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Selected AbstractsHost factor Ebp1: Selective inhibitor of influenza virus transcriptaseGENES TO CELLS, Issue 2 2007Ayae Honda Influenza virus RNA polymerase is composed of three virus-coded proteins, and is involved in both transcription and replication of the negative-strand genome RNA. Subunit PB1 plays key roles in both the RNA polymerase assembly and the catalytic function of RNA polymerization. Using yeast two-hybrid screening, a HeLa cell protein with the molecular mass of 45 kDa was identified. After cloning and sequencing, this protein was identified to be Ebp1, ErbB3-binding protein. Epb1 specifically interacts with PB1 both in vitro and in vivo, and Epb1 contact site on PB1 was mapped at its binding site of transcription primers. Ebp1 was found to interfere with in vitro RNA synthesis by influenza virus RNA polymerase (3P complex), but no inhibition was observed for capped RNA endonuclease and RNA-cap binding, the intrinsic activities of RNA polymerase. Since inhibition was not observed against other nucleic acid polymerases tested, we propose that Ebp1 is a selective inhibitor of influenza viral RNA polymerase. Accordingly over-expression of Ebp1 interfered with virus production. The PB1-contact site on Ebp1 overlaps with the interaction site with ErbB3 (epidermal receptor tyrosine kinase), androgen receptor (AR) and retinoblastoma gene product (Rb), which are involved in controlling cell proliferation and differentiation. [source] Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1PROTEIN SCIENCE, Issue 10 2006Vladislav V. Kiselyov Abstract The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1,Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR,ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1,Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF,Ig2 and Ig2,heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR,ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2,Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR,ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2,Ig2 interaction) in the absence of FGF. [source] The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formationTHE JOURNAL OF PHYSIOLOGY, Issue 1 2007Peter Gardzinski Synaptic vesicles aggregate at the presynaptic terminal during synapse formation via mechanisms that are poorly understood. Here we have investigated the role of the putative calcium sensor synaptotagmin I in vesicle aggregation during the formation of soma,soma synapses between identified partner cells using a simple in vitro synapse model in the mollusc Lymnaea stagnalis. Immunocytochemistry, optical imaging and electrophysiological recording techniques were used to monitor synapse formation and vesicle localization. Within 6 h, contact between appropriate synaptic partner cells up-regulated global synaptotagmin I expression, and induced a localized aggregation of synaptotagmin I at the contact site. Cell contacts between non-synaptic partner cells did not affect synaptotagmin I expression. Application of an human immunodeficiency virus type-1 transactivator (HIV-1 TAT)-tagged peptide corresponding to loop 3 of the synaptotagmin I C2A domain prevented synaptic vesicle aggregation and synapse formation. By contrast, a TAT-tagged peptide containing the calcium-binding motif of the C2B domain did not affect synaptic vesicle aggregation or synapse formation. Calcium imaging with Fura-2 demonstrated that TAT,C2 peptides did not alter either basal or evoked intracellular calcium levels. These results demonstrate that contact with an appropriate target cell is necessary to initiate synaptic vesicle aggregation during nascent synapse formation and that the initial aggregation of synaptic vesicles is dependent on loop 3 of the C2A domain of synaptotagmin I. [source] Mitochondrial clustering at the vertebrate neuromuscular junction during presynaptic differentiationDEVELOPMENTAL NEUROBIOLOGY, Issue 6 2006Chi Wai Lee Abstract During vertebrate neuromuscular junction (NMJ) development, presynaptic motor axons differentiate into nerve termini enriched in synaptic vesicles (SVs). At the nerve terminal, mitochondria are also concentrated, but how mitochondria become localized at these specialized domains is poorly understood. This process was studied in cultured Xenopus spinal neurons with mitochondrion-specific probe MitoTracker and SV markers. In nerve-muscle cocultures, mitochondria were concentrated stably at sites where neurites and muscle cells formed NMJs, and mitochondria coclustered with SVs where neurites were focally stimulated by beads coated with growth factors. Labeling with a mitochondrial membrane potential-dependent probe JC-1 revealed that these synaptic mitochondria were with higher membrane potential than the extrasynaptic ones. At early stages of bead-stimulation, actin-based protrusions and microtubule fragmentation were observed in neurites at bead contact sites, suggesting the involvement of cytoskeletal dynamics and rearrangement