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At least 577 records · Page 32Linked to original sources
PubMed2003

A theoretical study of the large Hg-Hg spin-spin coupling constants in Hg(2)(2+), Hg(3)(2+), and Hg(2)(2+)-crown ether complexes.

Nuclear spin-spin coupling constants (1)J(Hg-Hg) in the systems Hg(2)(2+) and Hg(3)(2+) represent the largest coupling constants so far observed in NMR experiments. We have performed a computational study on these ions, on Hg(2)(2+) complexes with 18-crown-6 and 15-crown-5, and on Hg(3)(2+) with solvent molecules and counterions. The results obtained with our recently developed program for the density functional computation of heavy nucleus spin-spin coupling constants are in good agreement with experiments. The data reveal that the bare ions Hg(2)(2+) and Hg(3)(2+) would afford much larger coupling constants than those experimentally observed, with an upper limit of approximately 0.9 MHz for Hg(2)(2+). This limit is much larger than that previously estimated by Hückel theory. It is demonstrated that in solution or due to complexation the experimentally determined values are much smaller than the free ion's coupling constants. With the help of intuitive MO arguments, it is illustrated how the environment strongly reduces the coupling constants in Hg(2)(2+) and Hg(3)(2+). The two-bond coupling constant (2)J(Hg-Hg) in Hg(3)(2+) is also examined.

Journal Article↗
PubMed2002

14-3-3 protein regulation of proton pumps and ion channels.

In addition to their regulation of cytoplasmic enzymes, the 14-3-3 proteins are important regulators of membrane localised proteins. In particular, many of the cells' ion pumps and channels are either directly or indirectly modulated by 14-3-3 proteins. Binding of 14-3-3 can lead to the activation of pump activity as in the case of the plasma membrane H+-ATPase or inhibition as in the case of the F-type ATP synthase complexes. 14-3-3 binding can also lead to surprising results such as the recruitment of 'sleepy' outward rectifiying K+ channels in tomato cells. Our present knowledge extends to an initial understanding of isoform-specific binding of 14-3-3 to certain membrane proteins and a perception of the protein kinases and phosphatases that maintain the regulatory process in a state of flux.

14-3-3 Proteins↗
PubMed2004

Transgenic TCR expression: comparison of single chain with full-length receptor constructs for T-cell function.

Genetic modification of T lymphocytes with T-cell receptor (TCR) genes provides a novel tool for adoptive immunotherapy. However, the efficiency of full-length TCR (flTCR)-transduced T cells could be limited by factors such as incorrect pairing between exogenous and endogenous TCR chains and downregulation of the CD3 complex. To overcome these hurdles, one promising strategy is to use three-domain single-chain TCRs (3D-scTCR), in which TCR Valpha and Vbeta chains are joined by a linker with signal transduction domains fused at the carboxyl termini as signal transducers and amplifiers. Our results showed that surface expression of scTCRs on T cells after retroviral transduction was affected by the origin of the transmembrane (TM) region and placement of signaling domains. scTCR-modified T cells were functional as shown by cytokine (IL-2 and IFN-gamma) release in response to antigen stimulation and cytolytic activity against specific target cells. CD8 and CD28, but not the complete CD3 complex, could enhance the scTCR-induced T cell activation. Compared with flTCR-modified T cells and native CTLs, scTCR-modified T cells require higher thresholds of antigen stimulation (approximately 10(-8) M peptide) to be functional. Despite the low efficiency of scTCRs, our data provide insight into further improvements in generating efficient scTCRs for in vivo applications.

Animals↗
PubMed2004

Global inhibition of Lef1/Tcf-dependent Wnt signaling at its nuclear end point abrogates development in transgenic Xenopus embryos.

Analysis of canonical Wnt signaling during vertebrate development by means of knock-out or transgenic approaches is often hampered by functional redundancy as well as pathway bifurcations downstream of the manipulated components. We report the design of an optimized chimera capable of blocking transcriptional activation of Lef1/Tcf-beta-catenin target genes, thus enabling intervention with the canonical Wnt pathway at its nuclear end point. This construct was made hormone-inducible, both functionally and transcriptionally, and was transgenically integrated in Xenopus embryos. Down-regulation of target genes was clearly observed upon treatment of these embryos with dexamethasone. In addition, exposure of variously aged transgenic embryos to dexamethasone caused complex phenotypes with many new but also several recognizable features stemming from inhibition of canonical Wnt signaling. At least in some tissues, a significant reduction in cell proliferation and an increase in programmed cell death appeared to underlie these phenotypes. Our inducible transgenic system can serve a broad range of experimental settings designed to unveil new functional aspects of Lef1/Tcf-beta-catenin signaling during vertebrate embryogenesis.

Animals↗
PubMed2003

Airway vascular remodeling in asthma.

