Observation via one-dimensional 13Calpha NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper.

Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported. Histidine and arginine residues are mutated to lysine to reduce the unfolding temperature. CD and ultracentrifugation studies indicate that the molecule is a two-stranded coiled coil under benign conditions. Versions of the same peptide are made with 99% 13Calpha at selected sites. One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73 degrees C. Spectra at the temperature extremes establish the approximate chemical shifts for folded and unfolded forms at each labeled site. Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfolded forms interconvert slowly (> >2 ms) on the NMR time scale. Moreover, near room temperature, the structured form's resonance is double at several, but not all, sites, indicating at least two slowly interconverting, structured, local conformational substates. Analysis of the dynamics is possible. For example, near room temperature at the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant for interconversion of the two structured forms is near unity and the time scale is > or= 10-20 ms.

Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily.

We report the three-dimensional structure of osteogenic protein 1 (OP-1, also known as bone morphogenetic protein 7) to 2.8-A resolution. OP-1 is a member of the transforming growth factor beta (TGF-beta) superfamily of proteins and is able to induce new bone formation in vivo. Members of this superfamily share sequence similarity in their C-terminal regions and are implicated in embryonic development and adult tissue repair. Our crystal structure makes possible the structural comparison between two members of the TGF-beta superfamily. We find that although there is limited sequence identity between OP-1 and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-1/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-1 and TGF-beta 2 structures has revealed striking differences that provide insights into how these growth factors interact with their receptors.

Structure determination of murine mitochondrial carbonic anhydrase V at 2.45-A resolution: implications for catalytic proton transfer and inhibitor design.

The three-dimensional structure of murine mitochondrial carbonic anhydrase V has been determined and refined at 2.45-A resolution (crystallographic R factor = 0.187). Significant structural differences unique to the active site of carbonic anhydrase V are responsible for differences in the mechanism of catalytic proton transfer as compared with other carbonic anhydrase isozymes. In the prototypical isozyme, carbonic anhydrase II, catalytic proton transfer occurs via the shuttle group His-64; carbonic anhydrase V has Tyr-64, which is not an efficient proton shuttle due in part to the bulky adjacent side chain of Phe-65. Based on analysis of the structure of carbonic anhydrase V, we speculate that Tyr-131 may participate in proton transfer due to its proximity to zinc-bound solvent, its solvent accessibility, and its electrostatic environment in the protein structure. Finally, the design of isozyme-specific inhibitors is discussed in view of the complex between carbonic anhydrase V and acetazolamide, a transition-state analogue. Such inhibitors may be physiologically important in the regulation of blood glucose levels.

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells.

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Definition of a metal-dependent/Li(+)-inhibited phosphomonoesterase protein family based upon a conserved three-dimensional core structure.

Inositol polyphosphate 1-phosphatase, inositol monophosphate phosphatase, and fructose 1,6-bisphosphatase share a sequence motif, Asp-Pro-(Ile or Leu)-Asp-(Gly or Ser)-(Thr or Ser), that has been shown by crystallographic and mutagenesis studies to bind metal ions and participate in catalysis. We compared the six alpha-carbon coordinates of this motif from the crystal structures of these three phosphatases and found that they are superimposable with rms deviations ranging from 0.27 to 0.60 A. Remarkably, when these proteins were aligned by this motif a common core structure emerged, defined by five alpha-helices and 11 beta-strands comprising 155 residues having rms deviations ranging from 1.48 to 2.66 A. We used the superimposed structures to align the sequences within the common core, and a distant relationship was observed suggesting a common ancestor. The common core was used to align the sequences of several other proteins that share significant similarity to inositol monophosphate phosphatase, including proteins encoded by fungal qa-X and qutG, bacterial suhB and cysQ (identical to amtA), and yeast met22 (identical to hal2). Evolutionary comparison of the core sequences indicate that five distinct branches exist within this family. These proteins share metal-dependent/Li(+)-sensitive phosphomonoesterase activity, and each predicted tree branch exhibits unique substrate specificity. Thus, these proteins define an ancient structurally conserved family involved in diverse metabolic pathways including inositol signaling, gluconeogenesis, sulfate assimilation, and possibly quinone metabolism. Furthermore, we suggest that this protein family identifies candidate enzymes to account for both the therapeutic and toxic actions of Li+ as it is used in patients treated for manic depressive disease.

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.

