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131.
132.
Krzysztof Pluta William Diehl Xian-Yang Zhang Robert Kutner Agnieszka Bialkowska Jakob Reiser 《BMC biotechnology》2007,7(1):41
Background
RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes. 相似文献133.
J Diao H Pantua H Ngu L Komuves L Diehl G Schaefer SB Kapadia 《Journal of virology》2012,86(20):10935-10949
While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR. 相似文献
134.
135.
Sicklick JK Choi SS Bustamante M McCall SJ Pérez EH Huang J Li YX Rojkind M Diehl AM 《American journal of physiology. Gastrointestinal and liver physiology》2006,291(4):G575-G583
Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells. 相似文献
136.
Jehle S Hiller M Rehbein K Diehl A Oschkinat H van Rossum BJ 《Journal of biomolecular NMR》2006,36(3):169-177
A simple spectroscopic filtering technique is presented that may aid the assignment of 13C and 15N resonances of methyl-containing amino-acids in solid-state magic-angle spinning (MAS) NMR. A filtering block that selects methyl resonances is introduced in two-dimensional (2D) 13C-homonuclear and 15N–13C heteronuclear correlation experiments. The 2D 13C–13C correlation spectra are recorded with the methyl filter implemented prior to a 13C–13C mixing step. It is shown that these methyl-filtered 13C-homonuclear correlation spectra are instrumental in the assignment of Cδ resonances of leucines by suppression of Cγ–Cδ cross peaks. Further, a methyl filter is implemented prior to a 15N–13C transferred-echo double resonance (TEDOR) exchange scheme to obtain 2D 15N–13C heteronuclear correlation spectra. These experiments provide correlations between methyl groups and backbone amides. Some of the observed sequential 15N–13C correlations form the basis for initial sequence-specific assignments of backbone signals of the outer-membrane protein G. 相似文献
137.
Vincent Compagnon Patrik Diehl Irne Benveniste Denise Meyer Hubert Schaller Lukas Schreiber Rochus Franke Franck Pinot 《Plant physiology》2009,150(4):1831-1843
Suberin composition of various plants including Arabidopsis (Arabidopsis thaliana) has shown the presence of very long chain fatty acid derivatives C20 in addition to the C16 and C18 series. Phylogenetic studies and plant genome mining have led to the identification of putative aliphatic hydroxylases belonging to the CYP86B subfamily of cytochrome P450 monooxygenases. In Arabidopsis, this subfamily is represented by CYP86B1 and CYP86B2, which share about 45% identity with CYP86A1, a fatty acid ω-hydroxylase implicated in root suberin monomer synthesis. Here, we show that CYP86B1 is located to the endoplasmic reticulum and is highly expressed in roots. Indeed, CYP86B1 promoter-driven β-glucuronidase expression indicated strong reporter activities at known sites of suberin production such as the endodermis. These observations, together with the fact that proteins of the CYP86B type are widespread among plant species, suggested a role of CYP86B1 in suberin biogenesis. To investigate the involvement of CYP86B1 in suberin biogenesis, we characterized an allelic series of cyp86B1 mutants of which two strong alleles were knockouts and two weak ones were RNA interference-silenced lines. These root aliphatic plant hydroxylase lines had a root and a seed coat aliphatic polyester composition in which C22- and C24-hydroxyacids and α,ω-dicarboxylic acids were strongly reduced. However, these changes did not affect seed coat permeability and ion content in leaves. The presumed precursors, C22 and C24 fatty acids, accumulated in the suberin polyester. These results demonstrate that CYP86B1 is a very long chain fatty acid hydroxylase specifically involved in polyester monomer biosynthesis during the course of plant development. 相似文献
138.
Young-Min Kang M. Lynn Prewitt Susan V. Diehl 《International biodeterioration & biodegradation》2009,63(8):1036-1044
Proteins expressed by the brown-rot fungus Gloeophyllum trabeum were characterized from inoculated southern yellow pine sapwood undergoing decay, from pure cultures of the fungus and from uninoculated pinewood. Analysis was carried out by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF/TOF/MS. No proteins were detected from the clean uncontaminated wood. The inoculated wood undergoing active brown-rot decay produced 76 proteins, including the Fenton-chemistry related enzymes, alcohol oxidase, lipoxygenase, and catalase. One hundred and eleven proteins were detected from the pure culture and most were common metabolic proteins. A majority of proteins in both samples were identified as hypothetical proteins. A surprising result is that there was very little overlap between proteins found in both sets of samples, indicating a very different mechanism in action when the fungus is growing on a cellulose-based nutrient source (wood) versus glucose media. This study also highlights a current limitation of this approach, which is the limited protein and genomic sequence information annotated on the public databases. Of the 187 proteins characterized, only 36 were identified with confidence. To our knowledge, this is the first reported proteomic analysis of pinewood decayed by a brown-rot fungus and provides the initial characterization of proteins involved in this type of wood biodeterioration. Although significant limitations still exist in identifying the proteins, this limitation will diminish as functional proteins are identified and added to the databases. 相似文献
139.
Leonardo M. Crema Luisa A. Diehl Ana P. Aguiar Lúcia Almeida Fernanda U. Fontella Letícia Pettenuzzo Deusa Vendite Carla Dalmaz 《Neurochemical research》2010,35(11):1700-1707
Previous studies have shown sex-specific oxidative changes in spinal cord of rats submitted to chronic stress, which may be
due to gonadal hormones. Here, we assessed total radical-trapping potential (TRAP), superoxide dismutase (SOD) and glutathione
peroxidase (GPx) activities and lipid peroxidation (evaluated by the TBARS test) in the spinal cord of ovariectomized (OVX)
female rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided
into controls and chronically stressed (for 40 days). Our findings demonstrate that chronic stress decreased TRAP, and increased
SOD activity in spinal cord homogenates from ovariectomized female rats and had no effect on GPx activity. On the other hand,
groups receiving 17β-estradiol replacement presented a decreased GPx activity, but no alteration in TRAP and in SOD activity.
No differences in the TBARS test were found in any of the groups analyzed. In conclusion, our results support the idea that
chronic stress induces an imbalance between SOD and GPx activities, additionally decreasing TRAP. Estradiol replacement did
not reverse the effects of chronic stress, but induced a decrease in GPx activity. Therefore, estradiol replacement in ovariectomized
chronically stressed rats could make the spinal cord more susceptible to oxidative injury. 相似文献
140.
Lindman S Bauer MC Lund M Diehl C Mulder FA Akke M Linse S 《Biophysical journal》2010,99(10):3365-3373
Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pKa values. However, pKa values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pKa values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pKa values were determined for carboxyl groups by monitoring their pH-dependent 13C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pKa values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pKa values of the folded and unfolded states and compared to experimental stability data. The use of experimental pKa values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone. 相似文献