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991.
Karl Georg Haeusler Lydia Koch Juliane Ueberreiter Matthias Endres Heinz-Peter Schultheiss Peter U Heuschmann Alexander Schirdewan Jochen B Fiebach 《BMC neurology》2010,10(1):63
Background
Catheter ablation of the pulmonary veins has become accepted as a standard therapeutic approach for symptomatic paroxysmal atrial fibrillation (AF). However, there is some evidence for an ablation associated (silent) stroke risk, lowering the hope to limit the stroke risk by restoration of rhythm over rate control in AF. The purpose of the prospective randomized single-center study "Mesh Ablator versus Cryoballoon Pulmonary Vein Ablation of Symptomatic Paroxysmal Atrial Fibrillation" (MACPAF) is to compare the efficacy and safety of two balloon based pulmonary vein ablation systems in patients with symptomatic paroxysmal AF. 相似文献992.
Robert Huber Thomas G Palmen Nadine Ryk Anne-Kathrin Hillmer Karina Luft Frank Kensy Jochen Büchs 《BMC biotechnology》2010,10(1):22
Background
High-throughput cultivations in microtiter plates are the method of choice to express proteins from recombinant clone libraries. Such processes typically include several steps, whereby some of them are linked by replication steps: transformation, plating, colony picking, preculture, main culture and induction. In this study, the effects of conventional replication methods and replication tools (8-channel pipette, 96-pin replicators: steel replicator with fixed or spring-loaded pins, plastic replicator with fixed pins) on growth kinetics of Escherichia coli SCS1 pQE-30 pSE111 were observed. Growth was monitored with the BioLector, an on-line monitoring technique for microtiter plates. Furthermore, the influence of these effects on product formation of Escherichia coli pRhotHi-2-EcFbFP was investigated. Finally, a high-throughput cultivation process was simulated with Corynebacterium glutamicum pEKEx2-phoD-GFP, beginning at the colony picking step. 相似文献993.
Jochen M. Schwenk Ulrika Igel Maja Neiman Hanno Langen Charlotte Becker Anders Bjartell Fredrik Ponten Fredrik Wiklund Henrik Gr?nberg Peter Nilsson Mathias Uhlen 《Molecular & cellular proteomics : MCP》2010,9(11):2497-2507
There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format.There is a great need for protein biomarkers for early diagnosis of disease as well as for prognostic markers in which the outcome of a particular disease or treatment can be predicted (1). In particular, biomarkers that make it possible to monitor the progress of treatment or the reoccurrence of a particular disease are of great clinical value. However, there are still few protein biomarkers in clinical practice today, and despite many biomarker discovery efforts by many laboratories using many different approaches, a limited number have been introduced into the clinical routine during the last 10 years (2). The complexity of serum or plasma proteomes with their broad dynamic range of protein concentrations and the lack of high throughput methods with high sensitivity have hampered such discovery and validation efforts.The most common approach for protein biomarker discovery today is the use of proteomics methods in which samples from case-control groups are compared using biochemical and biophysical methods, most notably with mass spectrometry (3). The introduction of more and more sophisticated instrumentation has increased the sensitivity and throughput of mass spectrometry during the last years (4). One of the advantages with mass spectrometry is that the method also allows for the detection of differences in protein modifications, such as glycosylation or phosphorylation, which have been found useful for some applications (5). Although many potential biomarkers have been discovered using mass spectrometry, the approach is yet limited to the analysis of a relatively small number of patient samples.The alternative approach for biomarker discovery is to use affinity probes, usually antibodies but also other reagents, such as aptamers (6) or Affibody molecules (7). The advantage of such probe-based methods is the possibility to analyze many samples in parallel, and many assays based on antibodies, such as ELISA, are very sensitive in the sub-ng/ml range. In