Coupling between transcription termination and RNA polymerase inchworming

AbstractAdvancement of RNA polymerase of E. coli occurs in alternating laps of monotonic and inchworm-like movement. Cycles of inchworming are encoded in DNA and involve straining and relaxation of the ternary complex accompanied by characteristic leaping of DNA and RNA footprints. We demonstrate that the oligo(T) tract that constitutes a normal part of transcription terminators acts as an inchworming signal so that the leap coincides with the termination event. Prevention of leaping with a roadblock of cleavage-defective EcoRI protein results in suppression of RNA chain release at a termination site. The results indicate that straining and relaxation of RNA polymerase are steps in the termination mechanism

Variation in guided streamer propagation along a DBD plasma jet by tailoring the applied voltage waveform

Experimental data on the evolution of a helium atmospheric pressure plasma jet driven by two different voltage waveforms are presented. The characteristics of directed ionization waves (guided streamers) were compared for a sinusoidal voltage waveform with a frequency of 52 kHz and a voltage waveform that was formed via the superposition of 41.6 kHz bipolar square pulses and 300 kHz oscillations. With the sinusoidal voltage, two consecutive ionization waves were observed. With a special tailoring voltage, control of the guided streamer propagation in a stepwise mode was achieved. The observed second streamer and the second step of propagation could be regarded as a secondary ionization wave for both voltages. A change in the voltage waveform led to significant variations in the secondary ionization wave formation and streamer parameters. The voltage waveform enabled the number of ionization waves and their propagation to change, which provided the possibility of controlling the plasma parameters of the jet

The conformational manifolds of drug-like molecules as studied in combination of experimental and computational techniques

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Solvation of conformationally flexible molecules. Experiment and computer simulations

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Purine nucleoside phosphorylase controls nicotinamide riboside metabolism in mammalian cells

Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD+ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.publishedVersio

The MANGUA Project : A Population-Based HIV Cohort in Guatemala

Introduction. The MANGUA cohort is an ongoing multicenter, observational study of people living with HIV/AIDS in Guatemala. The cohort is based on the MANGUA application which is an electronic database to capture essential data from the medical records of HIV patients in care. Methods. The cohort enrolls HIV-positive adults ≥16 years of age. A predefined set of sociodemographic, behavioral, clinical, and laboratory data are registered at entry to the cohort study. Results. As of October 1st, 2012, 21 697 patients had been included in the MANGUA cohort (median age: 33 years, 40.3% female). At enrollment 74.1% had signs of advanced HIV infection and only 56.3% had baseline CD4 cell counts. In the first 12 months after starting antiretroviral treatment 26.9% (n = 3938) of the patients were lost to the program. Conclusions. The implementation of a cohort of HIV-positive patients in care in Guatemala is feasible and has provided national HIV indicators to monitor and evaluate the HIV epidemic. The identified percentages of late presenters and high rates of LTFU will help the Ministry to target their current efforts in improving access to diagnosis and care

Depletion of Deoxyribonucleotide Pools is an Endogenous Source of DNA Damage in Cells Undergoing Oncogene-Induced Senescence

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response (DDR). Oxidative stress and hyper-replication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHF) undergoing HRAS[superscript G12V]-induced senescence. NHF-HRAS[superscript G12V] cells under-expressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with RB tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic co-expression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes and proliferation arrest in two types of NHF expressing HRAS[superscript G12V]. Reciprocally, shRNA-mediated suppression of TS and RR caused DNA damage and senescence in NHF although less efficiently than HRAS[superscript G12V]. However, overexpression of TS and RR in quiescent NHF did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of oncogene-induced senescence.This is the author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/the-american-journal-of-pathology/

Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C‐MYC depletion

The down‐regulation of dominant oncogenes, including C‐MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC‐depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC‐depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribonucleosides to culture media substantially inhibited DNA damage and senescence‐associated phenotypes caused by C‐MYC depletion. Our data demonstrate the essential role of TS and RR in C‐MYC‐dependent suppression of senescence in melanoma cells.Keywords: ribonucleotide reductase, oncogene‐induced senescence, dNTP, myc, melanoma, thymidylate synthas

Фенольные соединения этанольных извлечений Lemna minor L., Lemna trisulca L. и Lemna polyrrhiza L. Schleid. и их иммуномодулирующая активность