TextMess 2.0: las tecnologías del lenguaje humano ante los nuevos retos de la comunicación digital

El objetivo general de este proyecto se centra en el estudio, desarrollo y experimentación de diferentes técnicas y sistemas basados en Tecnologías del Lenguaje Humano (TLH) para el desarrollo de la próxima generación de sistemas de procesamiento inteligente de la información digital (modelado, recuperación, tratamiento, comprensión y descubrimiento) afrontando los actuales retos de la comunicación digital. En este nuevo escenario, los sistemas deben incorporar capacidades de razonamiento que descubrirán la subjetividad de la información en todos sus contextos (espacial, temporal y emocional) analizando las diferentes dimensiones de uso (multilingualidad, multimodalidad y registro).

Polarized interacting exciton gas in quantum wells and bulk semiconductors

We develop a theory to calculate exciton binding energies of both two- and three-dimensional spin polarized exciton gases within a mean field approach. Our method allows the analysis of recent experiments showing the importance of the polarization and intensity of the excitation light on the exciton luminescence of GaAs quantum wells. We study the breaking of the spin degeneracy observed at high exciton density (5×1010 cm2). Energy level splitting between spin +1 and spin -1 is shown to be due to many-body interexcitonic exchange while the spin relaxation time is controlled by intraexciton exchange. © 1996 The American Physical Society.

Characterization of soluble and bound EPS obtained from 2 submerged membrane bioreactors by 3D-EEM and HPSEC

This research study deals with the quantification and characterization of the EPS obtained from two 25 L bench scale membrane bioreactors (MBRs) with micro-(MF-MBR) and ultrafiltration (UF-MBR) submerged membranes. Both reactors were fed with synthetic water and operated for 168 days without sludge extraction, increasing their mixed liquor suspended solid (MLSS) concentration during the experimentation time. The characterization of soluble EPS (EPSs) was achieved by the centrifugation of mixed liquor and bound EPS (EPSb) by extraction using a cationic resin exchange (CER). EPS characterization was carried out by applying the 3-dimensional excitation–emission matrix fluorescence spectroscopy (3D-EEM) and high-performance size exclusion chromatography (HPSEC) with the aim of obtaining structural and functional information thereof. With regard to the 3D-EEM analysis, fluorescence spectra of EPSb and EPSs showed 2 peaks in both MBRs at all the MLSS concentrations studied. The peaks obtained for EPSb were associated to soluble microbial by-product-like (predominantly protein-derived compounds) and to aromatic protein. For EPSs, the peaks were associated with humic and fulvic acids. In both MBRs, the fluorescence intensity (FI) of the peaks increased as MLSS and protein concentrations increased. The FI of the EPSs peaks was much lower than for EPSb. It was verified that the evolution of the FI clearly depends on the concentration of protein and humic acids for EPSb and EPSs, respectively. Chromatographic analysis showed that the intensity of the EPSb peak increased while the concentrations of MLSS did. Additionally, the mean MW calculated was always higher the higher the MLSS concentrations in the reactors. MW was higher for the MF-MBR than for the UF-MBR for the same MLSS concentrations demonstrating that the filtration carried out with a UF membrane lead to retentions of lower MW particles.

Estudo da alteração dimensional de um silicone de adição após um método de desinfecção e esterilização

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Three-Dimensional Structure and Disposition of the Air Conducting and Gas Exchange Conduits of the Avian Lung: The Domestic Duck (Cairina moschata)

The anatomy of the domestic duck lung was studied macroscopically, by casting and by light, transmission, and scanning electron microscopy. The lung had four categories of secondary bronchi (SB), namely, the medioventral (MV, 4-5), laterodorsal (LD, 6-10), lateroventral (LV, 2-4), and posterior secondary bronchi (PO, 36-44). The neopulmonic parabronchi formed an intricate feltwork on the ventral third of the lung and inosculated those from the other SB. The lung parenchyma was organized into cylindrical parabronchi separated by thin septa containing blood vessels. Atria were shallow and well-fortified by epithelial ridges reinforced by smooth muscle bundles and gave rise to 2-6 elongate infundibulae. Air capillaries arose either directly from the atria or from infundibulae and were tubular or globular in shape with thin interconnecting branches. The newly described spatial disposition of the conducting air conduits closely resembles that of the chicken. This remarkable similarity between the categories, numbers, and 3D arrangement of the SB in the duck and chicken points to a convergence in function-oriented design. To illuminate airflow dynamics in the avian lung, precise directions of airflow in the various categories of SB and parabronchi need to be characterized.

Mechanism of dimensional instability /

"Classification cancelled or changed to UNCLASSIFIED by authority of TIS Change Notice No. 1849 by ASB TISOR, date 8/14/55."