Structural Modeling and DNA Binding Autoinhibition Analysis of Ergp55, a Critical Transcription Factor in Prostate Cancer

BackgroundThe Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins are highly conserved in their DNA binding domain and involved in various development processes and regulation of cancer metabolism. To study the structure and DNA binding autoinhibition mechanism of Ergp55 protein, we have produced full length and smaller polypeptides of Ergp55 protein in E. coli and characterized using various biophysical techniques.ResultsThe Ergp55 polypeptides contain large amount of ?-helix and random coil structures as measured by circular dichorism spectroscopy. The full length Ergp55 forms a flexible and elongated molecule as revealed by molecular modeling, dynamics simulation and structural prediction algorithms. The binding analyses of Ergp55 polypeptides with target DNA sequences of E74 and cfos promoters indicate that longer fragments of Ergp55 (beyond the Ets domain) showed the evidence of auto-inhibition. This study also revealed the parts of Ergp55 protein that mediate auto-inhibition.SignificanceThe current study will aid in designing the compounds that stabilize the inhibited form of Ergp55 and inhibit its binding to promoter DNA. It will contribute in the development of drugs targeting Ergp55 for the prostate cancer treatment.

Shanti P. Gangwar¹, Sharmistha Dey², Ajay K. Saxena^1*

1 Structural Biology Lab, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, 2 Department of Biophysics, All India Institutes of Medical Sciences, New Delhi, India

Background

The Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins are highly conserved in their DNA binding domain and involved in various development processes and regulation of cancer metabolism. To study the structure and DNA binding autoinhibition mechanism of Ergp55 protein, we have produced full length and smaller polypeptides of Ergp55 protein in E. coli and characterized using various biophysical techniques.

Results

The Ergp55 polypeptides contain large amount of ?-helix and random coil structures as measured by circular dichorism spectroscopy. The full length Ergp55 forms a flexible and elongated molecule as revealed by molecular modeling, dynamics simulation and structural prediction algorithms. The binding analyses of Ergp55 polypeptides with target DNA sequences of E74 and cfos promoters indicate that longer fragments of Ergp55 (beyond the Ets domain) showed the evidence of auto-inhibition. This study also revealed the parts of Ergp55 protein that mediate auto-inhibition.

Significance

The current study will aid in designing the compounds that stabilize the inhibited form of Ergp55 and inhibit its binding to promoter DNA. It will contribute in the development of drugs targeting Ergp55 for the prostate cancer treatment.

Citation: Gangwar SP, Dey S, Saxena AK (2012) Structural Modeling and DNA Binding Autoinhibition Analysis of Ergp55, a Critical Transcription Factor in Prostate Cancer. PLoS ONE 7(6): e39850. doi:10.1371/journal.pone.0039850

Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America

Received: May 5, 2012; Accepted: May 31, 2012; Published: June 28, 2012

Copyright: ? 2012 Gangwar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The work was supported by grant from the Department of Science and Technology (DST) New Delhi, India (No-SR/SO/HS-05/2009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: ajaysaxena@mail.jnu.ac.in

Introduction?Top

The Ets family proteins share highly conserved winged helix-turn-helix DNA-binding domain and bind to consensus DNA core sequence 5?-GGA (A/T)-3? [1]. The Erg proteins belong to Ets family of transcription factor. Erg gene is rearranged in human myeloid leukemia [2] and in 5?10% of patients with Ewing's sarcoma [3]. In both cases, chromosomal translocations results in the expression of oncogenic fusion proteins composed of Erg and member of Tet subfamily of RNA binding proteins. Erg protein is essential for definitive hematopoiesis, adult hematopoietic stem cell function and maintenance of normal peripheral blood platelet numbers [4]. The TMPRSS2-Erg fusion oncogene transcripts observed in prostate cancer cells are significantly associated with aggressive cancer, metastatic spread and increased probability of death [5].

The Erg gene encodes five proteins, Erg-1, Erg-2, Ergp55, Ergp49 and Ergp38 as a result of different splicing, polyadenylation or initiation codon. The Ergp55 isoform contains four functional domains, which are involved in DNA binding, transcriptional activation and negative regulation of transactivation [6]. The Ergp55 protein forms dimer with itself and with two other isoforms Ergp49 and Ergp38, via PNT and Ets domain [7]. The central domain of Ergp55 behaves as inhibitory domain on dimerization and its removal enhances the transactivation property (7). The critical residues of Ets domain of Ergp55, which mediate Ergp55-jun/fos-DNA ternary complex formation, have been identified and characterized [8].

So far, tertiary structure of any full-length Ets protein is not determined. However, structures of DNA-binding domains of several Ets proteins have been determined using X-ray crystallography and nuclear magnetic resonance techniques [9]?[16]. The gonome-wide analysis of Ets-family DNA-binding in vitro and in vivo has been studied recently [17].

The precise mechanism by which, Ergp55 protein acts on transcription is not understood. To understand the structure and DNA binding autoinhibition mechanism of Ergp55, we performed circular dichorism, molecular modeling and theoretical structural prediction analysis on Ergp55 polypeptides. To understand the DNA binding autoinhibition mechanism, the binding studies of Ergp55 polypeptides with DNA sequences of E74 and cfos promoters were carried out. Our results indicated that (i) Ergp55 polypeptides contains high percentage of ?-helix and random coil structures (ii) full length Ergp55 is a flexible and elongated molecule (iii) longer fragments beyond the canonical Ets domain of Ergp55 showed the evidence of autoinhibition.

Materials and Methods?Top

Construction of Ergp55 plasmids

The full length Erg_1?479 and Erg_112?399 genes were cloned in pET28a (+) vector. The Erg_1?399 and Erg_307?399 genes were cloned in pRSETA and pET21a (+) expression vector. All deletion mutant genes were amplified from full-length Erg_1?479 plasmid using polymerase chain reaction.

Figure 1. The human Ergp55 protein sequence.

(A) Shown here the amino acid sequences full length Ergp55 without 6xHis tag and cleavage site. The residues were highlighted according to the size of Ergp55 domains, NTD (yellow), PNT (green), CAE/CD (blue/cyan), Ets (red) and CTD (black). (B) Shown here the Ergp55 constructs used in the experiments (coloring scheme same as in Fig. 1A). The labeled residues defining the sequence of beginning and end of constructs. Aestericks denote the position of 6xHis tag on various Ergp55 constructs.

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Purification of Ergp55 polypeptides

The Erg_1?479, Erg_1?399, Erg_112?399 and Erg_307?399 plasmids were transformed into E. coli. BL21 (DE3) cells. The cells were grown in 3 liters luria bertani media containing appropriate antibiotics at 37?C, until OD₆₀₀ reached to 0.6. The 0.125 mM IPTG was induced at 37?C and culture further shaked for 4 h at 220 rpm. The cells were harvested by centrifugation at 6000x g for 10?min at 4?C. The cells were suspended in 50 ml of lysis buffer containing (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM benzamidine-HCl, 0.1% triton X-100, 5% glycerol, 3 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and 0.5 mg/ml lysozyme) and disrupted by sonication at 4?C. The crude lysate was centrifuged at 25000? g for 20 min at 4?C. The supernatant was loaded on Ni-NTA column, pre-equilibrated with binding buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM benzamidine-HCl, 5% glycerol, 2 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and 10 mM imidazole). The proteins were eluted from column with elution buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM benzamidine-HCl, 5% glycerol, 3 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and 250 mM imidazole). The eluted fractions were pooled, concentrated and loaded on Sephacryl S-200 HR gel-filtration column pre-equilibrated in buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 3 mM 2-mercaptoethanol and 5% glycerol). The purified proteins were concentrated to 10 mg/ml using amicon ultra centrifugal filter device (Mw cutoff~ 10 kD). The protein concentration was measured by UV radiation at 280 nm by using extinction coefficient calculated with EXPASY software (http://us.expasy.org/tools/protparam.htm?l).

