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A relatively new therapy in the treatment of hemorrhoids is transanal hemorrhoidal dearterialization (THD). We report a case of brain abscess caused by Streptococcus milleri following THD. Although a brain abscess after drainage of a perianal abscess has been described in the literature, no report exists of a brain abscess following treatment of hemorrhoids. A healthy 51-year-old man with hemorrhoids underwent THD. Two weeks later he presented with a headache, bradyphrenia, flattened behavior and a left hemiplegia. No perianal complaint and/or perianal abscess was present. A contrast CT scan of the cerebrum showed a right temporoparieto-occipital abscess, with edema and compression of the surrounding tissue and lateral ventricles. MRI showed an abscess with leakage in the right lateral ventricle. Treatment with dexamethasone and intravenous antibiotics was started. Because of progression of symptoms, 3 weeks later ventriculoscopy was performed and the abscess was drained. Culture of the punctuate showed S. milleri. Because of developing hydrocephalus 3 days after ventriculoscopy, first an external ventricle drain and later a ventriculoperitoneal drain was placed. Hereafter the hemiplegia and cognitive disorders improved. This case report describes a severe complication following treatment of hemorrhoids with THD which until now, to our knowledge, has never been described in the literature.

GOCE will be the first satellite gravity mission measuring gravity gradients in space using a dedicated instrument called a gradiometer. High resolution gravity field recovery will be possible from these gradients. Such a recovery requires a proper description of the gravity gradient errors, where the a priori error model is for example based on end-to-end instrument simulations. One way to test the error model against real data, i.e. to see if the a priori model really describes the actual error, is to compare along track interpolated gradients with the measured gradients. The difference between the interpolated and measured gravity gradients is caused by, among others, the interpolation error and the measurement errors. The idea is that if the interpolation error is small enough, then the differences should be predicted reasonably well by the error model. This paper discusses a simulation study where the gravity gradient errors are generated with an end-to-end instrument simulator. The measurement error will be compared with the interpolation error and we will assess the latter as a function of the sampling interval.

The Earth Cloud, Aerosol and Radiation Explorer (EarthCARE) is a satellite mission implemented by the European Space Agency (ESA), in cooperation with the Japan Aerospace Exploration Agency (JAXA), to measure global profiles of aerosols, clouds and precipitation properties together with radiative fluxes and derived heating rates. The simultaneous measurements of the vertical structure and horizontal distribution of cloud and aerosol fields, together with outgoing radiation, will be used in particular to evaluate their representation in weather forecasting and climate models and to improve our understanding of cloud and aerosol radiative impact and feedback mechanisms. To achieve the objective, the goal is that a retrieved scene with footprint size of 10 km × 10 km is measured with sufficiently high resolution that the atmospheric vertical profile of short-wave (solar) and long-wave (thermal) flux can be reconstructed with an accuracy of 10 W m−2 at the top of the atmosphere. To optimise the performance of the two active instruments, the platform will fly at a relatively low altitude of 393 km, with an equatorial revisit time of 25 d. The scientific payload consists of four instruments: an atmospheric lidar, a cloud-profiling radar with Doppler capability, a multi-spectral imager and a broadband radiometer. Co-located measurements from these instruments are processed in the ground segment, which produces and distributes a wide range of science data products. As well as the Level 1 (L1) product of each instrument, a large number of multiple-instrument L2 products have been developed, in both Europe and Japan, benefiting from the data synergy. An end-to-end simulator and several test scenes have been developed that simulate EarthCARE observations and provide a development and test environment for L1 and L2 processors. Within this paper the EarthCARE observational requirements are addressed. An overview is given of the space segment with a detailed description of the four science instruments, demonstrating how the observational requirements will be met. Furthermore, the elements of the space segment and ground segment that are relevant for science data users are described and the data products are introduced.

The mechanisms driving metabolic dysfunction in obesity remain incompletely understood. Here, the authors show that the levels of Hippo pathway effector YAP are reduced in muscle from individuals with insulin resistance and obese-diabetic mice, and that YAP promotes skeletal muscle lipid metabolism and limits adiposity in obese mice.

