various noncatalytic functions. In this work, we have performed expression the genetic construct encoding RNLS lacking its N-terminal signal peptide (tRNLS) in E. coli Rosetta (DE3) cells. The recombinant protein was accumulated in inclusion bodies in an insoluble form, which could be solubilized in the presence of a high concentration of urea or guanidine chloride. In contrast to full-length RNLS, which was effectively solubilized in the presence of 8 M urea, tRNLS was preferentially solubilized in the presence of 6 M guanidine chloride.Реналаза (RNLS) – флавопротеин, N-концевой пептид которого (1-17 аминокислотных остатка (а.о.)) выполняет несколько важных функций. В клетках он участвует в формировании так газываемой укладки Россмана (2-35 а.о.), необходимой для «размещения» кофактора FAD и выполнения каталитических функций RNLS в качестве FAD-зависимой оксидоредуктазы (КФ 1.6.3.5). При секреции RNLS во внеклеточное пространство этот пептид отщепляется, а образующаяся укороченная внеклеточная RNLS не может связывать FAD и поэтому выполняет различные некаталитические функции. В данной работе мы осуществили экспрессию генетической конструкции, кодирующей лишенную N-концевого сигнального пептида RNLS (tRNLS), в клетках E. coli Rosetta (DE3). Как и в случае полноразмерной RNLS, рекомбинантная tRNLS накапливается в тельцах включения в нерастворимой форме, которая может быть переведена в растворимую форму в присутствии высокой концентрации мочевины или гуанидинхлорида. При этом, в отличие от полноразмерной RNLS, которая солюбилизировалась в присутствии 8 М мочевины, более эффективная солюбилизация tRNLS была достигнута в присутствии 6 М гуанидинхлорида

    Phosphorus-based compounds for EUV multilayer optics materials

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    We have evaluated the prospects of phosphorus-based compounds in extreme ultraviolet multilayer optics. Boron phosphide (BP) is suggested to be used as a spacer material in reflective multilayer optics operating just above the L-photoabsorption edge of P (λ ≈9.2 nm). Mo, Ag, Ru, Rh, and Pd were considered for applications as reflector materials. Our calculations for multilayer structures with perfect interfaces show that the Pd/BP material combination suggests the highest reflectivity values, exceeding 70% within the 9.2 – 10.0 nm spectral range. We also discuss the potential of fabrication of BP-based multilayer structures for optical applications in the extreme ultraviolet rang

    Multiple Palaeoproterozoic carbon burial episodes and excursions

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    Organic-rich rocks (averaging 2–5% total organic carbon) and positive carbonate-carbon isotope excursions (View the MathML source and locally much higher, i.e. the Lomagundi-Jatuli Event) are hallmark features of Palaeoproterozoic successions and are assumed to archive a global event of unique environmental conditions following the c. 2.3 Ga Great Oxidation Event. Here we combine new and published geochronology that shows that the main Palaeoproterozoic carbon burial episodes (CBEs) preserved in Russia, Gabon and Australia were temporally discrete depositional events between c. 2.10 and 1.85 Ga. In northwest Russia we can also show that timing of the termination of the Lomagundi-Jatuli Event may have differed by up to 50 Ma between localities, and that Ni mineralisation occurred at c. 1920 Ma. Further, CBEs have traits in common with Mesozoic Oceanic Anoxic Events (OAEs); both are exceptionally organic-rich relative to encasing strata, associated with contemporaneous igneous activity and marked by organic carbon isotope profiles that exhibit a stepped decrease followed by a stabilisation period and recovery. Although CBE strata are thicker and of greater duration than OAEs (100 s of metres versus metres, ∼106 years versus ∼105 years), their shared characteristics hint at a commonality of cause(s) and feedbacks. This suggests that CBEs represent processes that can be either basin-specific or global in nature and a combination of circumstances that are not unique to the Palaeoproterozoic. Our findings urge circumspection and re-consideration of models that assume CBEs are a Deep Time singularity