Figure 2. Size exclusion chromatography of Ergp55 polypeptides.

The chromatogram of (A) full length Erg_1?479 (B) Erg_1?399 (C) Erg_112?399 and (D) Erg_307?399 polypeptides are shown. The SDS-PAGE analysis and calculated molecular weight of each protein are denoted on each chromatogram.

doi:10.1371/journal.pone.0039850.g002

N-terminal protein sequencing and ion spray mass spectrometry confirmed identity and purity of Ergp55 polypeptides. Protein concentration was determined using absorbance at 280 nm. Coomassie brilliant blue stained SDS-PAGE analysis indicated that all Ergp55 polypeptides were purified greater than 95% purity. All proteins were stored at ?20?C.

Surface plasmon resonance experiment

Biosensor studies were performed using BIAcore 2000 (Biacore Pharmacia Biosensor AB, Uppsala Sweeden) equipment [18]. All experiments were performed at 25?C in HBS-buffer containing [10 mM HEPES buffer pH 7.4, 150 mM NaCl, 50 mM EDTA, 0.005% P20 as surfactant). Since all Ergp55 polypeptides contain 6xHis tag either on N- or C-terminal, the sensor chip containing high density of immobilized Ni-NTA is an ideal tag to immobilize these proteins. Initially, the flow cells of the chip were activated by passing nickel chloride solution over it. 40 ul of each Ergp55 polypeptide e.g., Erg_1?479 (0.14 ug/ul), Erg_1?399 (0.19 ug/ul), Erg_112?399 (0.92 ug/ul) and Erg_307?399 (0.17 ug/ul) were injected in different flow cells of Ni-NTA chip at flow rate of 10 ul/min.

Figure 3. Binding analysis of DNA sequence of E74 promoter to immobilized Ergp55 polypeptides.

In figures (A) Erg_1?479 (B) Erg_1?399 (C) Erg_112?399 and (D) Erg_307?399 polypeptides were immobilized on Ni-NTA chip. Three concentration (1, 2, 3 ?M) of DNA sequence of E74 promoter injected on each immobilized Ergp55 polypeptide.

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In different flow cells of Ni-NTA chip, following RU unit of each Ergp55 polypeptide was immobilized e.g., Erg_1?479 (2904 RU), Erg_1?399 (2848 RU), Erg_112?399 (2825 RU) and Erg_307?399 (247 RU), where 1 RU corresponds to immobilized protein of concentration ~1 pg/mm. Binding experiments were performed with three different concentration [1, 2, 3 ?M) of DNA sequence of E74 promoter (5? TACCGGAAGT 3?) in HBS-buffer and injected over immobilized Ergp55 polypeptides at flow rate of 10 ?l/min. Similar experiment was performed using DNA sequence of cfos promoter sequence (5? GACAGGATGTG 3?). The sensogram allowed to run for another 4 min. The regeneration of biosensor surface was done using 30 s pulse of 1 mM NaOH at flow rate of 10 ?l/min. Associate and dissociation kinetic constants were calculated by BIAeveluation 3.0 software using simple 1:1 Langmuir model with the assumption, that density of Ergp55 polypeptides on the sensor chip were not high enough to support bivalent DNA binding.

Figure 4. Binding analysis of DNA sequence of cfos promoter to immobilized Ergp55 polypeptides.

In figures (A) Erg_1?479 (B) Erg_1?399 (C) Erg_112?399 and (D) Erg_307?399 polypeptides are immobilized on Ni-NTA chip. Three different concentration (1, 2, 3 ?M) of DNA sequence of cfos promoter injected on each immobilized Ergp55 polypeptide.

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Circular dichorism

CD measurements were recorded using Chirascan^TM CD spectropolarimeter (Applied Photophysics) with a water bath to maintain the constant temperature. The Ergp55 polypeptides were diluted to 0.2 mg/ml in 10 mM sodium phosphate buffer, pH 8.0 and loaded on 0.1 cm quartz cuvette. The blank of all experiments was 10 mM sodium phosphate buffer, pH 8.0. The final spectrum was an average of three sequential scan.

Figure 5. CD spectra of Ergp55 polypeptides.

(A) CD spectra of Erg_1?479, Erg_1?399, Erg_112?399 and Erg_307?399 polypeptides recorded from 200 nm to 260 nm. (B) Temperature induced unfolding of full length Ergp55 protein. The plot (inner) containing mean residue ellipticity versus temperature indicates the denaturation of ?-helices of folded full length Ergp55.

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For thermal denaturation study of full length Ergp55, the CD spectra were recorded at 10?C increment starting from 10?C to 90?C. Before measurement, the sample cuvette was equilibrated at each temperature. Temperature readings were taken within cuvette holder agreed with temperature of water bath. All CD data were converted to mean residue ellipticity (deg. cm²/dmol). The Dichroweb server [19] was used to estimate the amount of secondary structure in Ergp55 polypeptides from CD spectra.

Secondary structure prediction

The SOPMA [20], GOR [21] and PSIPRED [22] algorithms were used to predict the secondary structure contents in full length Ergp55, which showed ~21% ?-helix, 12?15% ?-sheet and 65?69% random coil structures.

Figure 6. Model of Ergp55 polypeptides.

(A) PSIPRED program analysis of full length Ergp55. (B) Structural model of full length Ergp55 after molecular modeling and dynamics simulation analysis using GROMACS program (C) The disordered prediction made by DISOPRED for (a), NTD (b), PNT (c), CAE/CD (d), ETS and (e), CTD regions of Ergp55.

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Modeling of Ergp55 polypeptides

Phyre server [23] was used to obtain the structure of full length Ergp55 protein (1?479 residues). The server yielded the structure of PNT domain (95?221 residues) of Ergp55 using template PDB-2YTU (solution structure of SAM-PNT domain of human friend leukemia integration factor-1 transcription factor, not yet published). The NMR solution structure of PNT domain (108?201 residues) was obtained from protein data bank (PDB-1SVO) [24]. The Phyre server also yielded the structure of Ets domain (284?412 residues) of Ergp55 using the template PDB-2NNY (Regulation of transcription factor Ets-1 by DNA mediated homodimerization) [13]. The NMR structure of Ets domain of Fli1 (306?403 residues) was obtained from protein data bank (PDB-1FliA) [25]. The structural modeling of N- terminal (1?94 residues), central domain (222?283 residues) and C- terminal (413?479 residues) of Ergp55 were not attempted due to lack of input model.