during presynaptic differentiation. Treating the cultures with an actin polymerization blocker, latrunculin A (Ltn A), almost completely abolished the formation of actin-based protrusions and partially inhibited bead-induced mitochondrial and SV clustering, whereas the microtubule disrupting agent nocodazole was ineffective in inhibiting the clustering of mitochondria and SVs. Lastly, in contrast to Ltn A, which blocked bead-induced clustering of both mitochondria and SVs, the ser/thr phosphatase inhibitor okadaic acid inhibited SV clustering but not mitochondrial clustering. These results suggest that at developing NMJs, synaptogenic stimuli induce the clustering of mitochondria together with SVs at presynaptic terminals in an actin cytoskeleton-dependent manner and involving different intracellular signaling molecules. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] The CD200 and CD200 receptor cell surface proteins interact through their N-terminal immunoglobulin-like domainsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2004Deborah Hatherley Abstract CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) involved in regulation of macrophage function. Both CD200 and CD200R contain two Ig superfamily domains like many other leukocyte membrane proteins. Site-directed mutagenesis of CD200R showed that, like CD200, it interacted through its N-terminal domain. This indicated that the cell-cell interaction spans four Ig superfamily domains and this distance is similar to many interactions found between T,cells and antigen-presenting cells. This suggests that this topology is also important in interactions of CD200 on a variety of cells with CD200R on myeloid cells, and comparable contact sites may be important mediating regulation in other cell-cell interactions. The mutagenesis showed that the binding involved the predicted GFCC, face of its N-terminal domain, like that of CD200, suggesting that the interaction evolved from a homotypic interaction. [source] Interaction of the alpha-helical H6 peptide from the pro-apoptotic protein tBid with cardiolipinFEBS JOURNAL, Issue 21 2009Patrice X. Petit BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices ,H6 and ,H7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c -releasing activity. In the present study, we focused on the interaction between the ,H6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the ,H6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of ,H6 into the phospholipid monolayer. The modification of two charged amino acid residues in ,H6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and ,H6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that ,H6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via ,H6. [source] Mortuary patterns in burial caves on Mangaia, Cook IslandsINTERNATIONAL JOURNAL OF OSTEOARCHAEOLOGY, Issue 3 2003S. C. Antón Abstract The behavioural, cultural, and political implications of archaeological human remains in non-mortuary, possibly culinary, contexts requires that we understand the range of mortuary practices in a particular region. Although several rockshelter sites on Mangaia, Cook Islands have yielded burned, fragmentary human bones in earth ovens that seem to support archaeological models and ethnohistoric accounts of ritual sacrifice and cannibalism, the absence of data on the range of Mangaian mortuary patterns obscures these interpretations. We describe burial patterns based on 40 above-ground interments representing at least 92 individuals in caves of Mangaia, Cook Islands, in order to begin to develop an island-wide perspective on mortuary patterns. Sampling both pre- and post-European contact sites we found that multiple interments dominate probable pre-contact burials (73%, 19 of 26) and single interments dominate post-contact contexts (80%, eight of ten burials), probably reflecting the influence of Christianity on mortuary ritual. Subadults were more frequent in all post-contact contexts suggesting alternative burial places, probably church cemeteries, for adults. Burial cave remains are broadly consistent with ethnohistoric accounts of interment in caves, however, they also illustrate additional burial practices and differences between time periods, such as primary body position and the role of multiple-individual interments, which are not discussed ethnohistorically. The mortuary practices in Mangaian burial caves differ from burials associated with marae and seem completely unrelated to the presence of highly fragmentary and burnt human remains in pre-contact rockshelter middens elsewhere on the island. Copyright © 2003 John Wiley & Sons, Ltd. [source] Membrane potential and endocytic activity control disintegration of cell,cell adhesion and cell fusion in vinculin-injected MDBK cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Riitta Palovuori Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961,974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell,cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin,membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin,membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local