    Сравнительный протеомный анализ изатин-связывающих белков печени и мозга мышей

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    Isatin (indol-2,3-dione) is an endogenous indole, exhibiting various biological activities that are realized via its interacts with numerous target proteins (so-called isatin-binding proteins). To date, isatin-binding proteins have been characterized in the brain of mice and rats. In this study we have performed a comparative proteomic analysis of the isatin-binding proteins of the mouse liver and brain. Proteomic profiling of clarified lysates of membrane and soluble fractions of liver and brain homogenates was performed using 5-aminocaproyl-isatin as an affinity ligand. During affinity based separation of isatin-binding proteins of soluble and membrane fractions of mouse brain homogenates lysed with Triton X-100, 63 individual proteins were identified. A similar separation of mouse liver homogenate fractions during affinity chromatography resulted in identification of 80 proteins. All identified liver and brain proteins belonged to the following functional groups: (I) Carbohydrate metabolism and energy generation; (II) Lipid metabolism; (III) Metabolism of nucleotides and amino acids; (IV) Formation of the cytoskeleton, exocytosis; (V) Regulation of gene expression, cell division and differentiation; (VI) Antioxidant and protective proteins; (VII) Signal transmission and regulation of enzyme activity. The total number of isatin-binding proteins common for the brain and liver was only 12. The most common for the brain and liver of isatin-binding proteins was found in group VI (antioxidant and protective proteins), complete absence of coincidence in group II (lipid metabolism) and group IV (formation of the cytoskeleton, exocytosis). The observed differences in the profile of isatin-binding proteins appear to play an important role in the specific effects of isatin in certain organs.Изатин (индол-2,3-дион) – эндогенный индол, проявляющий разнообразные виды биологической активности, которая реализуется при его взаимодействии с многочисленными белками-мишенями (т.н. изатин-связывающими белками). На сегодняшний день изатин-связывающие белки охарактеризованы в мозге мышей и крыс. Целью настоящего исследования был сравнительный протеомный анализ изатин-связывающих белков печени и мозга мышей. Протеомное профилирование осветленных лизатов мембранной и растворимой фракций гомогенатов печени и мозга выполнено с использованием 5-аминокапроилизатина в качестве аффинного лиганда. При аффинном разделении изатин-связывающих белков растворимых и мембранных фракций гомогенатов мозга мыши, лизированных тритоном Х-100, было идентифицировано 63 индивидуальных белка. Аналогичное разделение фракций гомогената печени мыши в ходе аффинной хроматографии позволило идентифицировать 80 белков. Все идентифицированные белки печени и мозга принадлежали к следующим функциональным группам: (I) Метаболизм углеводов и генерация энергии; (II) Метаболизм липидов; (III) Метаболизм нуклеотидов и аминокислот; (IV) Формирование цитоскелета, экзоцитоз; (V) Регуляция экспрессии генов, клеточного деления и дифференцировки; (VI) Антиоксидантные и протекторные белки; (VII) Передача сигнала и регуляция активности ферментов. При этом общее число изатин-связывающих белков, совпадающих для мозга и печени, было невелико - всего 12. Наибольшее число общих для мозга и печени изатин-связывающих белков обнаружено в группе VI (aнтиоксидантные и протекторные белки), полное отсутствие совпадений – в группе II (метаболизм липидов) и группе IV (формирование цитоскелета, экзоцитоз). Обнаруженные различия в профиле изатин-связывающих белков, по-видимому, играют важную роль в специфических эффектах изатина в определенных органах

    The grandest of them all : the Lomagundi-Jatuli Event and Earth's oxygenation

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    Funding: K.K., A.L. and T.K. received funding from Estonian Science Agency Project PRG447 and Yu.D., A.R., D.R. and P.M. were supported by the state assignment of the Institute of Geology, Karelian Research Centre of the Russian Academy of Sciences.The Paleoproterozoic Lomagundi–Jatuli Event (LJE) is generally considered the largest, in both amplitude and duration, positive carbonate C-isotope (δ13Ccarb) excursion in Earth history. Conventional thinking is that it represents a global perturbation of the carbon cycle between 2.3–2.1 Ga linked directly with, and in part causing, the postulated rise in atmospheric oxygen during the Great Oxidation Event. In addition to new high-resolution δ13Ccarb measurements from LJE-bearing successions of NW Russia, we compiled 14 943 δ13Ccarb values obtained from marine carbonate rocks 3.0–1.0 Ga in age and from selected Phanerozoic time intervals as a comparator of the LJE. Those data integrated with sedimentology show that, contra to consensus, the δ13Ccarb trend of the LJE is facies (i.e. palaeoenvironment) dependent. Throughout the LJE interval, the C-isotope composition of open and deeper marine settings maintained a mean δ13Ccarb value of +1.5 ± 2.4‰, comparable to those settings for most of Earth history. In contrast, the 13C-rich values that are the hallmark of the LJE are limited largely to nearshore-marine and coastal-evaporitic settings with mean δ13Ccarb values of +6.2 ± 2.0‰ and +8.1 ± 3.8‰, respectively. Our findings confirm that changes in δ13Ccarb are linked directly to facies changes and archive contemporaneous dissolved inorganic carbon pools having variable C-isotopic compositions in laterally adjacent depositional settings. The implications are that the LJE cannot be construed a priori as representative of the global carbon cycle or a planetary-scale disturbance to that cycle, nor as direct evidence for oxygenation of the ocean–atmosphere system. This requires rethinking models relying on those concepts and framing new ideas in the search for understanding the genesis of the grandest of all positive C-isotope excursions, its timing and its hypothesized linkage to oxygenation of the atmosphere.Publisher PDFPeer reviewe

