54,018 research outputs found
The Escherichia coli MutL Protein Physically Interacts with MutH and Stimulates the MutH-associated Endonuclease Activity
No full textAll possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins. A two-hybrid screen previously identified a physical interaction between MutL and UvrD. Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected. A series of truncations from the NH2 and COOH termini of MutL demonstrated that the COOH-terminal 218 amino acids were sufficient for the two-hybrid interaction with MutH. Removal of a small number of residues from either the NH2 or COOH termini of MutH eliminated the two-hybrid interaction with MutL. Protein affinity chromatography experiments confirmed that MutL, but not MutS, physically associates with MutH. Furthermore, MutL greatly stimulated the d(GATC)-specific endonuclease activity of MutH in the absence of MutS and a mispaired base. Stimulation of the MutH-associated endonuclease activity by MutL was dependent on ATP binding but not ATP hydrolysis. Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP. These results suggest that MutL activates the MutH-associated endonuclease activity through a physical interaction during methyl-directed mismatch repair in Escherichia coli
Mutational Analysis of the MutH from Escherichia Coli: a Dissertation
No full textSome images did not scan well. Please see print version for images. Page 130 missing in original document.DNA mismatch repair is one process in the preservation of genomic integrity. It has been found in Archeae, bacteria, plants, yeast and mammals. The mismatch repair system is highly conserved among species and allows the strand-specific elimination of base-base mispairs, chemical base modifications, as well as short insertion/deletion loops following DNA replication. The repair system also has important effects on homeologous recombination, contributing to the frequency of reciprocal exchanges. In humans, defects in the repair system have been found to be associated with tumorigenesis. In Escherichia coli, this pathway was originally called long patch repair before being renamed the methyl-directed mismatch repair system. It is unique in that it utilizes a DNA methylation pattern to discriminate between the parental DNA strand and the newly synthesized daughter DNA strand. The current model for the initiation of methyl-directed mismatch repair is that the mispaired bases are recognized and bound by the MutS protein with MutL as a helper protein for binding. MutL also assists the MutH protein to bind, thereby forming the completed initiation complex of MutS, MutL and MutH. In the presence of ATP, there is evidence for translocation ofthe complex along the DNA forming alpha loops. At a d(GATC) site the MutH protein binds and nicks the unmethylated daughter DNA strand 5' to the d(G) (by recognizing the N6-d(A) methylation of the parental DNA strand which it is unable to cut). This completes the initiation of the repair system and allows the hydrolysis and resynthesis of the daughter DNA strand. MutH is a monomer of 25.5 kD in solution and contains a latent Mg2+-dependent endonuclease activity. Unmethylated DNA is nicked without any discrimination on one of the two strands and fully methylated DNA is resistant to cleavage by MutH even though the protein is able to bind the d(GATC) site. The structure of MutH was recently solved and compared to a group of restriction endonucleases that share a structural common core domain with similarly placed catalytic residues. The MutH protein is comprised of two major domains that are able to pivot and rotate with respect to one another. The cleft between the two domains is large enough for double-strand DNA to bind. This research started with the determination of the MutH structure before it was known. After crystallizing the protein and collecting several heavy atom data sets, it was found that the electron density maps were too discontinuous to trace the structure of the protein. Following that work, site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA binding residues (in two flexible loop regions), to determine if MutH has the same mechanism for DNA binding and catalysis as PvuII (MutH histidines 112 and 115), and to localize the residues responsible for MutH stimulation by MutL (MutH C-terminal tail region). An in-vivoscreen based on the mutator phenotype was used to select for functionally defective MutH mutants. These bacteria accumulate mutations at a greater frequency than wild-type and this was monitored by selection on plates with rifampicin. Three MutH mutants were identified from this screen (K48A, G49A, and Δ214). They were purified and assayed for total activity and binding ability. Four other mutants with wild-type phenotypic screen results were also chosen to confirm they were not involved in any MutH function (D47A, H112A, H115A, and Δ224). No DNA binding residues (such as D47A) were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. The purified D47A MutH protein showed wild-type biochemical activity. Instead, the lysine residue (K48) in the first flexible loop was found to function in catalysis together with the three presumed catalytic amino acids (Asp70, Glu77, and Lys79). This purified MutH protein (K48A) had wild-type binding ability but no endonuclease activity without MutL. In the presence of MutL, the K48A protein had only a three-fold reduction in endonuclease activity. This research has shown that MutL stimulates the wild-type MutH activity by 1000-fold. The wild-type MutH stimulation by MutL for binding was only shown to be 16-fold. The G49A MutH mutant interferes with the proper functioning of the protein but is not informative about the mechanism of action. The binding