Trypanosoma brucei inosine-5′-monophosphate dehydrogenase (IMPDH) is an enzyme in the guanine nucleotide biosynthesis pathway and of interest as a drug target. Here the authors present the 2.8 Å room temperature structure of TbIMPDH determined by utilizing X-ray free-electron laser radiation and crystals that were grown in insect cells and find that ATP and GMP are bound at the canonical sites of the Bateman domains.

It is the aim of the GOCE mission to determine a model of the Earth’s gravity field with high accuracy and resolution. For this purpose, gravity gradients will be measured in combination with high–low satellite-to-satellite tracking. The gravity gradients are derived from pair-wise differenced accelerations as determined by the six three-axes accelerometers that form the GOCE gradiometer. Since the measured accelerations suffer from errors of a random and systematic nature, the gravity gradients may suffer from random and systematic errors as well. Systematic errors are, for example, a scale factor and a bias. The common accelerations of the paired accelerometers also are contaminated with such errors. The common accelerations are used in the drag-free control of the satellite and are important for the separation of the gravitational and non-gravitational forces in the gravity field determination. The checking of the gravity gradients and the common accelerations against independent data (i.e. external to the GOCE satellite) in order to free the observations as well as is possible from systematic errors is called external calibration. The possibilities and limitations of using terrestrial gravity data and global gravity models for external calibration of the gravity gradients are reviewed. It turns out that the determination of a gravity gradient scale factor and bias using just the accurate knowledge of the central term and the flattening ( J 2) of the Earth’s gravity field is not good enough. When global gravity field models are used for the calibration, higher degrees and orders should be taken into account as well. With today’s existing global models it seems to be possible to remove the greater part of the systematic errors of the GOCE gradients. A gravity gradient bias can accurately be recovered using terrestrial gravity data in a regional approach with least squares collocation. However, since regional data are used it may not be possible to determine calibration parameters valid for the whole (global) gravity gradient data set. Nevertheless, regional terrestrial gravity data could be used to validate the measured and calibrated gravity gradients. In addition, a possible use of GOCE high–low satellite-to-satellite tracking data to calibrate the common accelerations is explored; it is shown that this approach fails. If more accurate gravity field information becomes available then such a calibration may become feasible. [ABSTRACT FROM AUTHOR]

MIPAS, the Michelson Interferometer for Passive Atmospheric Sounding, is a mid-infrared emission spectrometer which is part of the core payload of ENVISAT. It is a limb sounder, i.e. it scans across the horizon detecting atmospheric spectral radiances which are inverted to vertical temperature, trace species and cloud distributions. These data can be used for scientific investigations in various research fields including dynamics and chemistry in the altitude region between upper troposphere and lower thermosphere. The instrument is a well calibrated and characterized Fourier transform spectrometer which is able to detect many trace constituents simultaneously. The different concepts of retrieval methods are described including multi-target and two-dimensional retrievals. Operationally generated data sets consist of temperature, H2O, O3, CH4, N2O, HNO3, and NO2 profiles. Measurement errors are investigated in detail and random and systematic errors are specified. The results are validated by independent instrumentation which has been operated at ground stations or aboard balloon gondolas and aircraft. Intercomparisons of MIPAS measurements with other satellite data have been carried out, too. As a result, it has been proven that the MIPAS data are of good quality. MIPAS can be operated in different measurement modes in order to optimize the scientific output. Due to the wealth of information in the MIPAS spectra, many scientific results have already been published. They include intercomparisons of temperature distributions with ECMWF data, the derivation of the whole NOy family, the study of atmospheric processes during the Antarctic vortex split in September~2002, the determination of properties of Polar Stratospheric Clouds, the downward transport of NOx in the middle atmosphere, the stratosphere-troposphere exchange, the influence of solar variability on the middle atmosphere, and the observation of Non-LTE effects in the mesosphere.

Stratospheric aerosols play an important role in a number of atmospheric issues such as midlatitude ozone depletion, atmospheric dynamics and the Earth radiative budget. Polar stratospheric clouds on the other hand are a crucial factor in the yearly Arctic and Antarctic ozone depletion. It is therefore important to quantify the stratospheric aerosol/PSC abundance. In orbit since March 2002, the GOMOS instrument onboard the European Envisat satellite has provided a vast aerosol extinction data set. In this paper we present aerosol/PSC zonal median values that were constructed from this data set, together with a discussion of the results.