    LED Monitoring System for the BTeV Lead Tungstate Crystal Calorimeter Prototype

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    We report on the performance of a monitoring system for a prototype calorimeter for the BTeV experiment that uses Lead Tungstate crystals coupled with photomultiplier tubes. The tests were carried out at the 70 GeV accelerator complex at Protvino, Russia.Comment: 12 pages, 8 figures, LaTeX2e, revised versio

    Application of gamma activation analysis for research of Cs and I diffusion into a glassceramic matrix

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    Nuclear reactions ¹³³Cs(γ,n)¹³²Cs, ¹²⁷I(γ,n)¹²⁶I were utilized for research of Cs and I diffusion in glassceramic matrices. The glassceramic matrix was manufactured with the help of hot isostatic pressing at 910°C and pressure 100 MPa. Diffusivities of cesium and iodine in a grain and through interphase boundary at 600°C were equal 10⁻¹¹ and 7.9⋅10⁻⁹ sm²/s, accordingly. The decrease of iodine diffusivity in a grain was observed at 750°C. A method of manufacture of glassceramic matrix for long-lived storage and nuclear-waste disposal ¹²⁹I is proposed.Ядерные реакции ¹³³Cs(γ,n)¹³²Cs, ¹²⁷I(γ,n)¹²⁶I использовались для исследования диффузии Cs и I в стеклокерамической матрице. Стеклокерамическая матрица изготовлена при помощи газостатического прессования при 910°С и давлении 100 МПа. Коэффициенты диффузии цезия и йода в зерне и по границам зерен при 600°С составили 10⁻¹¹ и 7,9⋅10⁻⁹ см²/с, соответственно. Обнаружено уменьшение коэффициента диффузии йода в зерне при 750°С. Предложен способ создания матрицы для захоронения ¹²⁹I.Ядерні реакції ¹³³Cs(γ,n)¹³²Cs, ¹²⁷I(γ,n)¹²⁶I використовувалися для дослідження дифузії Cs та I у склокерамічній матриці. Склокерамічна матриця виготовлена за допомогою газостатичного пресування при 910°С і тиску 100 МПа. Коефіцієнти дифузії цезію і йоду в зерні і по границях зерен при 600° С склали 10⁻¹¹ та 7,9⋅10⁻⁹ см²/с, відповідно. Виявлено зменшення коефіцієнта дифузії йоду в зерні при 750°С. Запропоновано спосіб створення матриці для поховання ¹²⁹I

    Magnetic Fields, Relativistic Particles, and Shock Waves in Cluster Outskirts

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    It is only now, with low-frequency radio telescopes, long exposures with high-resolution X-ray satellites and gamma-ray telescopes, that we are beginning to learn about the physics in the periphery of galaxy clusters. In the coming years, Sunyaev-Zeldovich telescopes are going to deliver further great insights into the plasma physics of these special regions in the Universe. The last years have already shown tremendous progress with detections of shocks, estimates of magnetic field strengths and constraints on the particle acceleration efficiency. X-ray observations have revealed shock fronts in cluster outskirts which have allowed inferences about the microphysical structure of shocks fronts in such extreme environments. The best indications for magnetic fields and relativistic particles in cluster outskirts come from observations of so-called radio relics, which are megaparsec-sized regions of radio emission from the edges of galaxy clusters. As these are difficult to detect due to their low surface brightness, only few of these objects are known. But they have provided unprecedented evidence for the acceleration of relativistic particles at shock fronts and the existence of muG strength fields as far out as the virial radius of clusters. In this review we summarise the observational and theoretical state of our knowledge of magnetic fields, relativistic particles and shocks in cluster outskirts.Comment: 34 pages, to be published in Space Science Review
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