particular, sandwich immunoassays in which two separate antibodies are used to increase the sensitivity and selectivity allow proteins to be assayed down to pg/ml (8). Recently, new assays based on amplification methods have been described, such as the proximity ligation method (9), and these have the potential to score protein on a single molecule level. However, the lack of validated antibodies to most human proteins (10) makes it impossible to use antibody-based protocols for a majority of the potential protein targets, and this is even more difficult for assays based on paired antibodies that require two distinct antibodies with separate and non-overlapping epitopes. Because of this limitation, current studies are directed by candidate target lists reported in the literature (11) or in associated gene expression studies (12) or built on collections of in-house binder libraries (13).Recently, new efforts have been described for the generation of antibodies on a whole-proteome level (14). Version 6 of the Human Protein Atlas contains validated antibodies toward proteins from 8,400 human genes, corresponding to 42% of the protein-encoded genes in man. All antibodies published in the Human Protein Atlas are publicly available and include a total of more than 40 antibody providers from the United States, Canada, Europe, Australia, and Asia. Several other efforts, such as the ProteomeBinder (15), the SH2 consortium (16), and the NCI affinity capture project (17), have recently been initiated with the aim to generate affinity reagents toward human protein targets. The objective of these efforts is to have publicly available antibodies to a representative protein from all of the protein-encoded genes by 2014 (18), and this emphasizes the need to develop high throughput methods for immunobased protein profiling to leverage this tool box of antibodies to allow high throughput biomarker discovery.We have shown earlier that antibodies utilized in suspension bead arrays can be used for profiling proteins in serum and plasma (19). Hereby, we found that the ability to detect proteins such as components of the complement system was enhanced by heat treatment, most likely because epitopes might be exposed at elevated temperatures and thus become available for antibody binding. In particular, this is likely to be the case for antibodies recognizing linear epitopes on the protein target. Here, we analyzed the functionality of antibodies following different epitope retrieval protocols at different temperatures, and we describe a method for multiplex analysis of plasma or serum using plasma from patients with elevated PSA1 levels as an example. A method suitable for analysis of large numbers of biobank samples is presented. 相似文献
994.
Detergent solubilization and purification of the E. coli heavy metal P-type ATPase ZntA yields an enzyme with reduced hydrolytic activity in vitro. Here, it is shown that the in vitro hydrolytic activity of detergent solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. The protein-lipid interaction of ZntA was characterized by enzyme-coupled ATPase assays and fluorescence spectroscopy. Among the most abundant naturally occurring phospholipids, only phosphatidyl-glycerol lipids (PG) enhance the in vitro enzymatic ATPase activity of ZntA. Re-lipidation of detergent purified ZntA with 1,2-dioleoylphosphatidyl-glycerol (DOPG) increases the ATPase activity four-fold compared to the purified state. All other E. coli phospholipids fail to activate the ATPase. Among the phosphatidyl-glycerol family, highest activity was observed for 1,2-dioleoyl-PG followed by 1,2-dimyristoyl-PG, 1,2-dipalmitoyl-PG and 1,2-distearoyl-PG. Increasing intrinsic Trp fluorescence quantum yield upon relipidation of ZntA was used to determine a pH maximum for lipid binding at pH 6.7. The pH dependence of the lipid binding was confirmed by pH-dependent ATPase assays showing maximum activity at pH 6.7. The biophysical characterization of detergent solubilized membrane proteins crucially relies on the conformational stability and functional integrity of the protein under investigation. The present study describes how the E. coli ZntA P-type ATPase can be stabilized and functionally activated in a detergent solubilized system. 相似文献
995.
