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131.
Annett Sandner Juliane Illert Sabine Koitzsch Susanne Unverzagt Ilona Schön 《Experimental cell research》2013
Gastroesophageal reflux disease has been implicated in the pathogenesis of adenocarcinoma of the oesophagus. The same applies to laryngopharyngeal reflux (LPR) and squamous cell cancer of the head and neck, but so far, this link has not been proven. The impact of low pH and bile acids has not been studied extensively in cells other than oesophageal cancer cell lines and tissue. The aims of this study were to investigate the pathogenic potential of reflux and its single components on the mucosa of the upper respiratory tract. We measured DNA stability in human miniorgan cultures (MOCs) and primary epithelial cell cultures (EpCs) in response to reflux by the alkaline comet assay. As matrix metalloproteinases (MMPs) are involved in extracellular matrix remodelling processes and may contribute to cancer progression, we studied the expression of MMP1, -9, and -14 in MOCs, EpC, UM-SCC-22B, and FADUDD. DNA strand breaks (DNA-SBs) increased significantly at low pH and after incubation with human or artificial gastric juice. Single incubation with glycochenodeoxycholic acid also showed a significant increase in DNA-SBs. In epithelial cell cultures, human gastric juice increased the number of DNA-SBs at pH 4.5 and 5.5. Artificial gastric juice significantly up regulated the gene expression of MMP9. Western blot analysis confirmed the results of gene expression analysis, but the up regulation of MMP1, -9, and -14 was donor-specific. Reflux has the ability to promote genomic instability and may contribute to micro environmental changes suitable for the initiation of malignancy. Further functional gene analysis may elucidate the role of laryngopharyngeal reflux in the development of head neck squamous cell carcinoma (HNSCC). 相似文献
132.
O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function. 相似文献
133.
Identification of a new membrane-permeable inhibitor against inositol-1,4,5-trisphosphate-3-kinase A
Dominik Schröder Christoph Rehbach Carola Seyffarth Martin Neuenschwander Jens V. Kries Sabine Windhorst 《Biochemical and biophysical research communications》2013
Ectopic expression of the neuron-specific inositol-1,4,5-trisphosphate-3-kinase A (ITPKA) in lung cancer cells increases their metastatic potential because the protein exhibits two actin regulating activities; it bundles actin filaments and regulates inositol-1,4,5-trisphosphate (InsP3)-mediated calcium signals by phosphorylating InsP3. Thus, in order to inhibit the metastasis-promoting activity of ITPKA, both its actin bundling and its InsP3kinase activity has to be blocked. In this study, we performed a high throughput screen in order to identify specific and membrane-permeable substances against the InsP3kinase activity. Among 341,44 small molecules, 237 compounds (0.7%) were identified as potential InsP3kinase inhibitors. After determination of IC50-values, the three compounds with highest specificity and highest hydrophobicity (EPPC-3, BAMB-4, MEPTT-3) were further characterized. Only BAMB-4 was nearly completely taken up by H1299 cells and remained stable after cellular uptake, thus exhibiting a robust stability and a high membrane permeability. Determination of the inhibitor type revealed that BAMB-4 belongs to the group of mixed type inhibitors. Taken together, for the first time we identified a highly membrane-permeable inhibitor against the InsP3kinase activity of ITPKA providing the possibility to partly inhibit the metastasis-promoting effect of ITPKA in lung tumor cells. 相似文献
134.
Kornelia Neveling Lilian?A. Martinez-Carrera Irmgard H?lker Angelien Heister Aad Verrips Seyyed?Mohsen Hosseini-Barkooie Christian Gilissen Sascha Vermeer Maartje Pennings Rowdy Meijer Margot te?Riele Catharina?J.M. Frijns Oksana Suchowersky Linda MacLaren Sabine Rudnik-Sch?neborn Richard?J. Sinke Klaus Zerres R.?Brian Lowry Henny?H. Lemmink Lutz Garbes Joris?A. Veltman Helenius?J. Schelhaas Hans Scheffer Brunhilde Wirth 《American journal of human genetics》2013,92(6):946-954
Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA. 相似文献
135.
Angela Feechan Claire Anderson Laurent Torregrosa Angelica Jermakow Pere Mestre Sabine Wiedemann‐Merdinoglu Didier Merdinoglu Amanda R. Walker Lance Cadle‐Davidson Bruce Reisch Sebastien Aubourg Nadia Bentahar Bipna Shrestha Alain Bouquet Anne‐Françoise Adam‐Blondon Mark R. Thomas Ian B. Dry 《The Plant journal : for cell and molecular biology》2013,76(4):661-674
The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor. 相似文献
136.
Peter Šilhár Nicholas R. Silvaggi Sabine Pellett Kateřina Čapková Eric A. Johnson Karen N. Allen Kim D. Janda 《Bioorganic & medicinal chemistry》2013,21(5):1344-1348
Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based and thus have a limited window of treatment opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a therapeutic strategy for the treatment of botulism as it may provide an effective post exposure remedy. Using a combination of crystallographic and modeling studies a series of hydroxamates derived from 1-adamantylacetohydroxamic acid (3a) were prepared. From this group of compounds, an improved potency of about 17-fold was observed for two derivatives. Detailed mechanistic studies on these structures revealed a competitive inhibition model, with a Ki = 27 nM, which makes these compounds some of the most potent small molecule, non-peptidic BoNT/A LC inhibitors reported to date. 相似文献
137.
138.
Peter Bellstedt Thomas Seiboth Sabine Häfner Henriette Kutscha Ramadurai Ramachandran Matthias Görlach 《Journal of biomolecular NMR》2013,57(1):65-72
NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ("scrambling”) of NH and CO groups in a standard Escherichia coli expression host is provided. 相似文献
139.
Manon Ruffin Mélanie Voland Solenne Marie Monique Bonora Elise Blanchard Sabine Blouquit-Laye Emmanuel Naline Philippe Puyo Philippe Le Rouzic Loic Guillot Harriet Corvol Annick Clement Olivier Tabary 《生物化学与生物物理学报:疾病的分子基础》2013,1832(12):2340-2351
Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca2 +-activated Cl? channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl? channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl? channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process. 相似文献
140.
Amanda J. Bell Timothy J. Satchwell Kate J. Heesom Bethan R. Hawley Sabine Kupzig Matthew Hazell Rosey Mushens Andrew Herman Ashley M. Toye 《PloS one》2013,8(4)
Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes. 相似文献