40 resultados para Imaging, Three-Dimensional


Relevância:

100.00% 100.00%

Publicador:

Resumo:

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

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.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Methods of structural and statistical analysis of the relation between the sequence and secondary and three-dimensional structures are developed. About 5000 secondary structures of immunoglobulin molecules from the Kabat data base were predicted. Two statistical analyses of amino acids reveal 47 universal positions in strands and loops. Eight universally conservative positions out of the 47 are singled out because they contain the same amino acid in > 90% of all chains. The remaining 39 positions, which we term universally alternative positions, were divided into five groups: hydrophobic, charged and polar, aromatic, hydrophilic, and Gly-Ala, corresponding to the residues that occupied them in almost all chains. The analysis of residue-residue contacts shows that the 47 universal positions can be distinguished by the number and types of contacts. The calculations of contact maps in the 29 antibody structures revealed that residues in 24 of these 47 positions have contacts only with residues of antiparallel beta-strands in the same beta-sheet and residues in the remaining 23 positions always have far-away contacts with residues from other beta-sheets as well. In addition, residues in 6 of the 47 universal positions are also involved in interactions with residues of the other variable or constant domains.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Although attention plays a significant role in vision, its spatial deployment and spread in the third dimension is not well understood. In visual search experiments we show that we cannot easily focus attention across isodepth loci unless they are part of a well-formed surface with locally coplanar elements. Yet we can easily spread our attention selectively across well-formed surfaces that span an extreme range of stereoscopic depths. In cueing experiments, we show that this spread of attention is, in part, obligatory. Attentional selectivity is reduced when targets and distractors are coplanar with or rest on a common receding stereoscopic plane. We conclude that attention cannot be efficiently allocated to arbitrary depths and extents in space but is linked to and spreads automatically across perceived surfaces.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

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.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

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.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Knowing how motile bacteria move near and along a solid surface is crucial to understanding such diverse phenomena as the migration of infectious bacteria along a catheter, biofilm growth, and the movement of bacteria through the pore spaces of saturated soil, a critical step in the in situ bioremediation of contaminated aquifers. In this study, a tracking microscope is used to record the three-dimensional motion of Escherichia coli near a planar glass surface. Data from the tracking microscope are analyzed to quantify the effects of bacteria-surface interactions on the swimming behavior of bacteria. The speed of cells approaching the surface is found to decrease in agreement with the mathematical model of Ramia et al. [Ramia, M., Tullock, D. L. & Phan-Tien, N. (1993) Biophys J. 65,755-778], which represents the bacteria as spheres with a single polar flagellum rotating at a constant rate. The tendency of cells to swim adjacent to the surface is shown in computer-generated reproductions of cell traces. The attractive interaction potential between the cells and the solid surface is offered as one of several possible explanations for this tendency.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

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.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Complex three-dimensional waves of excitation can explain the observed cell movement pattern in Dictyostelium slugs. Here we show that these three-dimensional waves can be produced by a realistic model for the cAMP relay system [Martiel, J. L. & Goldbeter, A. (1987) Biophys J. 52, 807-828]. The conversion of scroll waves in the prestalk zone of the slug into planar wave fronts in the prespore zone can result from a smaller fraction of relaying cells in the prespore zone. Further, we show that the cAMP concentrations to which cells in a slug are exposed over time display a simple pattern, despite the complex spatial geometry of the waves. This cAMP distribution agrees well with observed patterns of cAMP-regulated cell type-specific gene expression. The core of the spiral, which is a region of low cAMP concentration, might direct expression of stalk-specific genes during culmination.