The Modeler [26] and LOMETS threading [27] programs were used to build the structure of full length Ergp55 using following inputs (i) structural model of PNT domain (95?221 residues) of Ergp55 (ii) structural model of Ets domain (284?412 residues) of Ergp55 (iii) NMR structure of PNT domain (108?201 residues) of Ergp55 and (iv) NMR structure of Ets domain (306?403 residues) of Fli1. Energy minimization was performed on modeled Ergp55 using Gromacs program version 4.0.5 [28]. 100 steps of steepest decent and 500 steps of conjugated gradient algorithms were used in energy minimization calculation.

Molecular dynamics simulations

The 10 ns molecular dynamics (MD) simulation was performed on minimized Ergp55 model using GROMACS program (version 4.0.5) with Gromacs43a2 force field [28]. The Ergp55 model was immersed in a cubic box extending 0.5 nm from the protein surface and solvated with explicit SPC water molecules. Chloride and sodium ions were added to neutralize the systems, which were then simulated with periodic boundary conditions. The solvated Ergp55 model consists of 4832 protein atoms surrounded by ~390, 000 water molecules. Before running the simulation, whole system was energy minimized for 200 iterations of steepest descents and then equilibrated for 20 ps keeping protein atoms restrained. All restraints were removed from the protein and temperature was gradually increased in 10 distinct steps of 5 ps simulations each.

Berendsen coupling was employed to maintain a constant temperature of 300 K with a coupling constant ? of 0.1 ps. Van der Waals interactions were modeled using 6?12 Lennard-Jones potentials with 1 nm cutoff. The coulomb cut off was 1.0. The time step employed was 2 fs and coordinates were saved every 5 ps for analysis of MD trajectories.

The stereochemistry of simulated Ergp55 model was checked by PROCHECK program of CCP4 suite [29]. Secondary structure composition was measured by DSSP program [30] and structure visualization by PyMOL program [31].

Results?Top

Purification of recombinant Ergp55 polypeptides

We produced full length and smaller polypeptides (containing subset of predicted domains) of Ergp55 in E. coli and purified using standard chromatographic techniques (Fig. 1A?B). During size exclusion chromatography, the purified Erg_1?479, Erg_1?399, Erg_112?399 and Erg_307?399 polypeptides eluted at volumes corresponding to their molecular weights (Fig. 2A?D). The full length Erg_1?479 eluted at the size of 53.8 kD, Erg_1?399 polypeptide eluted at 51.1 kD, Erg_112?399 eluted at 44.9 kD and Erg_112?399 eluted at 15.1 kD. The Erg_1?479 and Erg_1?399 polypeptides have tendency to degrade if kept for 7 days at 4?C. The Erg_112?399 and Erg_307?399 polypeptides do not degrade over time and more stable than Erg_1?479 and Erg_1?399 polypeptides.

DNA binding studies using E74 and cfos promoter sequences

The functionality of purified Ergp55 polypeptides was assessed by DNA binding experiment using surface plasmon resonance technique. The observed K_D value of Ergp55 polypeptides with DNA sequence of E74 promoter were e.g., Erg_1?479 ~ 704 nM, Erg_1?399 ~ 217 nM, Erg_112?399 ~ 115 nM and Erg_307?399 ~ 65 nM (Fig. 3A?D). These results indicated that Ets domain of Ergp55 (Erg_307?399) has the highest affinity for E74 promoter DNA sequence. The DNA binding affinity decreases ~ 2 fold for Erg_112?399 polypeptide, ~3 fold for Erg_1?399 polypeptide and ~10 fold for full-length Erg_1?479, when compared with Erg_307?399 polypeptide (Ets domain). These results indicate that N- as well as C- terminal domains with respect to Ets domain are involved in auto-inhibition of DNA binding to Ergp55 protein.

The observed K_D value of Ergp55 polypeptides with cfos promoter sequence were e.g., Erg_1?479 ~ 232 ?M, Erg_1?399 ~ 196 ?M, Erg_112?399 ~ 38 ?M and Erg_307?399 ~ 0.45 ?M (Fig. 4A?D). These results also indicate that Ets domain has the highest affinity for DNA sequences of cfos promoter. Both N- and C- terminal domains with respect to Ets domain are involved in inhibition of cfos DNA binding to Ergp55 protein.

CD measurements of Ergp55 polypeptides

To identify the secondary structure contents in Ergp55 polypeptides, the far-UV CD spectroscopy was used (Fig. 5A). The CD data were de-convoluted using DICHROWEB web server [19] and percentage of ?-helix, ?-sheet and random coil structures were estimated. The CD spectra of full-length Erg_1?479 (Fig. 5A) showed two minima around 208 nm and 222 nm, a characteristic of ?-helical structure. Deconvolution of data predicts ~35% ?-helix, 15% ?-sheet and 49% random coil structures in full length Ergp55. The CD data of Erg_1?399 polypeptide predicts ~25% ?-helix, 17% ?-sheet and 57% random coil structures, which shows less ?-helix and ?-sheet structure compare to full length Erg_1?479 structure.

In case of Erg_112?399 polypeptide, the CD data estimates ~29% ?-helix, 15% ?-sheet and 55% random coil structures. This polypeptide contains less ?-helix, similar ?-sheet structure compared to full length Erg_1?479. For Erg_307?399 polypeptide, the CD data estimates ~31% ?-helix, 10% ?-sheet and 59% random coil structures, which has less ?-helix and ?-sheet structure compare to full length Erg_1?479 structure. However, these values are close to secondary structure contents in crystal structure of Ets domain of Fli-1 (35.7% ?-helix, 4.1% ?-sheet, 60.2% random coil). The Ets domain of Fli-1 is the closest homologous to Ets domain of Ergp55 [32].

Thermostability of full length Ergp55

To assess the thermostability of full length Ergp55, a far UV-CD spectrum of protein was measured from 10 to 90?C (Fig. 5B). It is clear from spectra that secondary structure of Ergp55 denatures as temperature increased. When mean residue ellipticity at 222 nm is plotted against temperature, the inflection point of sigmoidal curve indicates the T_m of 45?2?C of full length Ergp55.

Molecular modeling and dynamic simulation of full length Ergp55

The secondary structure prediction on full length Ergp55 using PSIPRED program is shown in (Fig. 6A). The Modeler and automatic threading LOMETS programs were used to construct full length Ergp55 model using (i) structure of 95?221 residues of Ergp55 using the template PDB-2YTU (not published) having 57% sequence identity (ii) the structure of 284?412 residues of Ergp55 using template PDB-2NNY [13] having 40% sequence identity and (iii) NMR structure of 108?201 residues of Ergp55 and (iv) NMR structure of 306?403 residues of Ets domain of Fli-1 having 90% sequence identity. Energy minimization and dynamics simulations analysis were performed on constructed Ergp55 model, which yielded a flexible and elongated structure (Fig. 6B). The Ergp55 structure remained very stable during whole simulation time, as confirmed by all the indicators commonly used to analyze MD simulation.

The 93% residues of Egp55 model lie in most favored region of Ramachandran plot and a Prosa Z-score of ?4.87. DISOPRED [33] analysis on Ergp55 model indicated that N-terminal (1?118 residues) and C-terminal (397?479 residues) are largely disordered (except N-terminal 4?23 residues) and remaining Ergp55 structure (119?396 residues) were ordered (Fig. 6C). The structured PNT domain contains tertiary arrangement of four ?-helices, characteristic of large group of SAM domain [34]. The Ets domain consists of four ?-helices and four?-sheets, a characteristic of Ets family proteins. In N-terminal domain, 15 residue stretch predicted to form ?-helix and 3 residue long helix (219?221 residues) are observed in in CAE/CD domain of Ergp55. The stretches of residues in C-terminal domain of Ergp55 predicted to have only random coil structure.

