69 research outputs found
Three-step mechanism of promoter escape by RNA polymerase II
No full texthttp://dx.doi.org/10.13039/501100004189 Max Planck Societ
Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening
No full textTranscription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30–35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase “clamp head loop” and the TFIIF “charged region” that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems
Semi-automatic classification of skeletal morphology in genetically altered mice using flat-panel volume computed tomography.
No full textRapid progress in exploring the human and mouse genome has resulted in the generation of a multitude of mouse models to study gene functions in their biological context. However, effective screening methods that allow rapid noninvasive phenotyping of transgenic and knockout mice are still lacking. To identify murine models with bone alterations in vivo, we used flat-panel volume computed tomography (fpVCT) for high-resolution 3-D imaging and developed an algorithm with a computational intelligence system. First, we tested the accuracy and reliability of this approach by imaging discoidin domain receptor 2- (DDR2-) deficient mice, which display distinct skull abnormalities as shown by comparative landmark-based analysis. High-contrast fpVCT data of the skull with 200 microm isotropic resolution and 8-s scan time allowed segmentation and computation of significant shape features as well as visualization of morphological differences. The application of a trained artificial neuronal network to these datasets permitted a semi-automatic and highly accurate phenotype classification of DDR2-deficient compared to C57BL/6 wild-type mice. Even heterozygous DDR2 mice with only subtle phenotypic alterations were correctly determined by fpVCT imaging and identified as a new class. In addition, we successfully applied the algorithm to classify knockout mice lacking the DDR1 gene with no apparent skull deformities. Thus, this new method seems to be a potential tool to identify novel mouse phenotypes with skull changes from transgenic and knockout mice on the basis of random mutagenesis as well as from genetic models. However for this purpose, new neuronal networks have to be created and trained. In summary, the combination of fpVCT images with artificial neuronal networks provides a reliable, novel method for rapid, cost-effective, and noninvasive primary screening tool to detect skeletal phenotypes in mice
Flat-panel-detector-based volumetric CT: performance evaluation of imaging for skeletal structures of small animals in comparison to multislice CT
No full textObjectives: The aim of this study was to compare the image performance of silicon-based flat-panel-detector-based volumetric computed tomography (fpVCT) to multislice spiral computed tomography (MSCT) for the visualization and detail delectability of skeletal structures in rodents of different development stages. Materials and Methods: Rodents of different development stages were imaged with fpVCT (GE prototype with circular gantry with two 1024 x 1024, 200-mu m pixel size, amorphous silicon/Cesium lodid (Csl) flat-panel detector) and eightslice MSCT (LightSpeed Ultra). Imaging parameters (80 kVp, 100 mA) and the position of the rodents were identical in both techniques. Image quality, detail delectability, and contour of skeletal structures were judged by two observers in consensus using a 4-point scale (1=unsatisfactory... 4=good). Findings were displayed and evaluated in axial slices, multiplanar reconstructions (MPR), maximum intensity projections (MIP) and volume rendering technique (VRT) in both modalities. Mean and standard of error of mean were calculated. Results: In axial slices, visualization and detail delectability of very subtle skeletal structures, e.g., the basis of the skull was better in fpVCT than in MSCT (4 vs. 2 points). The MPRs of fpVCT showed less artifacts and more details than those of the MSCT. The MIPs and VRTs of the fpVCT demonstrated best image quality in all rodents of different development stages, whereas MSCT showed significant artifacts. Conclusion: fpVCT outperformed MSCT in imaging of small rodents. Due to the truly isotropic volume data set with high spatial resolution, fpVCT is a powerful tool in evaluating detailed skeletal structures. (c) 2007 Elsevier Inc. All rights reserved
Structural basis of transcription reduction by a promoter-proximal +1 nucleosome
No full textAt active human genes, the +1 nucleosome is located downstream of the RNA polymerase II (RNA Pol II) pre-initiation complex (PIC). However, at inactive genes, the +1 nucleosome is found further upstream, at a promoter-proximal location. Here, we establish a model system to show that a promoter-proximal +1 nucleosome can reduce RNA synthesis in vivo and in vitro, and we analyze its structural basis. We find that the PIC assembles normally when the edge of the +1 nucleosome is located 18 base pairs (bp) downstream of the transcription start site (TSS). However, when the nucleosome edge is located further upstream, only 10 bp downstream of the TSS, the PIC adopts an inhibited state. The transcription factor IIH (TFIIH) shows a closed conformation and its subunit XPB contacts DNA with only one of its two ATPase lobes, inconsistent with DNA opening. These results provide a mechanism for nucleosome-dependent regulation of transcription initiation
Morphologic changes of mammary carcinomas in mice over time as monitored by flat-panel detector volume computed tomography
No full textAbstractNoninvasive methods are strongly needed to detect and quantify not only tumor growth in murine tumor models but also the development of vascularization and necrosis within tumors. This study investigates the use of a new imaging technique, flat-panel detector volume computed tomography (fpVCT), to monitor in vivo tumor progression and structural changes within tumors of two murine carcinoma models. After tumor cell inoculation, single fpVCT scans of the entire mice were performed at different time points. The acquired isotropic, high-resolution volume data sets enable an accurate real-time assessment and precise measurements of tumor volumes. Spreading of contrast agent-containing blood vessels around and within the tumors was clearly visible over time. Furthermore, fpVCT permits the identification of differences in the uptake of contrast media within tumors, thus delineating necrosis, tumor tissues, and blood vessels. Classification of tumor tissues based on the decomposition of the underlying mixture distribution of tissue-related Hounsfield units allowed the quantitative acquisition of necrotic tissues at each time point. Morphologic alterations of the tumor depicted by fpVCT were confirmed by histopathologic examination. Concluding, our data show that fpVCT may be highly suitable for the noninvasive evaluation of tumor responses to anticancer therapies during the course of the disease
Flat-panel detector-based volume computed tomography: a novel 3D imaging technique to monitor osteolytic bone lesions in a mouse tumor metastasis model.