Nerve growth factor (NGF), a member of the neurotrophin family, is an all-beta-sheet protein with a characteristic structure motif, the cystine knot. Unfolding of NGF in 6 M GdnHCl has been described previously to involve an initial partial loss of structure and a subsequent very slow conversion to a second, completely unfolded state. This latter conversion was postulated to represent a back-threading of the disulfide bond that passes through the cystine knot (loop threading hypothesis). Here, this hypothesis was questioned with the pro form of the protein (proNGF). In proNGF, the mature part is preceded by the 103-amino acid pro-peptide. Consequently, loop threading of the N-terminally extended protein should be significantly delayed. However, unfolding kinetics of proNGF monitored by RP-HPLC, intrinsic fluorescence, and NMR spectroscopy were comparable to those of mature NGF. Time-resolved (1)H-(15)N HSQC spectra revealed a slow time-dependent loss of residual structure of which the kinetics correlated well with the transition observed by RP-HPLC. Refolding from the completely unfolded state led to a partial recovery of natively folded proNGF. In summary, the sequential unfolding of proNGF only marginally differed from that of mature NGF. Therefore, it is very unlikely that a loop threading mechanism is the cause of the slow unfolding step. 相似文献
996.
Synaptotagmins (syt) form a large family of transmembrane proteins and some of its isoforms are known to regulate calcium-induced membrane fusion during vesicular traffic. In view of the reported implication of the isoform syt8 in exocytosis we investigated the expression, localisation and calcium-sensitivity of syt8 in secretory cells. An immunopurified antipeptide antibody was generated which is directed against a C-terminal sequence and devoid of crossreactivity towards syt1 to 12. Subcellular fractionation and immunocytochemistry revealed two forms of synaptotagmin 8 (50 and 40 kDa). Whereas the 40-kDa was present in the cytosol in brain, in PC12 and in clonal beta-cells, the 50-kDa form was localised in very typical clusters and partially colocalised with the SNARE protein Vti1a. Moreover, in primary hippocampal neurons syt8 was only found within the soma. Amplification of syt8 by RT-PCR indicated that the observed protein variants were not generated by alternative splicing of the 6th exon and are most likely linked to variations in the N-terminal region. In contrast to the established calcium sensor syt2, endogenous cytosolic syt8 and transiently expressed syt8-C2AB-eGFP did not translocate upon a raise in cytosolic calcium in living cells. Syt8 is therefore not a calcium sensor in exocytotic membrane fusion in endocrine cells. 相似文献
997.
We constructed a novel temperature-sensitive vector as a tool for gene disruption by insertion-duplication mutagenesis (IDM) in Salmonella enterica and related species. A phoN insertion mutant was proven highly stable during growth in LB medium and during infection of macrophage cells in the absence of selection pressure. By progressive shortening of a phoN fragment, the minimal length for effective insertional mutagenesis driven by homologous recombination was determined to be 50 bp, allowing to disrupt even short genes that could not yet be subjected to site-specific IDM. We also showed that plasmid excision from the chromosome restores the wild-type genotype with a reliability of 98%. Intracellular recovery of the excised vector provides the option to switch between two genotypes and thus to rapidly attribute the observed mutant phenotype to the targeted gene. In addition, a fragment library was used to measure the integration rate at various chromosomal sites that varies greatly by at least 2.5 magnitudes, independently from the length of the cloned fragment. 相似文献
998.
999.
A Disulfide Bond-Containing Alkaline Phosphatase Triggers a BdbC-Dependent Secretion Stress Response in Bacillus subtilis 总被引:1,自引:0,他引:1 下载免费PDF全文
Elise Darmon Ronald Dorenbos Jochen Meens Roland Freudl Haike Antelmann Michael Hecker Oscar P. Kuipers Sierd Bron Wim J. Quax Jean-Yves F. Dubois Jan Maarten van Dijl 《Applied microbiology》2006,72(11):6876-6885
The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for α-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli. 相似文献
1000.
Engel M Hindie V Lopez-Garcia LA Stroba A Schaeffer F Adrian I Imig J Idrissova L Nastainczyk W Zeuzem S Alzari PM Hartmann RW Piiper A Biondi RM 《The EMBO journal》2006,25(23):5469-5480
Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate phosphoinositide-dependent protein kinase 1 (PDK1) by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins. 相似文献