Heuristic Refinement Method for the Derivation of Protein Solution Structures: Validation on Cytochrome B562

A method is described for determining the family of protein structures compatible with solution data obtained primarily from nuclear magnetic resonance (NMR) spectroscopy. Starting with all possible conformations, the method systematically excludes conformations until the remaining structures are only those compatible with the data. The apparent computational intractability of this approach is reduced by assembling the protein in pieces, by considering the protein at several levels of abstraction, by utilizing constraint satisfaction methods to consider only a few atoms at a time, and by utilizing artificial intelligence methods of heuristic control to decide which actions will exclude the most conformations. Example results are presented for simulated NMR data from the known crystal structure of cytochrome b562 (103 residues). For 10 sample backbones an average root-mean-square deviation from the crystal of 4.1 A was found for all alpha-carbon atoms and 2.8 A for helix alpha-carbons alone. The 10 backbones define the family of all structures compatible with the data and provide nearly correct starting structures for adjustment by any of the current structure determination methods

An assessment of the readiness of ablative materials for preflight application to the shuttle orbiter

The shuttle orbiter relies primarily on a reusable surface insulation (RSI) thermal protection system (TPS). The RSI is very efficient in its thermal performance; however, the RSI tile system has shown poor mechanical integrity. The state-of-the-art of the ablative TPS is reviewed, and an assessment made of the ablator's readiness for use on the shuttle orbiter. Unresolved technical issues with regard to the ablative TPS are identified. Short time, highly focused analytical and experimental programs were initiated to: (1) identify candidate ablation materials; (2) assess the data base for these materials; (3) evaluate the need and kind of waterproof coating; (4) calculate thermal and other stresses in an ablator tile; (5) identify an acceptable ablator/RSI tile joint filler; and (6) assess the sensitivity of the ablator to sequential heat pulses. Results from some of these programs are discussed

CASPASE-12 and Rheumatoid Arthritis in African-Americans

CASPASE-12 (CASP12) has a downregulatory function during infection and thus may protect against inflammatory disease. We investigated the distribution of CASP12 alleles (#rs497116) in African-Americans (AA) with rheumatoid arthritis (RA). CASP12 alleles were genotyped in 953 RA patients and 342 controls. Statistical analyses comparing genotype groups were performed using Kruskal–Wallis non-parametric ANOVA with Mann–Whitney U tests and chi-square tests. There was no significant difference in the overall distribution of CASP12 genotypes within AA with RA, but CASP12 homozygous patients had lower baseline joint-narrowing scores. CASP12 homozygosity appears to be a subtle protective factor for some aspects of RA in AA patients

Scattering of 7^{7}Be and 8^{8}B and the astrophysical S17_{17} factor

Measurements of scattering of $^{7}$Be at 87 MeV on a melamine (C$_{3}$N$_{6}$H$_{6}$) target and of $^{8}$B at 95 MeV on C were performed. For $^{7}$Be the angular range was extended over previous measurements and monitoring of the intensity of the radioactive beam was improved. The measurements allowed us to check and improve the optical model potentials used in the incoming and outgoing channels for the analysis of existing data on the proton transfer reaction $^{14}$N($^{7}$Be,$^{8}$B)$^{13}$C. The resultslead to an updated determination of the asymptotic normalization coefficient for the virtual decay $^{8}$B $\to$ $^{7}$Be + $p$. We find a slightly larger value, $C_{tot}^{2}(^{8}B)=0.466\pm 0.047$ fm$^{-1}$, for the melamine target. This implies an astrophysical factor, $S_{17}(0)=18.0\pm 1.8$ eV$\cdot$b, for the solar neutrino generating reaction $^{7}$Be($p$,$\gamma$)$^{8}$B.Comment: 7 pages, 4 figure

Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients.

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed. Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 micrometers in diameter. Preabsorption of the antisera was attended using microtubule protein, purified tubulin, actin, and microtubule-associated proteins. None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region. In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle

Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified Xenopus laevis augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs

Esperanto for histones : CENP-A, not CenH3, is the centromeric histone H3 variant

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres