Displacement affinity chromatography of protein phosphatase one (PP1) complexes

Background  

Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif.     

New products

Arosy future was forecast for Brookhaven National Laboratory's (BNL) user facilities at the first-ever joint meeting of the user communities for the National Synchrotron Light Source (NSLS) and the Center for Functional Nanomaterials (CFN), held May 15–17, 2006.     

Formal synthesis of (±)-methyl rocaglate using an unprecedented acetyl bromide mediated Nazarov reaction

To date the prototype Nazarov cyclization of a cross-conjugated pentadienone to the core structure of the rocaglate natural products has not been successful (9 into 12). It has been found that this conversion can be achieved by the use of acetylbromide in excellent yield and results in a strategically very direct route to these antitumor agents.     

Diffraction‐based automated crystal centering

A fully automated procedure for detecting and centering protein crystals in the X‐ray beam of a macromolecular crystallography beamline has been developed. A cryo‐loop centering routine that analyzes video images with an edge detection algorithm is first used to determine the dimensions of the loop holding the sample; then low‐dose X‐rays are used to record diffraction images in a grid over the edge and face plane of the loop. A three‐dimensional profile of the crystal based on the number of diffraction spots in each image is constructed. The derived center of mass is then used to align the crystal to the X‐ray beam. Typical samples can be accurately aligned in ～2–3 min. Because the procedure is based on the number of `good' spots as determined by the program Spotfinder, the best diffracting part of the crystal is aligned to the X‐ray beam.     

Fast XANES fluorescence imaging using a Maia detector

A new fast X‐ray absorption spectroscopy scanning method was recently implemented at the Hard X‐ray Microprobe endstation P06, PETRA III, DESY, utilizing a Maia detector. Spectromicroscopy maps were acquired with spectra for X‐ray absorption near‐edge structure (XANES) acquisition in the sub‐second regime. The method combines XANES measurements with raster‐scanning of the sample through the focused beam. The order of the scanning sequence of the axes, one beam energy axis and two (or more) spatial axes, is a variable experimental parameter and, depending on it, the dwell at each location can be either single and continuous (if the energy axis is the inner loop) or in shorter discontinuous intervals (if a spatial axis is innermost). The combination of improved spatial and temporal resolution may be necessary for rapidly changing samples, e.g. for following in operando chemical reactions or samples highly susceptible to beam damage where the rapid collection of single XANES spectra avoids issues with the emergence of chemical changes developing from latent damage. This paper compares data sets collected on a specially designed test pattern and a geological thin‐section scanning the energy as inner, middle and outer axis in the sequence. The XANES data of all three scanning schemes is found to show excellent agreement down to the single‐pixel level.     

Quantitative comparison of preparation methodologies for x-ray fluorescence microscopy of brain tissue

X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper calibration of the XFM instrument under the relevant operating conditions. Here, we describe the manufacture, characterization, and utilization of multi-element thin-film reference foils for use in calibration of XFM measurements of biological and other specimens. We have used these internal standards to assess the two-dimensional distribution of trace metals in a thin tissue section of a rat hippocampus. The data used in this study was acquired at the XFM beamline of the Australian Synchrotron using a new 384-element array detector (Maia) and at beamline 2-ID-E at the Advanced Photon Source. Post-processing of samples by different fixation techniques was investigated, with the conclusion that differences in solvent type and sample handling can significantly alter elemental content. The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.