and transient pH gradients play a role. © 2004 Wiley-Liss, Inc. [source] Differential clustering of Caspr by oligodendrocytes and Schwann cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2009Menahem Eisenbach Abstract Formation of the paranodal axoglial junction (PNJ) requires the presence of three cell adhesion molecules: the 155-kDa isoform of neurofascin (NF155) on the glial membrane and a complex of Caspr and contactin found on the axolemma. Here we report that the clustering of Caspr along myelinated axons during development differs fundamentally between the central (CNS) and peripheral (PNS) nervous systems. In cultures of Schwann cells (SC) and dorsal root ganglion (DRG) neurons, membrane accumulation of Caspr was detected only after myelination. In contrast, in oligodendrocytes (OL)/DRG neurons cocultures, Caspr was clustered upon initial glial cell contact already before myelination had begun. Premyelination clustering of Caspr was detected in cultures of oligodendrocytes and retinal ganglion cells, motor neurons, and DRG neurons as well as in mixed cell cultures of rat forebrain and spinal cords. Cocultures of oligodendrocyte precursor cells isolated from contactin- or neurofascin-deficient mice with wild-type DRG neurons showed that clustering of Caspr at initial contact sites between OL processes and the axon requires glial expression of NF155 but not of contactin. These results demonstrate that the expression of membrane proteins along the axolemma is determined by the type of the contacting glial cells and is not an intrinsic characteristic of the axon. © 2009 Wiley-Liss, Inc. [source] Expression pattern of adhesion molecules in junctional epithelium differs from that in other gingival epitheliaJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2006S. Hatakeyama Background and Objective:, The gingival epithelium is the physiologically important interface between the bacterially colonized gingival sulcus and periodontal soft and mineralized connective tissues, requiring protection from exposure to bacteria and their products. However, of the three epithelia comprising the gingival epithelium, the junctional epithelium has much wider intercellular spaces than the sulcular epithelium and oral gingival epithelium. Hence, the aim of the present study was to characterize the cell adhesion structure in the junctional epithelium compared with the other two epithelia. Material and Methods:, Gingival epithelia excised at therapeutic flap surgery from patients with periodontitis were examined for expression of adhesion molecules by immunofluorescence. Results:, In the oral gingival epithelium and sulcular epithelium, but not in the junctional epithelium, desmoglein 1 and 2 in cell,cell contact sites were more abundant in the upper than the suprabasal layers. E-cadherin, the main transmembranous molecule of adherens junctions, was present in spinous layers of the oral gingival epithelium and sulcular epithelium, but was scarce in the junctional epithelium. In contrast, desmoglein 3 and P-cadherin were present in all layers of the junctional epithelium as well as the oral gingival epithelium and sulcular epithelium. Connexin 43 was clearly localized to spinous layers of the oral gingival epithelium, sulcular epithelium and parts of the junctional epithelium. Claudin-1 and occludin were expressed in the cell membranes of a few superficial layers of the oral gingival epithelium. Conclusion:, These findings indicated that the junctional epithelium contains only a few desmosomes, composed of only desmoglein 3; adherens junctions are probably absent because of defective E-cadherin. Thus, the anchoring junctions connecting junctional epithelium cells are lax, causing widened intercellular spaces. In contrast, the oral gingival epithelium, which has a few tight junctions, functions as a barrier. [source] Buried water molecules in helical transmembrane proteinsPROTEIN SCIENCE, Issue 2 2008Robert Renthal Abstract Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules. [source] Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1PROTEIN SCIENCE, Issue 10 2006Vladislav V. Kiselyov Abstract The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1,Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR,ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1,Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF,Ig2 and Ig2,heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR,ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2,Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR,ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2,Ig2 interaction) in the absence of FGF. [source] Lymphatic/Blood Endothelial Cell Connections at the Capillary Level in Adult Rat MesenteryTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 10 2010Jennifer L. Robichaux Abstract Analyses of microvascular networks with traditional tracer filling techniques suggest