Several characteristic changes occur in the bronchial wall in asthma, including specific changes to the vasculature. These result in an increase in vessel numbers per unit area, as well as increased vessel activity suggested by vasodilatation, vessel leakage, and cellular margination with transmigration to target tissues. This combined action in asthma leads to airway-wall thickening and reduced airflow. Each component of the vascular response has been shown to be controlled by a range of inflammatory mediators and growth factors. These factors are themselves regulated by a complex process initially involving gene expression, transcription, and translation at the molecular level, then subsequent protein release, binding to matrix elements, endothelial cell activation, and a proliferative endothelial response. Many commonly used airway medications are capable of modulating the vascular response to inflammatory stimuli. New therapies might improve airflow through better regulation of vessel growth, dilatation, and leakage in the airway wall.

Airway Resistance↗
PubMed2004

Topological characterization of the c, c', and c" subunits of the vacuolar ATPase from the yeast Saccharomyces cerevisiae.

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V(1) and V(0). Hydrolysis of ATP in the V(1) subcomplex is tightly coupled to proton translocation accomplished by the V(0) subcomplex, which is composed of five unique subunits (a, d, c, c', and c"). Three of the subunits, subunit c (Vma3p), c' (Vma11p), and c" (Vma16p), are small highly hydrophobic integral membrane proteins called "proteolipids" that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c' (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c" (Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c' have four transmembrane segments with their N and C termini in the lumen and that c" has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal.

Cytosol↗
PubMed2024

Cilia defects upon loss of WDR4 are linked to proteasomal hyperactivity and ubiquitin shortage.

The WD repeat-containing protein 4 (WDR4) has repeatedly been associated with primary microcephaly, a condition of impaired brain and skull growth. Often, faulty centrosomes cause microcephaly, yet aberrant cilia may also be involved. Here, we show using a combination of approaches in human fibroblasts, zebrafish embryos and patient-derived cells that WDR4 facilitates cilium formation. Molecularly, we associated WDR4 loss-of-function with increased protein synthesis and concomitant upregulation of proteasomal activity, while ubiquitin precursor pools are reduced. Inhibition of proteasomal activity as well as supplementation with free ubiquitin restored normal ciliogenesis. Proteasome inhibition ameliorated microcephaly phenotypes. Thus, we propose that WDR4 loss-of-function impairs head growth and neurogenesis via aberrant cilia formation, initially caused by disturbed protein and ubiquitin homeostasis.

Animals↗
PubMed2005

Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion.

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans,Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning.

Journal Article↗
PubMed2004

Contribution of JAM-1 to epithelial differentiation and tight-junction biogenesis in the mouse preimplantation embryo.

We have investigated the contribution of the tight junction (TJ) transmembrane protein junction-adhesion-molecule 1 (JAM-1) to trophectoderm epithelial differentiation in the mouse embryo. JAM-1-encoding mRNA is expressed early from the embryonic genome and is detectable as protein from the eight-cell stage. Immunofluorescence confocal analysis of staged embryos and synchronized cell clusters revealed JAM-1 recruitment to cell contact sites occurred predominantly during the first hour after division to the eight-cell stage, earlier than any other TJ protein analysed to date in this model and before E-cadherin adhesion and cell polarization. During embryo compaction later in the fourth cell cycle, JAM-1 localized transiently yet precisely to the apical microvillous pole, where protein kinase Czeta (PKCzeta) and PKCdelta are also found, indicating a role in cell surface reorganization and polarization. Subsequently, in morulae and blastocysts, JAM-1 is distributed ubiquitously at cell contact sites within the embryo but is concentrated within the trophectoderm apicolateral junctional complex, a pattern resembling that of E-cadherin and nectin-2. However, treatment of embryos with anti-JAM-1-neutralizing antibodies indicated that JAM-1 did not contribute to global embryo compaction and adhesion but rather regulated the timing of blastocoel cavity formation dependent upon establishment of the trophectoderm TJ paracellular seal.

Adherens Junctions↗
PubMed2006

Structural comparison of three crystalline complexes of a peptidic toxin with a synaptic acetylcholine recognition protein.

Many peptidic toxins from animal venoms target neuronal or peripheral synaptic receptors with high affinities and specificities. Hence, these toxins are not only potent natural weapons but also precise molecular tools for pharmacological studies of their receptors. Although they belong to various structural and/or functional subfamilies, they often share similar molecular features, such as a highly reticulated scaffold presenting specific binding determinants.

Acetylcholine↗
PubMedDate not supplied

[The early correction of the hand deformities in Apert's syndrome. A preliminary report].

The correction of cranial and limb malformations of the Apert's syndrome requires efforts of a multidisciplinary team. The esthetic and functional repercussions that this syndrome brings to their carriers are severe and so the treatment should aim tom improve the function of the hands in order to integrate these patients in a more normal social live. In our Service, in the period from 1989 to 1992 five patients carrying this syndrome had their hand correction completed before the age of three years. It was observed that surgery was possible in this early stage with no additional complication rate. Quadrangular flaps for the neocomissure and for the coverage of the fingers were successfully used even with skeletal deviations. There was improvement of the esthetics of the hands, but their function could not be evaluated properly. Anyway, because of complex abnormalities the results will never be perfect. The early treatment of these alterations allows the gain of a better hand function and moreover accelerates the physical and intellectual development of the children.