The N- terminal and C-terminal domain of Ergp55 are positioned away from Ets domain. The DNA binding groove of Ets domain is exposed to solvent and free to bind promoter DNA sequences. The CAE/CD domain is positioned between PNT and Ets domain to regulate the activity of Ergp55. The N- terminal and C-terminal domains of Ergp55 are positioned in a region that do not prevent the DNA binding activity of Ets domain and play a role in transcriptional activation and localization of Ergp55.

Discussion?Top

In current study, we have expressed full length and smaller polypeptides of Ergp55 in E. coli. The combinations of two chromatography steps (Ni-NTA affinity and size exclusion chromatography) have yielded more than 95% pure Ergp55 polypeptides based on mass spectrometry and SDS-PAGE analysis. Prior to structural studies, the activity of purified Ergp55 polypeptides were checked by binding studies using DNA sequences of E74 and cfos promoters. The surface plasmon resonance technique was used for binding analysis. These results indicated that Ergp55 polypeptides produced in E. coli were in good conformation and bind specifically DNA sequences of E74 and cFos promoters with different affinities.

DNA binding autoinhibition of Ergp55.

In case of E74 promoter sequence, following K_D values were observed for Ergp55 polypeptides (i) Erg_307?399, 65 nM (ii) Erg_112?399, 115 nM (iii) Erg_1?399, 217 nM and (iv) full length Erg_1?479, 704 nM. Comparison of (i) and (ii) indicated that N-terminal region (PNT+CAE/CD domains) preceding to Ets domain inhibit the E74 DNA binding to Ets domain. Comparison of (ii) and (iii) showed the evidence of increased DNA binding inhibition by having NTD domain in Erg_1?399 polypeptide. Comparison of (iii) and (iv) indicate that adding CTD domain in Erg_1?399 polypeptide showed enhanced inhibition in DNA binding to Ets domain. These results indicate that E74 DNA binding to Ergp55 is negatively influenced by CAE/CD, PNT and NTD domains located at N-terminal and CTD domain located C-terminal region in Ergp55.

With cfos promoter DNA sequence, following K_D values were obtained for Ergp55 polypeptides (i) Erg_307?399, 0.4 ?M (ii) Erg_112?399, 37 ?M (iii) Erg_1?399, 196 ?M and (iv) full length Erg_1?479, 232 ?M. These results indicate that cfos promoter sequence bind with different affinity to Ergp55 polypeptides than E74 promoter sequence, however mechanism of DNA binding inhibition was similar as observed in case of E74 promoter DNA sequence.

A cooperatively acting DNA inhibiting region (468?510 residues) was identified at C-terminal of Ets-1 transcription factor [34]. In case of ERM and PEA3 transcription factors, two main domains located at N- and C-terminal with respect to their ETS- domain inhibiting DNA binding affinity [35]?[38]. One domain corresponds to residue 280?360 residues of ERM transcription factor, involved in inhibition of ERM DNA binding capacity. These domains are rich in proline residues generally devoid ?-helical structures. The mechanism by which two domains cooperatively inhibit ERM DNA binding is different than observed in case of Ets-1 transcription factor. The DNA binding activity of Ets domain is dependent on autoinhibitory module [39]. The binding affinity of Ets domain Ergp55 to E74 and cfos promoter DNA was consistent to the observation obtained in case of above transcription factors.

Circular dichorism analysis of Ergp55 polypeptides.

The circular dichorism technique was used to identify the secondary and tertiary structures of Ergp55 polypeptides. The full length and smaller Ergp55 polypeptides contain high ?-helical and random coil structures. The CD data estimates 35% ?-helix and 49% random coil structures in full length Ergp55 protein. Examinations of thermal stability and temperature effect on full length Ergp55 protein indicated that protein underwent an alteration of secondary structure upon heating. The secondary structure is regained after cooling the protein from 80?C to 20?C. Short change of temperature is unlikely to have any effect on secondary structure of Ergp55 protein.

Modeling and dynamics simulation of full length Ergp55.

The molecular modeling and dynamics simulation analysis indicated that full length Ergp55 acquires a flexible and highly elongated structure. Only PNT and Ets domains are structured in protein and long flexible regions are observed at N- and C- terminus of Ergp55. The structure of PNT domain of Ergp55 consists of four-helix bundle Sam like structure (34). The Ets domain of Ergp55 is structured in a winged helix-turn-helix with scheme ?₁?₁?₂?₂?₃?₃?₄?₄[40]?[41]. The central CAE/CD domains contain one small helix at position 220. The secondary and disordered prediction analysis on Ergp55 also supported the finding observed in modeling and dynamics simulation studies of Ergp55. All these observations supported the flexible, non-globularity and highly elongated structure of Ergp55 protein.

In conclusion, we have characterized the recombinant full length and smaller polypeptides of Ergp55 produced in E. coli. The Ergp55 polypeptides were purified greater than 95% purity as determined by mass spectrometry and SDS-PAGE analysis. The structural data presented here showed the evidence of flexible and highly elongated structure of full length Ergp55 protein. The binding analysis using DNA sequences of E74 and cfos promoters indicate that longer fragments of Ergp55 (beyond the canonical Ets domain) showed the evidence of autoinhibition.

Acknowledgments?Top

The authors thank Sita R. Meena and Pratibha for their suggestions in current project. We acknowledge the help from advanced instrumentation research facility (AIRF) and central instrumentation facility (CIF) of Jawaharlal Nehru University for allowing us to conduct the CD experiment. The authors thank SPR equipment facility of AIIMS, Delhi for allowing us to conduct the DNA binding experiments.

Author Contributions?Top

Conceived and designed the experiments: AKS. Performed the experiments: SPG SD AKS. Analyzed the data: AKS. Contributed reagents/materials/analysis tools: SD AKS. Wrote the paper: AKS.