that the blood and lymphatic systems are distinct without direct communications, yet involvement of common growth factors during angiogenesis and lymphangiogenesis suggest that interactions at the capillary level are possible. To investigate the structural basis for lymphatic/blood endothelial cell connections during normal physiological growth, the objective of this study was to characterize the spatial relations between lymphatic and blood capillaries in adult rat mesenteric tissue. Using immunohistochemical methods, adult male Wistar rat mesenteric tissues were labeled with antibodies against PECAM (an endothelial marker) and LYVE-1, Prox-1, or Podoplanin (lymphatic endothelial markers) or NG2 (a pericyte marker). Positive PECAM labeling identified apparent lymphatic/blood endothelial cell connections at the capillary level characterized by direct contact or direct alignment with one another. In PECAM labeled networks, a subset of the lymphatic and blood capillary blind ends were connected with each other. Intravital imaging of FITC-Albumin injected through the femoral vein did not identify lymphatic vessels. At contact sites, lymphatic endothelial markers did not extend along blood capillary segments. However, PECAM positive lymphatic sprouts, structurally similar to blood capillary sprouts, lacked observable lymphatic marker labeling. These observations suggest that nonlumenal lymphatic/blood endothelial cell interactions exist in unstimulated adult microvascular networks and highlight the potential for lymphatic/blood endothelial cell plasticity. Anat Rec 293:1629,1638, 2010. © 2010 Wiley-Liss, Inc. [source] Using LiDAR to detect cultural resources in a forested environment: an example from Isle Royale National Park, Michigan, USAARCHAEOLOGICAL PROSPECTION, Issue 3 2008Julie M. Gallagher Abstract This article discusses the use of light detecting and ranging (LiDAR) technology as an effective remote sensing tool for the location of cultural resources. Its use, particularly in Europe, has proven successful in the identification of archaeological sites obscured by dense vegetation or surface disturbances. This study used LiDAR-derived imagery to detect pre- and post-European contact sites, and their related features, in densely forested environments on Isle Royale, Michigan, USA. LiDAR bare-Earth models were used to ,see through' the vegetation in an effort to: (i) identify cultural features prior to the implementation of a pedestrian reconnaissance survey; (ii) aid in the development of a more informed survey strategy; and (iii) produce an overall safer, more efficient and more cost-effective research design. Three study areas were selected for investigation. Within these three study areas, a total of seven investigation locales containing 32 separate features were identified using LiDAR-derived imagery. Eighteen of the 32 features were found to have been previously recorded. Of the remaining 14 features, seven were confirmed in the field as being cultural features and were recorded for the first time as a result of this investigation. The remaining seven could not be located on the ground or were found to be non-cultural. The results of this study support the use of LiDAR as a viable method for the detection of cultural resources, particularly in remote and heavily forested environments. Despite its positive contributions, there is a limited range of archaeological (surface) features that can be detected using this technology. As applied to archaeology, LiDAR is not an exclusive investigatory technique. It must be part of a comprehensive research strategy that integrates field, laboratory and archival investigation in order to achieve the best possible interpretation of the archaeological record. Copyright © 2008 John Wiley & Sons, Ltd. [source] Crystallization and preliminary X-ray diffraction studies of mutants of B1 IgG-binding domain of protein L from Peptostreptococcus magnusACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2000Keyji Johnsen The small 62-residue IgG-binding domain B1 of protein L from Peptostreptococcus magnus (Ppl-B1) has proven to be a simple system for the study of the thermodynamics and kinetics of protein folding. X-ray diffraction studies have been initiated in order to determine how the thermostability, folding and unfolding rates of a series of point mutations spanning Ppl-B1 correlate with the high-resolution structures. To this end, a tryptophan-containing variant of Ppl-B1 (herein known as wild type) and two mutants, Lys61Ala and Val49Ala, have been crystallized. Full data sets have been collected for the wild type and the Lys61Ala and Val49Ala mutants to resolutions of 1.7, 2.3 and 1.8,Ĺ, respectively. Interestingly, all three crystallize using different precipitants and in different space groups. This may be a consequence of the relatively large effects of single-site mutations on surface-charge distribution or structural conformation, which might affect crystal contact sites. [source] | ||||||||||||||||||||||||||||