Reconstitution of 3′ end processing of mammalian pre-mRNA reveals a central role of RBBP6

The 3′ ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity

assessment of pd 1 pd l1 colocalization in hepatocellular carcinoma hcc using bright field double labeling and quantitative digital image analysis

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Predictive factors for response to neoadjuvant therapy in patients with oesophageal cancer

Background: Preoperative radio-chemotherapy (RCX) was introduced to improve the outcome of patients with oesophageal cancer (EC), but conflicting results have been released. Some 20-30% of patients show a complete pathological response, however, the perioperative morbidity and mortality is increased. To search for factors indicating response prior to the onset of RCX we investigated the proliferative activity (MIB-1), the expression of vascular endothelial growth factor (VEGF), and the capillary density (CD34) in samples of EC obtained by endoscopy prior to the start of the treatment. Methods: Forty-six (MIB-1) and 21 (VEGF, CD34) tissue specimens of ECs were available from 56 patients undergoing pretherapeutic endoscopy, RCX and surgery. Perioperative morbidity was divided into surgery and non-surgery related morbidity. MIB-1, VEGF and CD34 expression were investigated immunohistochemically. Multivariate analysis was carried out to prove independence of investigated variables. Results: Postoperative morbidity was noticed in 54 of 56 operated patients. Eight of 56 patients who received RCX died in hospital. Survival was significantly different between the group of complete responders (n=14) and non-responders (n=23; P=0.0026). None of the investigated tumour samples from patients with a complete response (CR) had a proliferation index of less than 45. Tumour samples from patients with a CR showed a VEGF expression of 10.7 compared with 36.58 of tumours with no response (P=0.035). CD34 expression showed a correlation with VEGF expression. The relation of mean indices of VEGF expression and proliferative activity in tumours from patients with complete, partial or no response was 10.7:58.8, 18.3:53.8 and 36.6:43.5, respectively. Conclusions: According to these results, it may be expected that tumours with a VEGF/MIB-1 ratio of 1:6 or less prior to RCX will respond to this therapy. Copyright © 2002 Elsevier Science B.V

Проектирование контактора постоянного тока для коммутации цепи привода экскаватора

РЕФЕРАТ Выпускная квалификационная работа включает в себя 90 страниц, 22 рисунка, 11 таблиц, 119 формул, 24 источника. Объект исследования — контактор постоянного тока. Цель работы заключается в проектировании контактора постоянного тока для коммутации цепи привода экскаватора на заданные параметры тока, напряжения и режима работы. Для достижения этой цели, необходимо решить следующие задачи: •выбор прототипа контактора постоянного тока; •расчет токоведущего контура; •расчет дугогасительного устройства; •расчет механической характеристики; •расчет электромагнитной системы. Результатом проведенной работы является спроектированный контактор постоянного тока.ESSAY Final qualifying work includes 90 pages, 22 figures, 11 tables, 119 formulas, 24 source. The object of study - DC contactor. The aim of the work is to design a DC contactor for switching chain excavator drive on given current parameters, voltage and operating mode. To achieve this goal, you need to solve the following problems: • Selection of a prototype DC contactor; • calculation of the current-carrying path; • calculation Interrupters; • calculation of the mechanical characteristics; • calculation of electromagnetic systems. The result of this work is designed DC contactor

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m3G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m3G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m3G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA

A Simple Approach to Assign Disulfide Connectivity Using Extracted Ion Chromatograms of Electron Transfer Dissociation Spectra

Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of ETD facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds

Endothelium-derived endothelin-1

One year after the revelation by Dr. Furchgott in 1980 that the endothelium was obligatory for acetylcholine to relax isolated arteries, it was clearly shown that the endothelium could also promote contraction. In 1988, Dr. Yanagisawa’s group identified endothelin-1 (ET-1) as the first endothelium-derived contracting factor. The circulating levels of this short (21-amino acid) peptide were quickly determined in humans, and it was reported that, in most cardiovascular diseases, circulating levels of ET-1 were increased, and ET-1 was then tagged as “a bad guy.” The discovery of two receptor subtypes in 1990, ET(A) and ET(B), permitted optimization of the first dual ET-1 receptor antagonist in 1993 by Dr. Clozel’s team, who entered clinical development with bosentan, which was offered to patients with pulmonary arterial hypertension in 2001. The revelation of Dr. Furchgott opened a Pandora’s box with ET-1 as one of the actors. In this brief review, we will discuss the physiological and pathophysiological role of endothelium-derived ET-1 focusing on the regulation of the vascular tone, and as much as possible in humans. The coronary bed will be used as a running example in this review because it is the most susceptible to endothelial dysfunction, but references to the cerebral and renal circulation will also be made. Many of the cardiovascular complications associated with aging and cardiovascular risk factors are initially attributable, at least in part, to endothelial dysfunction, particularly dysregulation of the vascular function associated with an imbalance in the close interdependence of nitric oxide and ET-1