The purpose of the study is to determine the composition of the phenolic compounds of ethanol extracts isolated from three species of duckweed: Lemna minor L., Lemna trisulca L. and Lemna polyrrhiza L. (the synonym is Spirodella polyrrhiza Schleid.) and to study its effect on immune system activation.Materials and methods. The objects of the study are: air-dried grass samples of Lemna minor L., Lemna trisuica L. and Lemna polyrrhiza L. collected during their growing season in 2010–2011 in low-flow and stagnant ponds of Kozhevnikovsky and Tomsk districts of Tomsk region. Isolation of polyphenolic complexes (PFC) was carried out by extraction of air-dried raw material with ethyl alcohol. In qualitative and quantitative analysis of the samples studied the method of reversed-phase high-performance liquid chromatography on an Agilent 1100 Series instrument (USA) was used in isocratic mode. In the experiments, 200 male C57BL / 6 and BALB / C mice aged 8–12 weeks were used to determine immunomodulatory activity. Cell proliferation was assessed by a colorimetric method. The absorption of the obtained solutions was measured with a multi-channel spectrophotometer at the wavelength of 540 nm. The determination of antibody-forming cells in the spleen was performed by local hemolysis. The titer of antibodies in serum was evaluated in the hemagglutination reaction. The local hypersensitivity reaction of immediate type was assessed by the author’s modification.Results. For the first time the study of the qualitative composition and quantitative content of PFC of Lemna minor L. (LM) , Lemna trisulca L. (LT) trisulkas, and Lemna polyrrhiza L. (LP) : (4,7 ± 0,4)%, (3,3 ± 0,3)%, (12,8 ± 0,7)% was carried out. The highest content of phenolic acids (10,76%) and the sum of flavonoids, isoflavonoids and coumarins (14,7%) were found in the LP sample. The content of chlorogenic and 3,5-dihydroxybenzoic acids was 2–9 times higher in LP than in other species of duckweed. In LM and LT with concentrations of 5 μg/ml and LP in the range of 0,5–160 μg/ml did not have a toxic effect on antigen presenting cells. When incubated with LT (20 μg/ml), the proliferation of macrophages was reduced by 1,2 times, and when incubated with LM and LT (80 μg/ml), by 1,2 and 1,8 times, respectively. PFC duckweed (LP) did not have a similar effect. LM and LP had a stimulating effect on the production of nitrites in the concentrations of 5 and 20 μg/ml, increasing it by 1,3–1,6 times. Course introduction of LT and LP led to a significant decrease in the number of antibody-forming cells by 1,5 and 2,3 times and a decrease in the local hypersensitivity reaction by 1,9 and 1,5 times, respectively.Цель исследования. Определение состава фенольных соединений этанольных извлечений, выделенных из трех видов ряски: ряски малой (Lemna minor L.), ряски тройчатой (Lemna trisulca L.) и ряски многокорневой (Lemna polyrrhiza L., синоним Spirodella polyrrhiza L.) Schleid.), и изучение его влияния на активацию системы иммунитета.Материал и методы. Объект исследования: воздушно-сухие образцы травы ряски малой (Lemna minor L.), ряски тройчатой (Lemna trisuica L.) и ряски многокорневой (Lemna polyrrhiza L.), собранные в период их вегетации в 2010–2011 гг. в малопроточных и стоячих водоемах Кожевниковского и Томского районов Томской области. Выделение полифенольных комплексов (ПФК) проводили экстракцией воздушно-сухого сырья спиртом этиловым. При качественном и количественном анализе исследуемых образцов применяли метод обращенно-фазовой высокоэффективной жидкостной хроматографии на приборе Agilent 1100 Series (США) в изократическом режиме. В экспериментах для определения иммуномодулирующей активности использовали 200 самцов мышей линий С57ВL/6 и BALB/C в возрасте 8–12 нед. Пролиферацию клеток оценивали колориметрическим методом. Абсорбцию полученных растворов замеряли при помощи многоканального спектрофотометра при длине волны 540 нм. Определение антителообразующих клеток в селезенке проводили методом локального гемолиза. Титр антител в сыворотке крови оценивали в реакции гемагглютинации. Локальную реакцию гиперчувствительности немедленного типа оценивали по методике в авторской модификации.Результаты. Впервые проведено исследование качественного состава и количественного содержания ПФК ряски малой Lemna minor L. (LM), ряски трисульки Lemna trisulca L. (LT) и ряски многокорневой Lemna polyrrhiza L.(LP): (4,7 ± 0,4)%, (3,3 ± 0,3)%, (12,8 ± 0,7)% соответственно. Наибольшее содержание фенолокислот (10,76%) и суммы флавоноидов, изофлавоноидов и кумаринов (14,7%) обнаружено в образце LP. Содержание хлорогеновой и 3,5-дигидроксибензойной кислот в 2–9 раз больше в LP, чем других видах ряски. LM, LT в концентрации 5 мкг/мл и LP в диапазоне 0,5–160 мкг/мл не оказывают токсического действия на антигенпрезентирующие клетки. При инкубации с LT (20 мкг/мл) пролиферация макрофагов снижается в 1,2 раза, а при инкубации с LM и LT (80 мкг/мл) в 1,2 и 1,8 раза соответственно. ПФК ряски многокорневой (LP) не оказывают подобного эффекта. LM и LP оказывают стимулирующее действие на продукцию нитритов в концентрациях 5 и 20 мкг/мл, усиливая ее в 1,3–1,6 раза. Курсовое введение LT и LP приводит к достоверному снижению числа антителобразующих клеток в 1,5 и 2,3 раза и уменьшению величины локальной реакции гиперчувствительности в 1,9 и 1,5 раза соответственно