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101.
The identification of epitopes involved in protein-protein interactions is essential for understanding protein structure and function. Large scale efforts, although identifying the interactions, did not always yield these epitopes, could not confirm most of the known interactions, and seemed particularly unsuccessful for native intrinsic membrane proteins. We have developed a fluidics-based approach (non-steady-state kinetics) to obtain the broadest set of the epitopes interacting with a given target and applied it to a phage display methodology optimized for membrane proteins. Phages expressing a liver cDNA library were screened against a membrane protein (voltage-dependent anion channel) reconstituted into liposomes and captured on a chip surface. The controlled fluidics was obtained by a surface plasmon resonance (SPR) device that combined the advantages of working with minute reaction volumes and non-equilibrium conditions. We demonstrated selective enrichment of binders and could even select for different binding affinities by fractionation of the selected outputs at various elution times. With voltage-dependent anion channel as bait (a mitochondrial channel critical for cellular metabolism and apoptosis) we found at least 40% of its already reported ligands and independently confirmed 55 novel functional interactions, some of which fully blocked the channel. This highly efficient approach is generally applicable for any protein and could be automated and scaled up even without the use of a SPR device. The epitopes directly identified by this method are useful not only for unraveling interactomes but also for drug design and therapeutics. 相似文献
102.
Wirths O Erck C Martens H Harmeier A Geumann C Jawhar S Kumar S Multhaup G Walter J Ingelsson M Degerman-Gunnarsson M Kalimo H Huitinga I Lannfelt L Bayer TA 《The Journal of biological chemistry》2010,285(53):41517-41524
N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers. 相似文献
103.
Paeshuyse J Coelmont L Vliegen I Van hemel J Vandenkerckhove J Peys E Sas B De Clercq E Neyts J 《Biochemical and biophysical research communications》2006,348(1):139-144
We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin. 相似文献
104.
The chrysolaminaran from the marine diatom Chaetoceros debilis was isolated and characterized by NMR spectroscopy. Cells were harvested in the stationary phase of growth after the medium had been depleted of nitrate when the chrysolaminaran content was expected to be at its highest. The chrysolaminaran was isolated with an yield of 17.5 mg/L, which corresponds to 15.8 pg/cell. 1H NMR indicated that the structure was similar to that of a beta-(1-->3) main chain with beta-(1-->6)-linked side chains. The degree of polymerization was found to be 30, corresponding to a molecular weight of approximately 4900. Thirty-three percent of the residues were found to be beta-(1-->6)-linked branches. The characteristics of the beta-(1-->6) branching were examined by NOESY NMR, which suggested pustulan-like branches, being beta-(1-->6) linked chains connected to the main chain with few branch points. Confirmation of the 1H NMR data was done by 13C-DEPT, TOCSY, COSY and HMQC NMR spectroscopy. The assignment of the resonances of the main beta-(1-->3) and beta-(1-->6) chains is presented. The structure proposed from our analyses is compared against other chrysolaminaran structures. 相似文献
105.
The naturally occurring antioxidant Ascopyrone P (1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose, 1) was prepared from the rare sugar 1,5-anhydro-D-fructose (AF, 3) in three steps in an overall yield of 36%. Thus, acetylation of 3 afforded the enolone 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-en-2-ulopyranose (4), which could be isomerised to 2,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-1-ene-3-ulose (6). Deacetylation of 6 under mild conditions gave crystalline Ascopyrone P (1). 相似文献
106.
Neutrophil elastase sorting involves plasma membrane trafficking requiring the C-terminal propeptide
Tapper H Källquist L Johnsson E Persson AM Hansson M Olsson I 《Experimental cell research》2006,312(18):3471-3484
The primary granules/secretory lysosomes of neutrophils store mature neutrophil elastase (NE) as a luminal protein after proteolytic removal of N-terminal and C-terminal pro-peptides from a proform of NE. The N-terminal pro-peptide prevents premature activation that might be toxic to the cell, but the C-terminal pro-peptide has no defined function. In this study, we investigated the role of the C-terminal pro-peptide in trafficking of NE by expressing, in rat basophilic leukemia (RBL) cells, both wild-type NE and the mutant NE/Delta248-267, which lacks the C-terminal pro-peptide. Both transfected proteins were found to be targeted to secretory lysosomes. In addition, results from antibody ligation and cell-surface biotinylation indicated that proform of NE was targeted to the plasma membrane, and then subjected to endocytosis. The results were supported by the detection of targeting of the proform to the plasma membrane followed by internalization both in RBL cells and normal granulopoietic precursor cells. Targeting of NE to the plasma membrane required the C-terminal pro-peptide as NE/Delta248-267 expressed in RBL cells bypassed plasma membrane trafficking. Our results indicate targeting of a population of NE to the plasma membrane and internalization dependent on the C-terminal NE pro-peptide. 相似文献
107.
Harbeck M Dobberstein R Ritz-Timme S Schröder I Grupe G 《Anthropologischer Anzeiger; Bericht über die biologisch-anthropologische Literatur》2006,64(3):273-282
Modern bone samples were experimentally degraded by incubation into water at increased temperature and examined in terms of their collagen content, the stable C and N isotopic ratios, and the molar C/N ratio. The same analyses were carried out with archaeological human bone of varying age (300 up to 8000 years). The experimentally degraded samples exhibited changes of the collagen's integrity, which influence the stable isotope ratios. In the case of the archaeological material, a correlation between stable delta13C- and delta15N-values and collagen content could be demonstrated. The molar C:N ratio was no suitable criterion for the assessment of the state of preservation of extractable collagen. 相似文献
108.
Goekint M Bos I Heyman E Meeusen R Michotte Y Sarre S 《Journal of applied physiology (Bethesda, Md. : 1985)》2012,112(4):535-541
Hippocampal brain-derived neurotrophic factor (BDNF) protein is increased with exercise in rats. Monoamines seem to play a role in the regulation of BDNF, and monoamine neurotransmission is known to increase with exercise. The purpose of this study was to examine the influence of acute exercise on monoaminergic neurotransmission and BDNF protein concentrations. Hippocampal microdialysis was performed in rats that were subjected to 60 min of treadmill running at 20 m/min or rest. Two hours postexercise, the rats were killed, and the hippocampus was dissected. In experiments without microdialysis, hippocampus and serum samples were collected immediately after exercise. Exercise induced a twofold increase in hippocampal dopamine release. Noradrenaline and serotonin release were not affected. Hippocampal BDNF levels were not influenced, whether they were measured immediately or 2 h after the exercise protocol. Serum BDNF levels did not change either, but serum BDNF was negatively correlated to peripheral corticosterone concentrations, indicating a possible inhibitory reaction to the stress of running. Sixty minutes of exercise enhances dopamine release in the hippocampus of the rat in vivo. However, this increase is not associated with changes in BDNF protein levels immediately nor 2 h after the acute exercise bout. An increased corticosterone level might be the contributing factor for the absence of changes in BDNF. 相似文献
109.
Neubauer K Hühns M Hausmann T Klemke F Lockau W Kahmann U Pistorius EK Kragl U Broer I 《Journal of biotechnology》2012,158(1-2):50-58
A chimeric cyanophycin synthetase gene composed of the cphATe coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively. 相似文献
110.
Gualano B Everaert I Stegen S Artioli GG Taes Y Roschel H Achten E Otaduy MC Junior AH Harris R Derave W 《Amino acids》2012,43(1):21-24
Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle. 相似文献