Abstract Background The potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice. Methods Four trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P. yoelii, assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 h to 5½ months after injection. Results A single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4½ months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (

Objective: To examine the effect of tongue cleaning with a tongue scraper (TS) or toothbrush (TB) in patients with periodontitis., Background: The tongue is a possible reservoir for bacterial (re)colonization of the periodontal tissues in patients with periodontitis. To date, it is not known what the influence of tongue cleaning is on the tongue coating of patients with periodontitis., Material and Methods: Eighteen systemically healthy, untreated moderate to severe adult patients with periodontitis with some degree of tongue coating were randomly assigned to the use of a TS or TB for cleaning the tongue. Microbial load of the saliva and tongue dorsum, amount of tongue coating and patient perception about tongue cleaning were studied at baseline and 2 weeks later., Results: Two weeks of tongue cleaning with either a TB or a TS, did not influence the microbiological counts, neither in the saliva, nor in the tongue coating, even though tongue coating was significantly less. The patients themselves experienced no differences in breath odour or taste sensation after 2 weeks of tongue cleaning; however, they felt that their tongue was cleaner at the end of the study compared to baseline. No differences could be detected between the uses of a TS vs a TB., Conclusion: In patients with periodontitis, tongue cleaning does not influence the bacterial load in the saliva or on the tongue dorsum., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction. The aim of this study was to determine whether the use of primary health care differs between students enrolled in Belgian and German government-funded universities. The secondary aim of the study was to determine the factors that might explain such a difference. Methods. Participants were recruited through all Belgian and German government-funded universities. Because not all the universities agreed to participate, recruiting was also done through social media groups of the universities. An anonymous online survey was used for data collection. Results. In total, 2238 completed surveys were evaluated, of which 544 from students in Belgium and 1694 from students in Germany. In Belgium, more students had a family physician (87%) as compared to the students in Germany (73%) (p < 0.001). During the two months prior to the study, 37% of the Belgian students and 35% of the German students attended a family physician (p = 0.37). More German students attended a specialist (40%) as compared to the Belgian students (24%) (p<0.001). The German students also attended the emergency department more frequently (6%) as compared to their Belgian counterparts (3%) (p = 0.004). Conclusion. Belgian university students were more likely to attend a primary care physician than the German students. The health care seemed to be better organized for Belgian students and they were more satisfied with the delivered care.

Aim: Nitric oxide donors, such as isosorbide dinitrate ointment (ISDN), are considered as first-choice agents in the treatment of chronic anal fissure. Injection with botulinum toxin A in the internal anal sphincter is often used as a second-line therapy, although it may give better results and fewer side effects than nitric oxide donors. The aim of this randomized clinical trial was to investigate whether botulinum toxin A (Dysport) is more effective than ISDN in the primary treatment of chronic anal fissure., Method: From April 2005 until October 2009, 60 patients (32 men) with a median age of 42 (25-82) years were randomized to receive either ISDN 10 mg/ml (1%) (n = 33) or injection with 60 units of Dysport (n = 27). The primary end-point was the percentage of complete fissure healing after 8 weeks., Results: After a median of 9 weeks complete fissure healing was noted in 18 of 27 patients in the Dysport group and in 11 of 33 patients in the ISDN group (P = 0.010). Absolute improvement of pain scores after 9 weeks was similar in both groups (P = 0.733). Patients treated with Dysport had fewer side effects than patients treated with ISDN (P = 0.028). Of the patients with a healed fissure, 28% of the Dysport group and 50% of the ISDN group had a recurrence within 1 year (P = 0.286; hazard ratio 2.08; 95% CI = 0.54-7.97)., Conclusion: Dysport is more effective than ISDN ointment and has fewer side effects in the primary treatment of chronic anal fissure. The recurrence rate within 1 year in both treatment groups is high., (Colorectal Disease © 2014 The Association of Coloproctology of Great Britain and Ireland.)