No full textSkeletal metastasis is an important cause of mortality in patients with breast cancer. Hence, animal models, in combination with various imaging techniques, are in high demand for preclinical assessment of novel therapies. We evaluated the applicability of flat-panel volume computed tomography (fpVCT) to noninvasive detection of osteolytic bone metastases that develop in severe immunodeficient mice after intracardial injection of MDA-MB-231 breast cancer cells. A single fpVCT scan at 200-microm isotropic resolution was employed to detect osteolysis within the entire skeleton. Osteolytic lesions identified by fpVCT correlated with Faxitron X-ray analysis and were subsequently confirmed by histopathological examination. Isotropic three-dimensional image data sets obtained by fpVCT were the basis for the precise visualization of the extent of the lesion within the cortical bone and for the measurement of bone loss. Furthermore, fpVCT imaging allows continuous monitoring of growth kinetics for each metastatic site and visualization of lesions in more complex regions of the skeleton, such as the skull. Our findings suggest that fpVCT is a powerful tool that can be used to monitor the occurrence and progression of osteolytic lesions in vivo and can be further developed to monitor responses to antimetastatic therapies over the course of the disease
Flat-panel detector-based volume computed tomography: a novel 3D imaging technique to monitor osteolytic bone lesions in a mouse tumor metastasis model.
No full textSkeletal metastasis is an important cause of mortality in patients with breast cancer. Hence, animal models, in combination with various imaging techniques, are in high demand for preclinical assessment of novel therapies. We evaluated the applicability of flat-panel volume computed tomography (fpVCT) to noninvasive detection of osteolytic bone metastases that develop in severe immunodeficient mice after intracardial injection of MDA-MB-231 breast cancer cells. A single fpVCT scan at 200-microm isotropic resolution was employed to detect osteolysis within the entire skeleton. Osteolytic lesions identified by fpVCT correlated with Faxitron X-ray analysis and were subsequently confirmed by histopathological examination. Isotropic three-dimensional image data sets obtained by fpVCT were the basis for the precise visualization of the extent of the lesion within the cortical bone and for the measurement of bone loss. Furthermore, fpVCT imaging allows continuous monitoring of growth kinetics for each metastatic site and visualization of lesions in more complex regions of the skeleton, such as the skull. Our findings suggest that fpVCT is a powerful tool that can be used to monitor the occurrence and progression of osteolytic lesions in vivo and can be further developed to monitor responses to antimetastatic therapies over the course of the disease
Multislice spiral computed tomography of an orthotopic severe combined immunodeficient mouse model for lung adenocarcinoma
No full textBackground and Aim: In recent years, much research interest has been rightfully directed towards modelling human disease. The increasing demand of laboratory animals has led to a major impetus in small animal imaging. We evaluated the feasibility of using multislice spiral CT (MSCT) technology in a severe combined immunodeficiency (SCID) mouse model for non-small cell lung cancer. Materials and Methods: Mice (n = 14) were implanted orthotopically with the human non-small cell lung adenocarcinoma cell line A 549. Mice were scanned with MSCT and sacrificed 7 days (n = 3), 14 days (n = 3), and 21 days (n = 5) after tumor cell implantation, respectively. Histopathology was performed. Tumor size as revealed by imaging and pathology was correlated. A group of animals (n = 3) received anti-cancer treatment with seco-CBI-Q-galatoside. Those mice were scanned with MSCT 21 days after tumor implantation and sacrificed, and histopathologic work-up was performed. Results: A total of 14 mice with 14 macroscopically visible and 5 microscopically appreciable adenocarcinomas of the lung were examined. All macroscopically visible tumors were readily depicted with MSCT except from 2 lesions of 1 rum in diameter. MSCT accurately displayed the different tumor sizes in treated vs. non-treated animals. Discussion: MSCT proved to be a valuable tool for imaging of a murine lung cancer model. This evidences a potential use of this technology in small animal lung cancer imaging
“Faithfully loyal to Your Imperial Highness...” Letters of bishop Grigoriy (Grabbe) to grand prince Vladimir Kirillovich (1971–1991)
No full textThis article makes public letters of Bishop Grigoriy (Grabbe; prior to 1979, protopresbyter Georgiy). The author of these letters was for several decades secretary of the Synod of Bishops of the Russian Orthodox Church Abroad and Ruler of the affairs of the Synodal offi ce of the Russian Orthodox Church Abroad. In the 1960s — early 1980s, he had a great power that sometimes exceeded the power of the Chairman of the Synod of bishops, Metropolitan Filaret (Voznesensky). The recipient of the letters is Grand Prince Kirill Vladimirovich Romanov. Although in 1939, the Russian Church Abroad actually recognised this person as the spiritual leader of the future free Russia, in the post-war years the attitude to him deteriorated. The correspondence was conducted against the background of protests by representatives of the house of the Romanovs, who did not agree with Vladimir Kirillovich’s claims to the throne. The author of the letters, being a supporter of Vladimir Kirillovich, tried to support him. The correspondence is also interesting because it refl ects important events in the life of the Russian Orthodox Church Abroad in those years. This is the preparation of the canonisation of new martyrs and confessors of Russia, the scandalous marriage that Revd. Grabbe performed on M. Golenevsky, calling him Tsarevich Alexei, and internal confl icts within the Russian Church Abroad. The correspondence refl ected Bishop Grigoriy’s attitude to the changes in the home country in the late 1980s and early 1990s, to Vladimir Kirillovich’s trip to the Soviet Union and his meeting with Patriarch Alexy II
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