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871.
2,7‐DICHLOROFLUORESCIN OXIDATION AND REACTIVE OXYGEN SPECIES: WHAT DOES IT MEASURE? 总被引:10,自引:0,他引:10
Intracellular 2,7-dichlorofluorescin (H(2)DCF) oxidation is often used to measure the production of reactive oxygen species (ROS) within cells. The rate of H(2)DCF oxidation depends on the concentration of glutathione, which is an alternative target for ROS. Our results suggest that increased rate of H(2)DCF oxidation be interpreted as an indication of general oxidative stress due to a variety of reasons, including depletion of antioxidants, rather than as a specific proof of augmented ROS formation. 相似文献
872.
《Chronobiology international》2012,29(12):1752-1760
ABSTRACTWe compared performance of four popular interpretative algorithms (IAs), i.e., Cole–Kripke, Rescored Cole–Kripke, Sadeh, and UCSD, utilized to derive sleep parameters from wrist actigraphy data. We conducted in-home sleep study of 40 healthy adults (17 female/23 male; age 26.7 ± 12.1 years), assessing sleep variables both by Motionlogger® Micro Watch Actigraphy (MMWA) and Zmachine® Insight+ electroencephalography (EEG). Data of MMWA were separately scored per 30 sec epochs by each of the four popular IAs, and data of the Zmachine were also scored per 30 sec epochs by its proprietary IA. In reference to the EEG Zmachine method, all four of the MMWA algorithms showed high (~94 to 98%) sensitivity and moderate (~42 to 54%) specificity in detecting Sleep epochs. All of them significantly underestimated Sleep Onset Latency (SOL: ~9 to 20 min), and all of them, except the Sadeh IA, significantly underestimated Wake After Sleep Onset (WASO: ~22 to 25 min) and overestimated Total Sleep Time (TST: ~32 to 45 min) and Sleep Efficiency (SE: ~7 to 9%). The Sadeh IA showed significantly smaller bias than the other three IAs in deriving WASO, TST, and SE. Overall, application of ‘Rescoring Rules’ improved performance of the Cole–Kripke IA. The Sadeh and Rescored Cole–Kripke IAs exhibited highest agreement with the EEG Zmachine method (Cohen’s Kappa: ~51%), while the UCSD IA exhibited lowest agreement (Cohen’s kappa: ~47%). However, minimum detectable change across all sleep parameters was smallest with use of the UCSD IA and, except for SOL, largest with use of the Sadeh algorithm. Findings of this study indicate the Sadeh IA is most appropriate for deriving sleep parameters of healthy adults, while the UCSD IA is most appropriate for evaluating change in sleep parameters over time or in response to medical intervention. 相似文献
873.
The ST Pinch is a 12-membered hydrogen-bonded motif (Ser/Thr-Xaa-Ser/Thr) involving the side chain oxygen atoms of two Ser/Thr residues. We identified the ST Pinch in 104 proteins in a database containing high-resolution crystal structures. Conformational analysis of the ST Pinch in these proteins points to specific preferences for the Xaa residue and a high propensity of this residue to adopt positive φ angles. Our results suggest that this motif serves as a linker of secondary structural elements within proteins and is a new addition to the existing list of short hydrogen bond-stabilized motifs in proteins. 相似文献
874.
Protein-ligand docking is widely applied to structure-based virtual screening for drug discovery. This article presents a novel docking technique, PRL-Dock, based on hydrogen bond matching and probabilistic relaxation labeling. It deals with multiple hydrogen bonds and can match many acceptors and donors simultaneously. In the matching process, the initial probability of matching an acceptor with a donor is estimated by an efficient scoring function and the compatibility coefficients are assigned according to the coexisting condition of two hydrogen bonds. After hydrogen bond matching, the geometric complementarity of the interacting donor and acceptor sites is taken into account for displacement of the ligand. It is reduced to an optimization problem to calculate the optimal translation and rotation matrixes that minimize the root mean square deviation between two sets of points, which can be solved using the Kabsch algorithm. In addition to the van der Waals interaction, the contribution of intermolecular hydrogen bonds in a complex is included in the scoring function to evaluate the docking quality. A modified Lennard-Jones 12-6 dispersion-repulsion term is used to estimate the van der Waals interaction to make the scoring function fairly "soft" so that ligands are not heavily penalized for small errors in the binding geometry. The calculation of this scoring function is very convenient. The evaluation is carried out on 278 rigid complexes and 93 flexible ones where there is at least one intermolecular hydrogen bond. The experiment results of docking accuracy and prediction of binding affinity demonstrate that the proposed method is highly effective. 相似文献
875.
Short range side chain‐backbone hydrogen bonded motifs involving Asn and Gln residues have been identified from a data set of 1370 protein crystal structures (resolution ≤ 1.5 Å). Hydrogen bonds involving residues i ? 5 to i + 5 have been considered. Out of 12,901 Asn residues, 3403 residues (26.4%) participate in such interactions, while out of 10,934 Gln residues, 1780 Gln residues (16.3%) are involved in these motifs. Hydrogen bonded ring sizes (Cn, where n is the number of atoms involved), directionality and internal torsion angles are used to classify motifs. The occurrence of the various motifs in the contexts of protein structure is illustrated. Distinct differences are established between the nature of motifs formed by Asn and Gln residues. For Asn, the most highly populated motifs are the C10 (COδi …NHi + 2), C13 (COδi …NHi + 3) and C17 (NδHi …COi ? 4) structures. In contrast, Gln predominantly forms C16 (COεi …NHi ? 3), C12 (NεHi …COi ? 2), C15 (NεHi …COi ? 3) and C18 (NεHi …COi ? 4) motifs, with only the C18motif being analogous to the Asn C17structure. Specific conformational types are established for the Asn containing motifs, which mimic backbone β‐turns and α‐turns. Histidine residues are shown to serve as a mimic for Asn residues in side chain‐backbone hydrogen bonded ring motifs. Illustrative examples from protein structures are considered. Proteins 2012; © 2011 Wiley Periodicals, Inc. 相似文献
876.
Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field 总被引:1,自引:0,他引:1
Ab initio protein folding is one of the major unsolved problems in computational biology owing to the difficulties in force field design and conformational search. We developed a novel program, QUARK, for template-free protein structure prediction. Query sequences are first broken into fragments of 1-20 residues where multiple fragment structures are retrieved at each position from unrelated experimental structures. Full-length structure models are then assembled from fragments using replica-exchange Monte Carlo simulations, which are guided by a composite knowledge-based force field. A number of novel energy terms and Monte Carlo movements are introduced and the particular contributions to enhancing the efficiency of both force field and search engine are analyzed in detail. QUARK prediction procedure is depicted and tested on the structure modeling of 145 nonhomologous proteins. Although no global templates are used and all fragments from experimental structures with template modeling score >0.5 are excluded, QUARK can successfully construct 3D models of correct folds in one-third cases of short proteins up to 100 residues. In the ninth community-wide Critical Assessment of protein Structure Prediction experiment, QUARK server outperformed the second and third best servers by 18 and 47% based on the cumulative Z-score of global distance test-total scores in the FM category. Although ab initio protein folding remains a significant challenge, these data demonstrate new progress toward the solution of the most important problem in the field. 相似文献
877.
The hexapeptide Ser-Gly-Ala-Gly-Lys-Thr has been synthesized and characterized. It was designed as a minimal soluble peptide that would be likely to have the phosphate-binding properties observed in the P-loops of proteins that bind the β-phosphate of GTP or ATP. The β-phosphate in such proteins is bound by a combination of the side chain ε-amino group of the lysine residue plus the concavity formed by successive main chain peptide NH groups called a nest, which is favored by the glycines. The hexapeptide is shown to bind HPO(4) (2-) strongly at neutral pH. The affinities of the various ionized species of phosphate and hexapeptide are analyzed, showing that they increase with pH. It is likely the main chain NH groups of the hexapeptide bind phosphate in much the same way as the corresponding P-loop atoms bind the phosphate ligand in proteins. Most proteinaceous P-loops are situated at the N-termini of α-helices, and this observation has frequently been considered a key aspect of these binding sites. Such a hexapeptide in isolation seems unlikely to form an α-helix, an expectation in accord with the CD spectra examined; this suggests that being at the N-terminus of an α-helix is not essential for phosphate binding. An unexpected finding about the hexapeptide-HPO(4) (2-) complex is that the side chain ε-amino group of the lysine occurs in its deprotonated form, which appears to bind HPO(4) (2-) via an N···H-O-P hydrogen bond. 相似文献
878.
Fredy D.A. Silva Ilka M. Vasconcelos Marina D.P. Lobo Patrícia G. de Castro Vladimir G. Magalhães Cléverson D.T. de Freitas Célia R.R.S. Carlini Paulo M. Pinto Leila M. Beltramini José H.A. Filho Eduardo B. Barros Luciana M.R. Alencar Thalles B. Grangeiro José T.A. Oliveira 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).Methods
Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.Results
Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx.General significance
The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. 相似文献879.
Jitender Singh Guda Maheedhara ReddyAakrati Agarwal Kottakota ChandrasekharS.K. Sopory M.K. ReddyTanushri Kaul 《Gene》2012
Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO2 into organic acids for subsequent C4 or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC4PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers, which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation. 相似文献
880.
K Trchounian 《Gene》2012,506(1):156-160
Escherichia coli molecular hydrogen (H(2)) production was studied during mixed carbon (glucose and glycerol) fermentation at pH 6.5. Wild type cells in the assays supplemented with glucose produced H(2) at ~2 fold lower level than cells grown on glucose only. When compared to the wild type, H(2) production in the assays added with glucose was decreased by ~2 fold in fhlA, hyfG and double fhlA hyfG mutants and by ~1.5 fold in hyaB, hybC, and double hyaB hybC mutants. However, in the assays with glycerol, no measurable H(2) production was detected. Taken together, these results suggest that during mixed carbon fermentation, H(2) could be produced with low efficiency via Hyd-3 and Hyd-4. This is a novel finding for Hyd-4 activity at pH 6.5. The insignificant decrease of H(2) production in the strains with defects in Hyd-1 and Hyd-2 was probably due to an interaction between the Hyd enzymes and their organization in the bacterial membrane. In the glucose assays, H(2) production in the wild type cells was inhibited ~2 fold by 0.3mM N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase. This inhibition was the same for fhlA and hyfG fhlA mutants but not hyaB, hybC, hyfG or hyaB hybC mutants. The results indicate that the FhlA protein coded by the fhlA gene might interact with the F(0)F(1)-ATPase. We propose that this interaction is mediated by mixed carbon fermentation. 相似文献