Dynamics of hydrogen-deuterium exchange in Chlamydomonas centrin

Chlamydomonas reinhardtii centrin is a 169-amino acid residue calcium binding protein belonging to the EF-hand protein superfamily. Centrin is associated with the microtubule organizing center (MTOC) in all eukaryotes, and in Chlamydomonas, centrin is a component of the flagellar basal body apparatus. Recombinant full-length centrin, calmodulin, and terminal domain fragments [Ccen-N (residues 1-94) and Ccen-C (residues 99-169)] were used to examine hydrogen-deuterium (H --> D) exchange dynamics using combined attenuated total reflectance (ATR) Fourier transform-infrared (FT-IR) spectroscopy, curve fit, and two-dimensional correlation analysis. Analysis of the Ccen-N and Ccen-C fragments allowed separation of domain specific solvent exchange events and together with analysis of the full-length proteins provides novel insight into domain accessibility to the aqueous environment and the internal dynamics of the protein.     

Structural bases of unphosphorylated STAT1 association and receptor binding

The crystal structure has been determined at 3.0 A resolution for an unphosphorylated STAT1 (1-683) complexed with a phosphopeptide derived from the alpha chain of interferon gamma (IFNgamma) receptor. Two dimer interfaces are seen, one between the N domains (NDs) (amino acid residues 1-123) and the other between the core fragments (CFs) (residues 132-683). Analyses of the wild-type (wt) and mutant STAT1 proteins by static light scattering, analytical ultracentrifugation, and coimmunoprecipitation suggest that STAT1 is predominantly dimeric prior to activation, and the dimer is mediated by the ND interactions. The connecting region between the ND and the CF is flexible and allows two interconvertable orientations of the CFs, termed "antiparallel" or "parallel," as determined by SH2 domain orientations. Functional implications of these dimer conformations are discussed. Also revealed in this structure is the detailed interaction between STAT1 SH2 domain and its docking site on IFNgamma receptor.     

Culture of adult mouse neurons

Primary neuronal cells used to model physiology are generally limited to embryonic tissue. However, embryonic tissue is not optimal as a model for age-related changes in physiology or late-onset disease. Successful culturing of neurons from adult animals, however, has been historically difficult, if not impossible. Here, we report methodology for routine and reliable cultivation of healthy striatal neurons from adult mice. The new methodology is cost-effective and improves the speed and simplicity of neuronal isolation.     

To die or not to die: DNA repair in neurons

One of the critical emerging problems in modern pathobiology is how cells govern the decision to live or die, and the cost of making such a decision. Nowhere are these questions more poignant than in deciphering the tissue-specific responses to DNA damage. Mutations in DNA repair enzymes, malfunctions in cell cycle regulation, and genetic instability are associated with most somatic cancers. However, in many hereditary diseases arising from mutations in DNA repair proteins, the same dominant mutations that cause cancer in dividing cells are often associated with cell death in terminally differentiated neurons. Context dependent differences in the response to DNA damage are used to make fundamental choices as to cell fate, and are likely to shed light on the mechanisms underlying human disease.     

Effect of autophosphorylation on the catalytic and regulatory properties of protein tyrosine kinase Src.

The function of autophosphorylation in Src family protein tyrosine kinases is not fully understood. In this paper we compared the catalytic and ligand-binding properties of autophosphorylated and nonautophosphorylated (control) Src. The following are the main differences we found. First, while both forms had the same K(m) for ATP-Mg, autophosphorylated Src had significantly higher K(m) values for the phosphate-accepting substrates, polyE(4)Y, and RCM-lysozyme. The autophosphorylated form also had significantly higher V(max) values than the control. The substrate specificity, as measured by V(max)/K(m) ratio, was altered by autophosphorylation and was dependent on the phosphate-accepting substrate. Second, while autophosphorylation did not affect Src activation by free Mg(2+), Zn(2+), which inhibited Src by competing against an essential Mg(2+) activator, inhibited the control threefold more potently than the autophosphorylated form. Third, autophosphorylation significantly reduced the ability of its SH2 domain to bind phosphotyrosine. Fourth, a Pro-rich Src SH3 domain binding peptide activated the control, but not the autophosphorylated Src even though the apparent binding affinity was not significantly affected by autophosphorylation. These differences indicated that autophosphorylation induced significant and widespread changes in the catalytic and regulatory properties of Src. The implications of these findings relative to Src biological regulation are discussed.     

3-Dimensional Diffusion Tensor Imaging (DTI) Atlas of the Rat Brain

Anatomical atlases play an important role in the analysis of neuroimaging data in rodent neuroimaging studies. Having a high resolution, detailed atlas not only can expand understanding of rodent brain anatomy, but also enables automatic segmentation of new images, thus greatly increasing the efficiency of future analysis when applied to new data. These atlases can be used to analyze new scans of individual cases using a variety of automated segmentation methods. This project seeks to develop a set of detailed 3D anatomical atlases of the brain at postnatal day 5 (P5), 14 (P14), and adults (P72) in Sprague-Dawley rats. Our methods consisted of first creating a template image based on fixed scans of control rats, then manually segmenting various individual brain regions on the template. Using itk-SNAP software, subcortical and cortical regions, including both white matter and gray matter structures, were manually segmented in the axial, sagittal, and coronal planes. The P5, P14, and P72 atlases had 39, 45, and 29 regions segmented, respectively. These atlases have been made available to the broader research community.