References?Top

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14. Mo Y, Vaessen B, Johnston K, Marmorstein R (1998) Structures of SAP-1 bound to DNA targets from the E74 and c-fos promoters: insights into DNA sequence discrimination by Ets proteins. Mol Cell 2: 201?212. Find this article online
15. Pufall MA, Lee GM, Nelson ML, Kang HS, Velyvis A, et al. (2005) Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured region. Science 309: 142?145. Find this article online
16. Wang Y, Feng L, Said M, Balderman S, Fayazi Z, et al. (2005) Analysis of the 2.0 A crystal structure of the protein-DNA complex of the human PDEF Ets domain bound to the prostate specific antigen regulatory site. Biochemistry 44: 7095?7106. Find this article online
17. Wei GH, Badis G, Berger MF, Kivioja T, Palin K, et al. (2010) Genome-wide analysis of Ets?family DNA-binding in vitro and in vivo.EMBO J 29: 2147?2160. Find this article online
18. Nieba l, Nieba-Axmann SE, Person A, Hamalainen M, Edebratt F, et al. (1997) BIACORE Analysis of Histidine-Tagged Proteins Using a Chelating NTA Sensor Chip. Anal Biochem 252: 217?228. Find this article online
19. Whitmore L, Wallace B A (2004) DICHROWEB: an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucl Acid Res 32: W668?673. Find this article online
20. Geourjon C, Deleage G (1995) SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Bios 11: 681?684. Find this article online
21. Garnier J, Gibrat JF, Robson B (1996) GOR secondary structure prediction method version IV. Meth Enzymol 266: 540?553. Find this article online
22. Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292: 195?202. Find this article online
23. Kelley LA, Sternberg MJE (2009) Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols 4: 363?371. Find this article online
24. Gai D, Zhao R, Li D, Finkielstein CV, Chen XS (2004) Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen Cell. Cambridge Mass 119: 47?60. Find this article online
25. Liang H, Mao X, Olejniczak ET, Nettesheim DG, Yu L (1994) Solution structure of the ets domain of Fli-1 when bound to DNA. Nat Struct Biol 1: 871?875. Find this article online
26. Fiser A, Do RK, Sali A (2000) Modeling of loops in protein structures. Protein Science 9: 1753?1773. Find this article online
27. Wu S, Zhang Y (2007) LOMETS: LOMETS: A local meta-threading-server for protein structure prediction. Nucl Acid Res 35: 3375?3382. Find this article online
28. Lindahl E, Hess B, Spoel DVD (2001) GROMACS 3.0: A package for molecular simulation and trajectory analysis. J Mol Mod 7: 306?317. Find this article online
29. Collaborative Computational Project, Number 4 (1994) Acta Cryst D50: 760?763. Find this article online
30. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22: 2577?2637. Find this article online
31. DeLano WL (2002) The PyMOL Molecular Graphics System DeLano Scientific. San Carlos CA USA.
32. Prasad DD, Rao VN, Reddy ES (1992) Structure, and expression of human Fli-1 gene. Cancer Res 52: 5833?5837. Find this article online
33. Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF, Jones DT (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. Journal of Molecular Biology 337: 635?645. Find this article online
34. Mackereth CD, Scharpf M, Gentile LN, Maclntosh SE, Slupsky CM, et al. (2004) Diversity in Structure and Function of the Ets Family PNT Domains. J Mol Biol 342: 1249?1264. Find this article online
35. Laget MP, Defossez PA, Albagli O, Baert JL, Dewitte F (1996) Two functionally distinct domains responsible for transactivation by the Ets family member ERM. Oncogene 12: 1325?1336. Find this article online
36. Greenall A, Willingham N, Cheung E, Boam DS, Sharrocks AD (2001) DNA binding by the ETS-domain transcription factor PEA3 is regulated by intramolecular protein-protein interactions J Biol Chem 276: 16207?16215. Find this article online
37. Bojovic BB, Hassel JA (2001) The PEA3 Ets transcription factor comprises multiple domains that regulate transactivation and DNA binding. J Biol Chem 276: 4509?45021. Find this article online
38. Mauen S, Huvent I, Raussens V, Demonte D, Baert JL (2006) Expression, purification, and structural prediction of the Ets transcription factor ERM. Biochem Biophys Acta 1760: 1192?1201. Find this article online
39. Brown LA, Mores A, Schilling TF, Jowett T, Baert JA, et al. (1998) Oncogene 17: 93?104. Find this article online
40. Mo Y, Vaessen B, Johnston K, Marmorstein R (2000) Structure of the Elk-1?1 DNA complex reveals how DNA distal residues affect ETS domain recognition of DNA. Nat Struct Biol 7: 292?297. Find this article online
41. Pio F, Kodandapani R, Ni CZ, Shepard W, Klemsz M, et al. (1996) New Insights on DNA Recognition by ets Proteins from the Crystal Structure of the PU.1 ETS Domain-DNA Complex. J Biol Chem 271: 23329?23337. Find this article online

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Screenwriter and Director Nora Ephron Dies at 71 - The Hollywood ...

Astrid Stawiarz/Getty Images

Nora Ephron, the celebrated screenwriter and director, died of leukemia Tuesday evening in New York, according to The New York Times.

The news followed an afternoon in which it was reported that she was gravely ill, though her?agency CAA and publisher Knopf denied earlier reports that she had died.

PHOTOS: Hollywood's Notable Deaths of 2012?

Ephron, best known for penning the screenplay for When Harry Met Sally ??(1989) and then writing and directing Sleepless in Seattle?(1989)?and?You've Got Mail (1998), had not been reported as ill before a story from gossip columnist Liz Smith -- which repeatedly referred to the 71-year-old in the past tense while never pronouncing her dead.

Smith's story was soon edited, but when speaking with The Hollywood Reporter, Smith said that she'd spoken with Ephron's son Jacob Bernstein.

?I was told this morning that she was dying, but I can?t confirm it,? she said. Smith went on to say that Bernstein told her that the family was planning for the funeral. But when no confirmation of Ephron?s death was forthcoming, Smith revised what she wrote.

?I have had to put everything in the present tense,? she wrote. ?It?s bad enough that she?s dying, but I don?t want to be the cause for everyone to grieve in advance."

Passing along the news that she said she received from Smith, columnist Margo Howard tweeted, "Well, to those of you who can't find the news of Nora Ephron's death, the funeral is Thursday - and maybe that's the way she wanted it."

VIDEO: Nora Ephron's 5 Most Memorable Movies

The daughter of Hollywood screenwriters, Ephron established herself as an original voice with an idiosyncratic take on modern manners with the personal essays she wrote in the 1960s for such publications as New York, Esquire and the New York Times Magazine. She made her feature screenwriting debut in 1983 with Mike Nichols' Silkwood. Her screenplays for Silkwood, Harry and Sleepless all earned Oscar nominations.

Ephron moved into directing with the 1992 feature This Is My Life, the story of a stand-up comic starring Julie Kavner. The following year, Sleepless proved to be a huge hit, making Ephron one of Hollywood's few successful woman directors. Most recently, she directed 2009's Julie & Julia, which starred her frequent collaborator Meryl Streep as Julia Child.

Ephron was married to writer Nicholas Pileggi and has two sons, Jacob and Max Bernstein, from her previous marriage to investigative reporter Carl Bernstein.

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Learn The Basics Of Home Improvement Today - Juss Free Vector Art

Posted by 11Zachary | Posted on 22-06-2012

Category : fashion

Tags: general

Upgrade your kitchen to improve home value quickly. Kitchen improvements can be as simple as refreshing paint or wallpaper or as complex as installing new cabinetry, appliances or flooring. You can do most kitchen improvements yourself or contract with a professional for more difficult items. Investing in your kitchen makes your home more enjoyable for yourself and your family, and it also increases the home value should you ever decide to sell your house.

A well known rule for doing any kind of construction, repair, or renovation work is to measure twice and cut once. This age old saying still holds up today in every circumstance. Following this rule will allow you to avoid costly and time consuming mistakes associated with projects by using less materials and saving time and effort on labor.

If you find yourself falling in love with a particular pattern of wallpaper but find that it?s either out of your price range or not quite durable enough for high-traffic areas, consider buying a roll of it anyway for decorative purposes. You can easily have it matted and framed, or can keep it around the house for smaller, more craft-type projects.