Objectives: Recently, guided implant surgery has been introduced and several studies verified its accuracy. While those studies reported on the accuracy of the entire procedure, this experiment wanted to evaluate the degree of deviation that can occur during the drilling procedure alone, due to the tolerance of the drill in the sleeve insert., Material and Methods: Drilling was executed in a plexi-glass box with a maximal inclination of the drills within the sleeve insert. Different sleeve inserts, sleeve positions, sleeve heights, sleeve insert heights and diameters were evaluated., Results: The two tested sleeve inserts gave a maximum deviation in angulation of 5.2° and a maximum horizontal deviation of 1.3 mm at the implant shoulder and 2.4 mm at the apex for a 13 mm implant. These deviations decreased if the distance of the sleeve above the plexi-glass box became smaller and hand hold sleeve inserts gave less deviation than drill hold sleeve inserts. The deviation increased by longer implant length, larger drill key diameter, shorter sleeves and/or drill key heights., Conclusions: For a minimal deviation during the surgery with a stereolithographic guide, it is very important to use the drill in a centric position, parallel to the cylinder. The use of longer drill keys and sleeves are critical for optimal accuracy., (© 2012 John Wiley & Sons A/S.)

Background: Enamel matrix derivative (EMD) is commonly used in periodontal therapy. The aim of this systematic review is to give an updated answer to the question of whether the additional use of EMD in periodontal therapy is more effective compared with a control or other regenerative procedures., Methods: A literature search in MEDLINE (PubMed) for the use of EMD in periodontal treatment was performed up to May 2010. The use of EMD in treatment of intrabony defects, furcations, and recessions was evaluated. Only randomized controlled trials with ≥1 year of follow-up were included. The primary outcome variable for intrabony defects was the change in clinical attachment level (CAL), for furcations the change in horizontal furcation depth, and for recession complete root coverage., Results: After screening, 27 studies (20 for intrabony defects, one for furcation, and six for recession) were eligible for the review. A meta-analysis was performed for intrabony defects and recession. The treatment of intrabony defects with EMD showed a significant additional gain in CAL of 1.30 mm compared with open-flap debridement, EDTA, or placebo, but no significant difference compared with resorbable membranes was shown. The use of EMD in combination with a coronally advanced flap compared with a coronally advanced flap alone showed significantly more complete root coverage (odds ratio of 3.5), but compared with a connective tissue graft, the result was not significantly different. The use of EMD in furcations (2.6 ± 1.8 mm) gave significantly more improvement in horizontal defect depth compared with resorbable membranes (1.9 ± 1.4 mm) as shown in one study., Conclusions: In the treatment of intrabony defects, the use of EMD is superior to control treatments but as effective as resorbable membranes. The additional use of EMD with a coronally advanced flap for recession coverage will give superior results compared with a control but is as effective as a connective tissue graft. The use of EMD in furcations will give more reduction in horizontal furcation defect depth compared with resorbable membranes.

Introduction: Chemical sphincterotomy, the use of pharmacological agents to reduce anal sphincter resting pressure, has become more and more popular in the treatment of chronic anal fissures (CAFs). It offers the possibility to avoid a lateral internal sphincterotomy and its associated risk of incontinence. In our hospital, patient with a chronic anal fissure are consecutively treated with isosorbide dinitrate 1% ointment, applied 6 times a day for 8 weeks, followed by diltiazem 2% ointment, applied 2 times a day for 8 weeks and Botulin Toxin A injections (Dysport; Ipsen, Hoofddorp, the Netherlands) in the internal anal sphincter. In a previous study (1), we describe high healing rates with this regime. Objective The objective of this study is to evaluate the effect of the combination of fissurectomy and Botulin Toxin A in the treatment of CAFs., Methods: Twenty-one patients (10 male patients, median age 48 years) with persistent symptoms of chronic anal fissures after following the above mentioned treatment, were enrolled in this study. Fissurectomy was combined with Botulinum Toxin A (80 U of Dysport) under regional anaesthesia in day care. Results After 12 weeks 19/21 CAFs (90%) had healed. Median follow-up was 16 (9-30) months. No recurrences were seen., Conclusion: Fissurectomy in combination with Botulinum Toxin A injection in the internal anal sphincter is an effective treatment for medically resistant CAFs.

