Apoptosis resistance downstream of eIF4E: posttranscriptional activation of an anti-apoptotic transcript carrying a consensus hairpin structure

Aberrant activation of the translation initiation machinery is a common property of malignant cells, and is essential for breast carcinoma cells to manifest a malignant phenotype. How does sustained activation of the rate limiting step in protein synthesis so fundamentally alter a cell? In this report, we test the post transcriptional operon theory as a possible mechanism, employing a model system in which apoptosis resistance is conferred on NIH 3T3 cells by ectopic expression of eIF4E. We show (i) there is a set of 255 transcripts that manifest an increase in translational efficiency during eIF4E-mediated escape from apoptosis; (ii) there is a novel prototype 55 nt RNA consensus hairpin structure that is overrepresented in the 5′-untranslated region of translationally activated transcripts; (iii) the identified consensus hairpin structure is sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress, but only when eIF4E is deregulated; and (iv) that osteopontin, one of the translationally activated transcripts harboring the identified consensus hairpin structure functions as one mediator of the apoptosis resistance seen in our model. Our findings offer genome-wide insights into the mechanism of eIF4E-mediated apoptosis resistance and provide a paradigm for the systematic study of posttranscriptional control in normal biology and disease

mTOR signaling: implications for cancer and anticancer therapy

Mounting evidence links deregulated protein synthesis to tumorigenesis via the translation initiation factor complex eIF4F. Components of this complex are often overexpressed in a large number of cancers and promote malignant transformation in experimental systems. mTOR affects the activity of the eIF4F complex by phosphorylating repressors of the eIF4F complex, the eIF4E binding proteins. The immunosuppressant rapamycin specifically inhibits mTOR activity and retards cancer growth. Importantly, mutations in upstream negative regulators of mTOR cause hamartomas, haemangiomas, and cancers that are sensitive to rapamycin treatment. Such mutations lead to increased eIF4F formation and consequently to enhanced translation initiation and cell growth. Thus, inhibition of translation initiation through targeting the mTOR-signalling pathway is emerging as a promising therapeutic option

Implication of 4E-BP1 protein dephosphorylation and accumulation in pancreatic cancer cell death induced by combined gemcitabine and TRAIL

Pancreatic cancer cells show varying sensitivity to the anticancer effects of gemcitabine. However, as a chemotherapeutic agent, gemcitabine can cause intolerably high levels of toxicity and patients often develop resistance to the beneficial effects of this drug. Combination studies show that use of gemcitabine with the pro-apoptotic cytokine TRAIL can enhance the inhibition of survival and induction of apoptosis of pancreatic cancer cells. Additionally, following combination treatment there is a dramatic increase in the level of the hypophosphorylated form of the tumour suppressor protein 4E-BP1. This is associated with inhibition of mTOR activity, resulting from caspase-mediated cleavage of the Raptor and Rictor components of mTOR. Use of the pan-caspase inhibitor Z-VAD-FMK indicates that the increase in level of 4E-BP1 is also caspase-mediated. ShRNA-silencing of 4E-BP1 expression renders cells more resistant to cell death induced by the combination treatment. Since the levels of 4E-BP1 are relatively low in untreated pancreatic cancer cells these results suggest that combined therapy with gemcitabine and TRAIL could improve the responsiveness of tumours to treatment by elevating the expression of 4E-BP1

ПРЕССОВЫЙ УЗЕЛ УСТАНОВКИ CONFORM ДЛЯ НЕПРЕРЫВНОГО ПРЕССОВАНИЯ ЦВЕТНЫХ МЕТАЛЛОВ

The study covers technology features of making pressed items of non-ferrous metals by Conform continuous forming. The analysis of pressing equipment designs identified a disadvantage typical for many Conform units – the lack of connection between the system for securing the fixed part of the split container (shoe) and the impeller shaft. Therefore, it is impossible to ensure a controlled gap between the shoe and the groove bottom during the operation, which results in unstable bar stock feeding to the die. The paper aimed at the development of a new shoe attachment system to ensure a steady flow of continuous metal extrusion process. This provides for forming a rigid and strong connection between the shoe and the impeller shaft using the system of levers and rods. An upgraded die assembly was proposed to solve this problem. The drive components and working parts of the pilot Conform unit were designed based on power calculations made for extrusion of aluminum and copper alloys using the formulas provided in the technical literature. The obtained results were used for strength calculations of die assembly parts following the known methods. The practical use of the design proposed in the paper will provide new opportunities to make pressed steel items of non-ferrous metals with a consistently high level of properties. Рассмотрены технические особенности схемы получения пресс-изделий из цветных металлов непрерывным прессованием способом Conform. На основании анализа разновидностей конструкции прессового оборудования выявлен характерный для многих установок Conform недостаток – отсутствие связи между системой крепления неподвижной части разъемного контейнера (башмака) с валом приводного колеса. Это не позволяет в процессе работы установки соблюдать контролируемый зазор между башмаком и дном канавки, что, в свою очередь, нарушает стабильность подачи прутковой заготовки к матрице. Цель работы заключалась в создании новой системы крепления башмака, обеспечивающей стабильное протекание процесса непрерывного экструдирования металла. При этом ставилась задача создания жесткой и прочной связи между башмаком и валом рабочего колеса системой рычагов и тяг. Для ее решения предложена модернизированная конструкция прессового узла установки. Проектирование элементов привода и рабочих частей опытной установки Conform проводилось на основании расчетов энергосиловых параметров экструдирования алюминиевых и медных сплавов, значения которых определялись по формулам, приведенным в технической литературе. Полученные результаты использованы при прочностных расчетах деталей прессового узла по известным методикам. Практическое использование предложенной в работе конструкции откроет новые возможности в получении прессованной металлопродукции из цветных металлов со стабильно высоким уровнем свойств.