Improve your home in an environmentally friendly way by adding weatherstripping to keep your doors and windows from leaking warm or cool air. Not only does making your home weatherproof benefit the environment, it also helps to put many of your hard earned dollars back in your wallet.

Tie a t-shirt around your face! Don?t have a dust mask when you?re sanding? Shame on you! If you?re in a pinch though you can improvise with any close weave natural fiber. It isn?t the best solution but it certainly beats having no protection for your respiratory system whatsoever!

If you are doing any home improvements on the roof of your home, make sure you wear the proper shoes so that you keep yourself safe. Many accidents in home improvements happen from people falling off their roofs. Be careful up there by wearing the right shoes and tie yourself off whenever possible.

Let your neighbors know about any home improvements you?ll be undertaking that might affect them. If you warn them about noise, possible damage to their land, or the fact that big trucks will be coming through, they?ll appreciate the heads up. Many home renovations are marred by angry neighbors interfering.

To add a funky (and temporary) new look to a room without the hassle of painting a wall, decoupage your switchplates with pretty scrapbooking paper, adhering with decoupage medium. Choose patterned or solid paper that picks up an accent color from the room the switch is in. Since switchplates are inexpensive, you can easily change them back to plain versions when you are ready to move.

Use neutral colors when you paint. They will create the illusion of extra, open space, and make rooms appear to be larger than they are. A well painted home suggests to visitors that it is also a well cared for home. Don?t let the paint get too faded or warn before sprucing it up with fresh coats.

A great way to add a new look to any room of the home is to tile or re-tile the floors. Tile flooring comes in thousands of colors and designs and is relatively inexpensive when you do it yourself. For as little as $1 a square foot, new tile floors can be an excellent home improvement project.

When you are thinking about taking out a home improvement loan make sure you do your research. It has become increasingly harder to take out a home improvement loan that is unsecured which means you may have to use your home as collateral. Make sure this is something that you are prepared to do.

If you are thinking of buying a new tub, sit in the tub before purchasing it. You may feel embarrassed, but sometimes bathtubs may look much bigger than they actually are. Some tubs are can not be returned or have a very high restocking fee, so make sure you are pleased with the tub before buying.

When making outside cosmetic improvements to your home, don?t neglect the front door. A cheap, ugly front door can make the whole front appear less attractive. To replace a standard front door, costs less than $200. The improvement you will see in the appearance of your home is more than worth the cost.

Even if you have only a hundred dollars at your disposal, there are many affordable and accessible home-improvement projects that can have a great impact on the appearance of your house. Keep the advice from this article in mind as you begin to plan out your next home and garden project.

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Mysterious Air Force space plane lands after 15 months in orbit

An unmanned space plane built by Boeing, the second of its kind, returns to Earth after 463 days in orbit for a clandestine mission.

By Alicia Chang,?Associated Press / June 18, 2012

Image from video made available by the Vandenberg Air Force Base shows the X-37B unmanned spacecraft landing at Vandenberg Air Force Base. The spacecraft, which was launched from Cape Canaveral Air Force Station in Florida in March 2011, conducted in-orbit experiments during the 15-month clandestine mission, officials said.

AP

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An unmanned Air Force space plane steered itself to a landing early Saturday at a California military base, capping a 15-month clandestine mission.

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The spacecraft, which was launched from Cape Canaveral Air Force Station in Florida in March 2011, conducted in-orbit experiments during the mission, officials said. It was the second such autonomous landing at the Vandenberg Air Force Base, 130 miles northwest of Los Angeles. In 2010, an identical unmanned spacecraft returned to Earth after seven months and 91 million miles in orbit.

The latest homecoming was set in motion when the stubby-winged robotic X-37B fired its engine to slip out of orbit, then pierced through the atmosphere and glided down the runway like an airplane.

"With the retirement of the Space Shuttle fleet, the X-37B OTV program brings a singular capability to space technology development," said Lt. Col. Tom McIntyre, the X-37B's program manager. "The return capability allows the Air Force to test new technologies without the same risk commitment faced by other programs. We're proud of the entire team's successful efforts to bring this mission to an outstanding conclusion."

With the second X-37B on the ground, the Air Force planned to launch the first one again in the fall. An exact date has not been set.

The twin X-37B vehicles are part of a military program testing robotically controlled reusable spacecraft technologies. Though the Air Force has emphasized the goal is to test the space plane itself, there's a classified payload on board ? a detail that has led to much speculation about the mission's ultimate purpose.

Some amateur trackers think the craft carried an experimental spy satellite sensor judging by its low orbit and inclination, suggesting reconnaissance or intelligence gathering rather than communications.

Harvard astrophysicist Jonathan McDowell, who runs Jonathan's Space Report, which tracks the world's space launches and satellites, said it's possible it was testing some form of new imaging.

The latest X-37B was boosted into orbit atop an Atlas 5 rocket. It was designed to stay aloft for nine months, but the Air Force wanted to test its endurance. After determining the space plane was performing well, the military decided in December to extend the mission.

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Friendswood girls? softball coach arrested, accused of sexual assaulting child

by Shern-Min Chow / KHOU 11 News

khou.com

Posted on June 14, 2012 at 11:01 PM

Updated today at 11:01 PM

HOUSTON?A Friendswood girls? softball coach who is accused of sexually assaulting a 13-year-old girl has been arrested.

Friendswood police said there were two incidents in March and April.? A few weeks later the 13-year-old girl told her parents, who notified authorities.? Late Thursday, Ryan Jon Loofboro was taken into custody.

Over at the Friendswood softball field on Thursday night girls were busy practicing for an upcoming tournament.? Most parents had not yet heard the news, but Friendswood Girls? Softball Association President and dad Dan Lisbony just found out. ?He was in the car when he heard the news.

?(I) actually had to pull over and read the email,? he said.

Loofboro, an assistant coach at the Friendswood Girls Softball Association, worked with the 14 and under team this spring.? He is charged with aggravated sexual assault and indecency with a child.? Bail has been set at $40,000 for each count. ??

Lisbony said the league does require coaches go through a background check and go through a softball coaching class.?

?We require all background checks for assistants and team moms who are going to be on the fields,? he said.

Loofsboro, 34, is a realtor who lives in Dickinson and sits on the planning and zoning committee.

Friendswood police officer Lisa Price says she?s concerned that there may be other victims.

?He had access to children in his capacity as a coach and we always have to assume there may be other victims,? she said.

The association said it will notify parents, not the kids, but Amy Elguezabal said she plans to talk to her kids and have them watch the KHOU 11 News report.

?You have the news out there, you guys coming out. ?When I grew up my dad never wanted me to watch the news now-a-days, the more you prepare them, the better off they are for situations like this,? she said.

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Business Article Marketing: How It Works | Article Directory Blog

?

If you?re interested in using article marketing, most likely you have a business with a website, and you?d like to draw more viewers or customers to your site. When you do business article marketing, you will write and submit articles that teach readers how to do things associated with your field.

You may be wondering, why would a publisher want to publish your article? What does he have to gain from that?

Imagine how many websites are on the internet?there are tons! Each of these websites needs to publish content so that they have something to show their visitors. Also, having quality content on a website increases the value of the site in Google?s eyes.

There are other online publishers than just website owners?there are also people who publish ezines. Ezine editors need content on a regular basis, because they are sending out email newsletters every week or so.