Advanced biomarkers for improved prediction and monitoring of disease and toxicology mechanisms are needed to control the high clinical failure rates among new compounds. Along with other uses, biomarkers are currently used in the industry to screen for toxic side effects of drug candidates and to identify appropriate patient populations. The use of combinatorial biomarkers is a first step towards an effective systems biology approach to drug development. In combination with high content screening, and in particular a novel, high content in situ proteomic technology, combinatorial biomarkers will strongly support the knowledge-based decision-making process by providing crucial information on functional biology.

We describe a cell-free chromatin assembly system derived from the yeast Saccharomyces cerevisiae, which efficiently packages DNA into minichromosomes in a reaction dependent on exogenous core histones and an ATP-regenerating system. Both supercoiled and relaxed plasmid DNA serve as templates for nucleosomal loading in a gradual process that takes at least 6 h for completion at 30 degrees C. Micrococcal nuclease digestion of the assembled minichromosomes displays an extended nucleosomal ladder with a repeat length of 165 bp. The purified minichromosomes contain the four core histones in stoichiometric proportion and exhibit phased nucleosomes over the mouse mammary tumour virus (MMTV) promoter. The progesterone receptor and NF1 synergize on these minichromosomes resulting in efficient cell-free transcription. The ease of manipulation and the potential use of yeast strains carrying mutations in the chromatin handling machinery make this system suitable for detailed mechanistic studies.

Minichromosomes assembled on the mouse mammary tumor virus (MMTV) promoter in vitro exhibit positioned nucleosomes, one of which covers the binding sites for progesterone receptor (PR) and nuclear factor 1 (NF1). Incorporation of histone H1 into MMTV minichromosomes improves the stability of this nucleosome and decreases basal transcription from the MMTV promoter, as well as its response to either PR or NF1. However, histone H1-containing minichromosomes display better PR binding and support a more efficient synergism between PR and NF1, leading to enhanced transcription initiation. A mutant MMTV promoter lacking positioned nucleosomes does not display enhanced transcriptional synergism in the presence of H1. Binding of PR leads to phosphorylation of H1, which leaves the promoter upon transcription initiation. Thus, H1 assumes a complex and dynamic role in the regulation of the MMTV promoter.

Transcription from the mouse mammary tumor virus (MMTV) promoter can be induced by glucocorticoids or progestins. The corresponding receptors bind to a cluster of hormone responsive elements (HREs) and activate the promoter by synergistic interactions with ubiquitous transcription factors, in particular nuclear factor 1 (NF1). Synergism between hormone receptors and NF1 depends on the precise positioning of the promoter sequences on the surface of a histone octamer in chromatin, but how linker histones participate in the process is unclear. Asymmetric binding of histone H1 to chromatin organized MMTV promoter sequences compacts the nucleosomal structure and leads to repression of basal transcription and to reduced binding of NF1. In contrast, H1 containing MMTV chromatin binds PR with higher affinity and is transcribed more efficiently in the presence of PR and NF1 than chromatin free of linker histone. Thus histone H1 represses hormone independent transcription and enhances the synergism between PR and NF1 resulting in tighter hormonal regulation. This positive effect of H1 is likely due to a better defined nucleosome positioning over the MMTV promoter. Upon binding of PR to the promoter chromatin a hitherto unidentified kinase is recruited or activated that phosphorylates H1. This is not sufficient for transcriptional activation but is likely a requisite for the action of ATP-dependent chromatin remodelling complexes. Following remodelling and in the presence of NF1, which maintains the open nucleosome conformation, additional PR molecules bind, transactivation takes place and H1 is displaced from the promoter during transcription initiation. Therefore, H1 plays a key role during the initial hormonal activation of the MMTV promoter in native chromatin, which includes recruitment by PR of a histone H1 kinase and an ATP-dependent chromatin remodelling complex, followed by NF1 binding, increased PR binding, transcription initiation and H1 displacement.