Epigenetic Activation of a Subset of mRNAs by eIF4E Explains Its Effects on Cell Proliferation

BACKGROUND: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5′ cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. METHODOLOGY/PRINCIPAL FINDINGS: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. CONCLUSIONS/SIGNIFICANCE: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs

Comparative Structural Analysis of Human DEAD-Box RNA Helicases

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members

Five Nuclear Loci Resolve the Polyploid History of Switchgrass (Panicum virgatum L.) and Relatives

Polyploidy poses challenges for phylogenetic reconstruction because of the need to identify and distinguish between homoeologous loci. This can be addressed by use of low copy nuclear markers. Panicum s.s. is a genus of about 100 species in the grass tribe Paniceae, subfamily Panicoideae, and is divided into five sections. Many of the species are known to be polyploids. The most well-known of the Panicum polyploids are switchgrass (Panicum virgatum) and common or Proso millet (P. miliaceum). Switchgrass is in section Virgata, along with P. tricholaenoides, P. amarum, and P. amarulum, whereas P. miliaceum is in sect. Panicum. We have generated sequence data from five low copy nuclear loci and two chloroplast loci and have clarified the origin of P. virgatum. We find that all members of sects. Virgata and Urvilleana are the result of diversification after a single allopolyploidy event. The closest diploid relatives of switchgrass are in sect. Rudgeana, native to Central and South America. Within sections Virgata and Urvilleana, P. tricholaenoides is sister to the remaining species. Panicum racemosum and P. urvilleanum form a clade, which may be sister to P. chloroleucum. Panicum amarum, P. amarulum, and the lowland and upland ecotypes of P. virgatum together form a clade, within which relationships are complex. Hexaploid and octoploid plants are likely allopolyploids, with P. amarum and P. amarulum sharing genomes with P. virgatum. Octoploid P. virgatum plants are formed via hybridization between disparate tetraploids. We show that polyploidy precedes diversification in a complex set of polyploids; our data thus suggest that polyploidy could provide the raw material for diversification. In addition, we show two rounds of allopolyploidization in the ancestry of switchgrass, and identify additional species that may be part of its broader gene pool. This may be relevant for development of the crop for biofuels

Casting Technology of Aluminium Alloys in an Electromagnetic Mould for the Production of Long Workpieces of Small Diameter

Приведены результаты исследований структуры и свойств длинномерных слитков Ǿ 8 мм из алюминиевых сплавов, отлитых в электромагнитный кристаллизатор (ЭМК). Показано, что литье в ЭМК позволяет обеспечить чистоту металла по неметаллическим включениям без предварительной очистки расплава (рафинирования, дегазации и т. д.). Установлено, что слитки малых диаметров, отлитые в электромагнитный кристаллизатор, имеют диспергированную структуру с размером дендритной ячейки ~4 мкм, которая характерна для гранулируемых алюминиевых сплавов, полученных со скоростями охлаждения 103-104 °С/с. Получен литой материал, который по свойствам равен деформированномуIn this paper you see how casting speed in electromagnetic mould of aluminum alloy AMg6 effect to the structure and physical properties of the resulting workpiece diameter of 8 mm. Metallurgical defects in microstructure are not detected such as oxides, inclusions, porosity, despite the fact that the melt does not pass the pre-cleaning, i.e. not been processed refining flux, and other degassing. The used technology enhances the uniformity of the chemical composition of the ingots. It is established that ingots of small diameters cast into an electromagnetic crystallizer have a dispersed structure with a dendritic cell size of ~ 4 μm, which is characteristic for granulated aluminum alloys obtained with cooling rates of 103-104 °C/s. The cast material is obtained, which is equal to the deformed materia