When you receive an ezine in your inbox, there may be one or two articles in there. The articles bring value to the ezine?ezine editors really need the content. A person is very likely to read the articles in an email newsletter, because there is not a whole lot of other content to distract them. The content has been cherry picked for them, and it is likely that it is of interest to them.

So, if you submit your article and it is published in an ezine, it is basically delivered straight to the inbox of potentially tens of thousands of people who are interested in your niche. You could not ask for a better audience!

You might be wondering why a website owner or ezine editor doesn?t write his or her own content?

Well, he may not have the writing skills, or he may not have English has his first language. She may not have the time or the inclination, or maybe she doesn?t know enough about the subject matter to write intelligently about it.

Whatever the case, the publisher would rather use a well-written article that someone else has created. By publishing content from multiple authors, they will also end up with an interesting diversity in writing styles that can be attractive to their readers.

What do you get out of article marketing?

As the author of an educational article, you get many rewards:

First, article marketing can improve your website?s search engine ranking for specific words or phrases that your potential customers are typing into search engines. A high ranking in Google (and the other search engines) translates into more visitors being funneled into your website. That is the biggest payoff that a website owner receives from doing article marketing, but it?s not the only one?.

Your articles also bring attention to you as an knowledgeable person in your field. A well-written article can inspire trust in a reader, and a reader who trusts you is more likely to purchase your products or services.

Your articles can also direct ?traffic? to your website, straight from the article. Each article that you submit will list you as the author in an area called the ?resource box?. You get to write your own resource box, so you decide what you?ll say about yourself and your business.

The resource box is your one place where you can actively toot your own horn and try to get the reader to enlist your services or visit your website. It?s also the only spot in the article submission where you can list your website address as a clickable link.

As you can see, submitting free reprint articles is a win-win situation for both the publisher and the author. It?s all above board?the publishers really need and want the content, and they personally select the content that they deem appropriate for their site. As the author, you get the benefit of marketing your website and also establishing yourself as an expert in your niche.

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Millions watch Pacific eclipse

20 May 2012 Last updated at 23:29 ET

An "annular eclipse" has being viewed across a swathe of the Earth stretching across the Pacific from Asia to the western US.

The eclipse occurs when the Moon is at its farthest from the Earth and does not block out the Sun completely.

Millions of people witnessed the resulting "ring of fire" phenomenon.

The eclipse passed almost directly over Tokyo before sweeping just below Alaska's Aleutian islands and making landfall in the western US.

In Japan "eclipse tours" were held at schools and parks, on pleasure boats and even private airplanes. Similar events were also held in China and Taiwan.

TV in Tokyo broadcast the event live.

Light rain fell on Tokyo as the eclipse began, but the clouds thinned as it reached its peak, providing near perfect conditions.

"It was a very mysterious sight - I've never seen anything like it," said Kaori Sasaki, who joined a crowd in central Tokyo.

Japanese electronics giant Panasonic sent an expedition to the top of Mount Fuji to film the eclipse using solar-powered equipment.

"Our goal is to broadcast the world's most beautiful annular eclipse from the highest mountain in Japan," the company said.

However, in Hong Kong skywatchers were not so lucky. Hundreds had gathered along the Kowloon waterfront where the Space Museum had set up solar-filtered telescopes, but heavy clouds obstructed the view.

In the US, viewing parties were reported in Reno, Nevada; Oakland, California, and elsewhere.

Hundreds also travelled to the Petroglyph National Monument in Albuquerque, New Mexico, which was hailed as one of the best vantage points.

"That's got to be the prettiest thing I've ever seen," said Brent Veltri of Salida, Colorado.

The eclipse was fully visible across a 240 to 300km-wide swathe but partial views could be seen across much of east Asia and North America.

The Slooh series of space telescopes has been covering the event on its website.

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Merck ponders next step for troubled heart drug

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Source: http://news.yahoo.com/merck-ponders-next-step-troubled-heart-drug-131247086.html

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Change Management : clipclip

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Source: http://www.clipclip.org/victorsmith/clips/detail/1199493

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How to ask China's prime minister a question - and get a real answer

Since China's prime minister approves questions before a press conference, he has time to formulate bland answers. But sometimes a cheeky foreign reporter finds a workaround.

There is no better measure of Chinese officialdom?s control-freakery than Prime Minister Wen Jiabao?s encounter with the press at the close of the annual meeting of the National People's Congress, China?s ersatz parliament.

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This year?s performance, like last year, and the year before, was a grand set-piece event. Several hundred journalists gathered in a gaudily pillared reception room in the Great Hall of the People, photographers clicked madly whenever Mr. Wen made a photogenic gesture, and we all went through the motions of a press conference.

But if you thought the rubber-stamp National Press Club sessions were scripted, you should have been at the press event. Every question asked had been solicited, negotiated, and approved by Chinese officials. If you had not been through that process, there was no point in putting your hand up.

Chinese state-run media, whose reporters always get a chance to ask a question, can be trusted to serve up softballs. The official news agency Xinhua, for example, opened the proceedings today with a less-than-challenging request that Wen ?evaluate your work? since he took office nine years ago.

Foreign reporters generally make more of an effort to elicit something other than pabulum, but they can do so only if they play by the rules. And that means submitting questions in advance and negotiating their wording so as not to give offense.?

This can take several days of back and forth, and frankly I wonder whether it is worth it.

Foreign journalists asked Wen questions on sensitive topics such as when China would allow direct competitive elections to its national leadership, the recent spate of self-immolations by Tibetan monks, and China?s controversial refusal to back Western policies toward Syria.

But since the prime minister had seen the questions in advance, he and his aides had had plenty of time to formulate bland and unexceptional answers to all of them.

Luckily for us, though, at the end of the three-hour press conference at which the didactic Wen took an average of 13 minutes to answer a question, a cheeky Reuters correspondent ducked the rules. He asked the question that he had flagged in advance ? an anodyne inquiry about the level of local government debt ? and then snuck in a second question about a disgraced police chief who has sparked a scandal that has embroiled one of China?s most high-profile politicians, Bo Xilai.(?read more about that here)

Wen took the question, and he gave a newsworthy answer. It did not seem that hard, or painful for him. Why, we were left wondering, can he not do that all the time??

Source: http://rss.csmonitor.com/~r/csmonitor/globalnews/~3/FWXrrhFaVt8/How-to-ask-China-s-prime-minister-a-question-and-get-a-real-answer

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Marty2634: @tommyxtopher How have you been, Tommy? Do you think Romney will be the GOP Pres Nominee & what's the Big Media Story today on Mediaite?