The light state transition regulates the distribution of absorbed excitation energy between the two photosystems (PSs) of photosynthesis under varying environmental conditions and/or metabolic demands. In cyanobacteria, there is evidence for the redistribution of energy absorbed by both chlorophyll (Chl) and by phycobilin pigments, and proposed mechanisms differ in the relative involvement of the two pigment types. We assayed changes in the distribution of excitation energy with 77K fluorescence emission spectroscopy determined for excitation of Chl and phycobilin pigments, in both wild-type and state transition-impaired mutant strains of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. Action spectra for the redistribution of both Chl and phycobilin pigments were very similar in both wild-type cyanobacteria. Both state transition-impaired mutants showed no redistribution of phycobilin-absorbed excitation energy, but retained changes in Chl-absorbed excitation. Action spectra for the Chl-absorbed changes in excitation in the two mutants were similar to each other and to those observed in the two wild types. Our data show that the redistribution of excitation energy absorbed by Chl is independent of the redistribution of excitation energy absorbed by phycobilin pigments and that both changes are triggered by the same environmental light conditions. We present a model for the state transition in cyanobacteria based on the x-ray structures of PSII, PSI, and allophycocyanin consistent with these results.

The mechanism underlying the synergism between transcription factors in eukaryotic gene expression is not fully understood. In minichromosomes assembled in vitro the synergism between steroid hormone receptors (SHRs) and nuclear factor 1 (NF1) on the mouse mammary tumor virus (MMTV) promoter does not require the proline-rich transactivation domain (PRD) of NF1. Here we show that similar results are obtained in yeast. In contrast, replacing the native hormone-responsive elements (HREs) by a single HRE results in a more accessible chromatin and makes the synergism with SHR dependent on the PRD of NF1. Following hormone induction, in addition to glucocorticoid receptor, the DNA binding domain of NF1 is needed and sufficient for establishing an open chromatin conformation on the wild type MMTV promoter. Thus, NF1 acts as a classical transcription factor in a relaxed chromatin context, whereas in the context of the wild type chromatin DNA binding of NF1 is sufficient to cooperate with SHRs by stabilizing an open chromatin conformation.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Previous analyses showed that, unlike Sp1, Sp3 fails to activate transcription in certain promoter settings. This is due to the presence of an inhibitory domain located between the second glutamine-rich activation domain and the DNA-binding domain. To further analyze the transcriptional properties of Sp3, we have expressed and purified recombinant Sp3 and Sp1 as epitope-tagged proteins from stable transfected insect cells. We found that Sp3 does act as a strong activator similar to Sp1 in an in vitro transcription assay using Sp1/Sp3-depleted HeLa nuclear extract. However, on the same promoter Sp3 is almost inactive when transfected into cells. Mutational studies demonstrate that a single lysine residue is responsible for the low transcriptional activity of Sp3 in vivo. We show that Sp3, but not a mutant of Sp3 that lacks this lysine residue, is highly acetylated in vivo. Our results strongly suggest that the transcriptional activity of Sp3 is regulated by acetylation. The consequences of acetylation for the activity of Sp3 are discussed.

Negatively supercoiled plasmids can be assembled into dynamic minichromosomes using Drosophila embryo extract as a source of histones and chromatin assembly factors. However, analysis of such mini-chromosomes is often difficult due to the presence in the crude extract of a large excess of macromolecules and low molecular weight molecules including ATP. Several techniques have been used to partially purify the minichromosomes based on either sizing columns or centrifugation on sucrose gradients. We have developed a single-step method employing a 30 min ultracentrifugation through a glycerol cushion. In contrast to chromatin purified in sucrose-containing buffers, the minichromosomes obtained with this method are suitable for transcriptional analysis. This method is fast, quantitative, flexible, can deal with several samples simultaneously and leads to concentration of the chromatin. As centrifugation through glycerol yields chromatin free of ATP and several characterized chromatin remodeling complexes, this method should be useful for structural and functional studies in vitro.

In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.

The transcription factor IID (TFIID) complex is highly conserved between the Drosophila and mammalian systems. A mammalian homolog has been described for all the Drosophila TATA box-binding protein-associated factors (TAFs), with the exception of dTAF(II)150. We previously reported the identification of CIF, an essential cofactor for TFIID-dependent transcription from promoters containing initiator (Inr) elements. Here we describe the molecular cloning of CIF150, the human homolog of dTAF(II)150, and present biochemical evidence that this factor is involved in Inr activity. CIF150 is capable of mediating TFIID-dependent Inr activity in a complementation assay, and a protein fraction lacking Inr activity lacks detectable amounts of CIF150. Despite the striking similarity to dTAF(II)150, CIF150 does not appear to be associated with human TFIID. However, in vitro binding assays revealed a specific and direct interaction between CIF150 and hTAF(II)135. This interaction might be structurally important for the functional interaction between CIF150 and human TFIID, since CIF150 stabilizes TFIID binding to a core promoter.