Acrocephalosyndactylia↗
PubMed2004

Mechanism of silver-mediated di-tert-butylsilylene transfer from a silacyclopropane to an alkene.

Kinetic studies of the reactions of cyclohexene silacyclopropane 1 and monosubstituted alkenes in the presence of 5 mol % of (Ph3P)2AgOTf suggested a possible mechanism for silver-mediated di-tert-butylsilylene transfer. The kinetic order in cyclohexene silacyclopropane 1 was determined to be one. Inverse kinetic saturation behavior (rate inhibition) was observed in monosubstituted alkene and cyclohexene concentrations. Saturation kinetic behavior in catalyst concentration was observed. A reactive intermediate, a silylsilver complex, was observed using low temperature 29Si NMR spectroscopy. Competition experiments between substituted styrenes and a deficient amount of 1 correlated well with the Hammett equation and provided a rho value of -0.62 +/- 0.02 using sigmap constants. These data support a mechanism involving reversible silver-promoted di-tert-butylsilylene extrusion from 1 followed by irreversible concerted electrophilic attack of the silylsilver intermediate on the alkene.

Alkenes↗
PubMed2004

Axons in cat visual cortex are topologically self-similar.

The axonal arbors of the different types of neocortical and thalamic neurons appear highly dissimilar when viewed in conventional 2D reconstructions. Nevertheless, we have found that their one-dimensional metrics and topologies are surprisingly similar. To discover this, we analysed the axonal branching pattern of 39 neurons (23 spiny, 13 smooth and three thalamic axons) that were filled intracellularly with horseradish peroxidase (HRP) during in vivo experiments in cat area 17. The axons were completely reconstructed and translated into dendrograms. Topological, fractal and Horton-Strahler analyses indicated that axons of smooth and spiny neurons had similar complexity, length ratios (a measure of the relative increase in the length of collateral segments as the axon branches) and bifurcation ratios (a measure of the relative increase in the number of collateral segments as the axon branches). We show that a simple random branching model (Galton-Watson process) predicts with reasonable accuracy the bifurcation ratio, length ratio and collateral length distribution of the axonal arbors.

Animals↗
PubMed2006

Gift not commodity? Lay people deliberating social sex selection.

In this paper we explore lay people's discussions of the controversial topic of social sex selection (SSS). In the UK and many other countries, SSS is prohibited by law. In 2003 the UK Human Fertilisation and Embryology Authority, after an extensive public consultation, decided against changing the existing legislation. However, this initiative and similar consultation exercises have been criticised on the grounds that public opinion is poorly informed and reasoned. In our study, one of the most consistently expressed ideas was that children should be regarded as 'a gift' rather than 'a commodity'. In contrast, the 'gift not a commodity' argument is rarely cited positively in Anglo-American, secular-liberal bioethics. These metaphorical statements are condensed articulations of complex but coherent moral intuitions. Where much of the bioethics literature stresses parental autonomy, our lay discussants balanced this principle with ideas about the need to respect the personhood of the potential child, and the characteristics of a good parent. We conclude our analysis by considering the implications for expanding bioethics' knowledge base and improving the input of lay people in bioethical decision making.

Adult↗
PubMed1975

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.

Avidin↗
PubMed2004

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein.

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6.

Cell Line, Tumor↗
PubMed2005

Structural characterization of the regulatory proteins TenA and TenI from Bacillus subtilis and identification of TenA as a thiaminase II.

Bacillus subtilis gene products TenA and TenI have been implicated in regulating the production of extracellular proteases, but their role in the regulation process remains unclear. The structural characterization of these proteins was undertaken to help provide insight into their function. We have determined the structure of TenA alone and in complex with 4-amino-2-methyl-5-hydroxymethylpyrimidine, and we demonstrate that TenA is a thiaminase II. The TenA structure suggests that the degradation of thiamin by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I. Three active-site residues, Asp44, Cys135, and Glu205, are likely involved in substrate binding and catalysis based on the enzyme/product complex structure and the conservation of these residues within TenA sequences. We have also determined the structure of TenI. Although TenI shows significant structural homology to thiamin phosphate synthase, it has no known enzymatic function. The structure suggests that TenI is unable to bind thiamin phosphate, largely resulting from the presence of leucine at position 119, while the corresponding residue in thiamin phosphate synthase is glycine.

Alkyl and Aryl Transferases↗
PubMed2006

Visualization of molecular interactions by fluorescence complementation.

The visualization of protein complexes in living cells enables the examination of protein interactions in their normal environment and the determination of their subcellular localization. The bimolecular fluorescence complementation assay has been used to visualize interactions among multiple proteins in many cell types and organisms. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin-family peptides.

Microscopy, Fluorescence↗