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@tommyxtopher How have you been, Tommy? Do you think Romney will be the GOP Pres Nominee & what's the Big Media Story today on Mediaite? about 2 hours ago via web in reply to tommyxtopher

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Source: http://twitter.com/Marty2634/statuses/183309506523045888

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100% This Is Not a Film

This is as a fascinating bit of minimalism as there has been in quite a long time, not only for its insights into the state of censorship in Iran(from what I can infer, Jafar Panahi's arrest sounds like something out of "Hawaii 5-0,") but for its symbolism in capturing a day in the life of Jafar Panahi, trapped inside waiting for loved ones to return on New Year's Day with fireworks going off just outside. So it may come as no surprise that the only identifiable movie on his DVD rack is "Buried" starring Ryan Reynolds. In all honesty, I don't think 'This is Not a Film' was really meant to be a title in a conventional sense. Rather, it is meant to possibly to work around the Iranian authorities' sentencing Panahi to six years of prison and 20 years without directing, writing a film or giving interviews.(His lawyer thinks there is a chance he could have the prohibitions thrown out and his sentence reduced but he is still going to jail.) All of which he bares up under with humor and fatalism. So, that only leaves him to talk about his old films in pointing out how little control he has had over his amateur casts in the past. To accentuate this, his pet iguana(which is bigger than most cars, by the way) takes center stage at times. So, he stages scenes from what would have been his latest film about a young woman who wants to attend university in Tehran over her traditional parents' wishes. This all comes about when Mojtaba Mirtahmasb conceives of a behind the scenes series on banned directors.(Another way of looking at it, is as they put it, when hairdressers get bored, they do each others' hair.) Even though Panahi does not want to appeal to other Iranian directions which might get them in trouble also, Mirtahmasb also is currently in hot water, too. And finally, the real reason for this being made, is as Panahi puts it, to petition for international pressure, or in other words, us.

March 11, 2012

Source: http://www.rottentomatoes.com/m/this_is_not_a_film_2011/

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AJC Spring 2012 Dining Guide | Food and More with John Kessler

Beyond The Atlanta 50: Our print book and e-book versions are loaded with extras, including expanded reviews and excellent options in every category. How to preorder your copies.

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OTP or ITP, there?s a whole lot of dining out there to explore

By John Kessler

The last time I summoned the sheer chutzpah to propose a list of metro Atlanta?s top 50

John Kessler is the AJC's chief dining critic

restaurants, the year was 2004. Back then many of the best restaurants were also the most ambitious ones. Scouting out the talented chefs who used top-tier ingredients meant going to a lot of places with tablecloths and ice-filled wine buckets.

That hierarchy has collapsed, and I have to say it makes this quest a lot more rewarding. Today you can eat great food in scores of casual joints, in bars where you shout over head-banger music, in revitalized neighborhoods throughout the metro area, in farmers markets and at food truck rallies.

All across Atlanta, I have witnessed the doctrine of manifest yumminess. (Find an ?Atlanta 50? restaurant near you with our interactive map.)

So how do you choose 50 from this bounty?

You eat and eat, and eat some more. But first you must decide what doesn?t belong.

I chose to stay away from restaurants outside the metro area (sorry, Athens) and restaurants that only serve late at night (like Octopus Bar) or on weekends (Quinones at Bacchanalia).

I avoided some very good places that suffer from inconsistency and others that are too similar to better spots nearby. I tried as best as I could to provide a variety of styles and cuisines.

I?m still looking for a French, Thai or Middle Eastern spot to merit a place on this list. I?m sorry to the worthy Italian and Japanese restaurants that were edged out.

I did not limit it to only those places that we?ve rated three stars and above but instead made room for two-star restaurants ? such as Roswell?s Table & Main and Lilburn?s Three Blind Mice ? that add so much to their communities and connect so well with their fans. It?s not an ITP or an OTP thing, it?s a looking-like-Atlanta thing.

Some restaurants that I had not previously rated, but have visited at least twice, now get a rating.

Two previously rated places got a bump. Atlanta?s Heirloom Market BBQ deserves three stars as a citywide destination for its inimitable fusion barbecue. Miller Union has grown into a defining restaurant for today?s Atlanta and it deserves a fourth star. (A guide to our star ratings and a list of the restaurants we?ve rated.)

No restaurant on this list lost a star. Yay!

I hope you enjoy this list and can use it as a guide when you travel throughout metro Atlanta. As you can see, there?s lots to explore.

The 2012 Atlanta 50:

(Restaurant capsule reviews listed in alphabetical order.)

REVIEWS FOR RESTAURANTS A-B:

> 4th & Swift, Atlanta; Abattoir, Atlanta; Antico Pizza Napoletana, Midtown; Aria, Buckhead; Bacchanalia, Midtown; Barcelona Wine Bar, Atlanta; Bistro VG, Roswell; Bocado, Midtown; Bone?s, Buckhead; Busy Bee Cafe, Atlanta.

REVIEWS FOR RESTAURANTS C-G:

> Cafe Restaurant Dominicano, Norcross; Cakes & Ale (restaurant and bakery), Decatur; Canoe, Atlanta; Cardamom Hill, Atlanta; Community Q BBQ, Decatur; Empire State South, Midtown; Desta, Atlanta; Ecco, Atlanta; Golden House, Duluth.

REVIEWS FOR RESTAURANTS H-P:

> Heirloom Market BBQ, Atlanta; Honey Pig, Duluth; Holeman and Finch, Atlanta; Kevin Rathbun Steak, Atlanta; Kyma, Buckhead; McKendrick Steak House, Dunwoody; Miller Union, Midtown; Nam Phuong, Norcross; One Eared Stag, Atlanta; Pura Vida Tapas, Atlanta.

REVIEWS FOR RESTAURANTS R-Ta:

> Rathbun?s, Atlanta; Restaurant Eugene, Atlanta; Rumi?s Kitchen, Sandy Springs; Seed Kitchen & Bar, Marietta; Sotto Sotto, Atlanta; Sound Table, Atlanta; Sushi House Hayakawa, Doraville; Table & Main, Roswell; Taqueria del Sol, three metro locations; Taqueria Oaxaquena, Jonesboro; Tasty China, Marietta.

REVIEWS FOR RESTAURANTS Th-W:

> Three Blind Mice, Lilburn; Tofu Village, Marietta; Tomo Japanese Restaurant, Buckhead; Umaido, Suwanee; Varasano?s, Atlanta; Viande Rouge Steakhouse , Johns Creek; Vingenzo?s, Woodstock; Woodfire Grill, Atlanta; Woo Nam Jeong Stone Bowl House, Doraville; World Peace Cafe, Sandy Springs.

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> BEHIND THE ATLANTA 50: AJC chief dining critic John Kessler has spent the past three months researching this dining guide by revisiting potential ?top 50? restaurants at a breakneck pace that would put members of any competitive eating league to shame.

Many of the places he revisited made the ?Atlanta 50? cut, and many others did not. He downed Sichuan dan dan noodles in his car, ate Ethiopian food for breakfast and brought his editor leftover steak in exchange for a busted deadline or two.

See what else John brings to the table.

Previously:

> Where We?ve Eaten: All Our Restaurant Reviews Since 2010

> First Look: Recently Opened Restaurants

> Fall 2011 Dining Guide: Distinctive Culinary Voices

> Spring 2011 Dining Guide: Splurges and Destinations

> A Night On The Town: Our Complete Food, Restaurant and Nightclub Guide

Source: http://blogs.ajc.com/food-and-more/2012/03/22/spring-dining-guide-the-atlanta-50/?cxntfid=blogs_food_and_more

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How is the market in the Los Feliz & Franklin Hills areas of Los ...

[unable to retrieve full-text content]ChristopheChoo.com Christophe Choo of the Christophe Choo Real Estate Group at Coldwell Banker Previews International in Beverly Hills gives you 90-day stats for Single Family Homes, Condominiums & Townhomes in ...

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