Adenovirus large E1A, Epstein-Barr virus Zebra, and herpes simplex virus VP16 were studied as models of animal cell transcriptional activators. Large E1A can activate transcription from a TATA box, a result that leads us to suggest that it interacts with a general transcription factor. Initial studies showed that large E1A binds directly to the TBP subunit of TFIID. However, analysis of multiple E1A and TBP mutants failed to support the significance of this in vitro interaction for the mechanism of activation. Recent studies to be reported elsewhere indicate that conserved region 3 of large E1A, which is required for its activation function, binds to one subunit of a multisubunit protein that stimulates in vitro transcription in response to large E1A and other activators. A method was developed for the rapid purification of TFIID approximately 25,000-fold to near homogeneity from a cell line engineered to express an epitope-tagged form of TBP. Purified TFIID contains 11 major TAFs ranging in mass from approximately 250 to 20 kD. Zta and VP16, but not large E1A, greatly stimulate the rate and extent of assembly of a TFIID-TFIIA complex on promoter DNA (DA complex). For VP16, this is a function of the carboxy-terminal activation subdomain. An excellent correlation was found between the ability of VP16C mutants to stimulate DA complex assembly and their ability to activate transcription in vivo. Consequently, for a subset of activation domains, DA complex assembly activity is an important component of the overall mechanism of activation.

The 2-phenylindole system has proved to be a versatile structure for the design of potent antiestrogens, especially when functional groups have been introduced into the alkyl side chain in position 1. In analogy to steroidal structures such as ICI 164,384 a number of 2-phenylindoles with carbamoylalkyl and aminoalkyl side chains were synthesized. They bind to the calf uterine estrogen receptor with relative binding affinities between 2.1 and 21 (estradiol = 100). The antiestrogenic effect of these compounds was demonstrated by the inhibition of transcriptional activity which was measured in a new luciferase assay with the EREwtc luc as reporter plasmid. The derivative with a methyl-n-propyldodecanamide side chain (4h) antagonized the effect of estradiol (10(-9) M) completely at concentrations of 10(-7) M and higher. As a sensitive model for quantification of estrogenic and antiestrogenic effects in vitro we used HeLa-cells cotransfected both with the reporter plasmid and estrogen receptor expression vectors HEG0 and HE0. In cells transfected with these vectors transcriptional activity was strongly dependent on side chain structure. With mutated receptors we were able to show that this activity was mainly due to TAF-1 whereas TAF-2 remained silent. When we studied the effect of some of the new compounds in vivo using the mouse uterine weight assay, we observed a correlation between transcriptional activity in transfected HeLa cells and estrogenic effects in mice. Two of the 1-carbamoylalkyl-2-phenylindoles (4f, 4h) proved to be "pure" antiestrogens both in vitro and in vivo. In estrogen-sensitive MCF-7 breast cancer cells, they strongly inhibit cellular growth. Some of the IC50-values were close to 10(-8) M.

A rapid assay system for measuring the potential of estrogenic drugs is introduced. Luciferase induction could be measured in estrogen-receptor-positive human MCF-7 breast cancer cells, which had been transfected with a novel luciferase reporter plasmid ERE luc. The minimal requirement was 1 h exposure to the inducing drug and 3.5 h of incubation after removal of the drug. The assay system was used to measure the stability of the drug diaqua-[1,2-bis (2,6-dichloro-4-hydroxyphenyl) ethylenediamine] platinum(II) sulfate, containing an estrogenic ligand and reactive platinum. Luciferase activity was observed only when the drug was in the culture medium and cells for short times, whereas the estrogenic ligand alone remained active. It is assumed that binding of the platinum moiety to macromolecular constituents of the culture or cells renders the drug inaccessible for binding to the estrogen receptor.