ability of this mutant was the same as wild-type and the endonuclease activity was down 30-fold with a 10-fold stimulation by MutL. The extra methyl group of the alanine may cause slight structural changes in the lysine 48 side chain that slows catalysis. The two histidines (H112 and H115) in MutH that are in a similar position as the two histidines (H84 and H85) in PvuII (that signal for DNA binding and catalysis) were changed to alanines, but had wild-type activity both in-vivo and in-vitro. These results indicate that the MutH signal for DNA binding and catalysis remains unknown. The two deletion mutations (MutHΔ224 and MutHΔ214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala220, Leu221, Leu222, Ala223, and Arg224). Mutant MutHΔ224 had wild-type MutL stimulation activity, while MutHΔ214 showed no MutL stimulation. Another deletion mutant, MutHΔ119, from another laboratory was shown to have wild-type MutL stimulation also. This leaves one (or more) of the remaining five residues as important for MutL stimulation.Biochemistry and Molecular Pharmacolog
Synthesis of a group of novel Xanomeline/77-LH-28-1 hybrid ligands and their FRET investigation at muscarinic acetylcholine receptor subtypes
No full textIn connection with our interest in investigating novel rationally designed bitopic (i.e., orthosteric/allosteric) derivatives targeting muscarinic acetylcholine receptor (mAChR) subtypes (1,2,3), in this study we designed and synthesized a new set of ligands that integrate in the same molecular skeleton the pharmacophoric moieties of Xanomeline and of 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone). Xanomeline is a well-known M1/M4-preferring orthosteric agonist, which ameliorated cognitive impairments in Alzheimer’s disease patients and showed activity in various models of schizophrenia, thus being potentially beneficial for treatment of positive, negative and cognitive symptoms (4). On the other hand, 77-LH-28-1 was characterized as an M1-selective, positive allosteric modulator, thus representing an interesting pharmacological tool with cognition enhancing properties (5). As illustrated below, we planned the novel bipharmacophoric derivatives as merged structures, with the tetrahydropyridine nucleus of Xanomeline as the central core.
In the last years, different receptor sensors, based on the fluorescence resonance energy transfer (FRET), were generated for various G protein-coupled receptors, and represented a valuable tool to investigate real time receptor activation as well as ligand-receptor interactions. Recently, this analysis was performed also on a set of bitopic ligands designed for a selective interaction with M1 mAChRs (6). Our preliminary results on the group of Xanomeline/77-LH-28-1 hybrid compounds indicate, for the M1 sensor, a reproducible activation response, which depends on the linker length. Conversely, no FRET-related effect could be detected at the M2 sensor. Thus, a critical spacer length of the hybrid compounds induces conformational changes with a degree of selectively for the M1 muscarinic receptor. The synthesis and the results of pharmacological investigation will be presented and discussed.
References: 1. J. Antony, K. Kellershohn, M. Mohr-Andrä, A. Kebig, S. Prilla, M. Muth, E. Heller, T. Disingrini, C. Dallanoce et al., FASEB J 2009, 23, 442-450. 2. A. Bock, B. Chirinda, F. Krebs, R. Messerer, J. Bätz, M. Muth, C. Dallanoce et al., Nat. Chem. Biol. 2014, 10, 18-20. 3. A. Bock, M. Bermudez, F. Krebs, C. Matera, B. Chirinda, D. Sydow, C. Dallanoce et al., J. Biol. Chem. 2016, 291, 16375-16389. 4. S. Barak, I. Weiner, Int. J. Neuropsychoph. 2011, 14, 1233-1246. 5. C. J. Langmead, N. E. Austin, C. L. Branch, J. T. Brown, K. A. Buchanan, C. H. Davies, I. T. Forbes et al., Br. J. Pharmacol. 2008, 154, 1104-1115. 6. R. Messerer, M. Kauk, D. Volpato, M. C. Alonso Canizal, J. Klöckner, U. Zabel, S. Nuber, C. Hoffmann, U. Holzgrabe, ACS Chem. Biol. 2017, 12, 833-843
"Closing the R&D Gap, Evaluating the Sources of R&D Spending"
No full textBoth spending and tax policies have been implemented in the United States with the goal of stimulating private sector research and development (R&D). Karier questions whether current R&D policy, especially the research and experimentation tax credit, can contribute to closing the gap between nondefense expenditures on R&D in the United States and such expenditures in other countries, such as Japan and Germany. He also explores possible changes to our current R&D policy to make it more effective.
Urban Structure and Housing Prices: Some Evidence from Australian Cities
No full textThis paper studies determinants of some aspects of the structure of cities, including density and the price of land and housing. We use a version of the Alonso-Muth-Mills model, calibrated to broadly match some of the features of a representative large city. While the calibrated model omits many real-world features, it can nonetheless be used to explore the impact of factors such as: (i) the provision of transport infrastructure; (ii) zoning policies that limit housing density; (iii) frictions on the production of housing; and (iv) population size. The empirical section of the paper shows that the model is consistent with some empirical regularities for large Australian cities. The results of the paper draw attention to structural factors that may have contributed to developments in the Australian housing market in recent years.housing; housing prices; land prices; zoning; land use
Letter from C. D. Dawson, Tusayan Copper Mining and Smelting, to Carl Hayden
No full textLetter from C. D. Dawson to Carl Hayden urging him to consider the rights of miners and farmers when drawing up the boundaries for the proposed park
Measurement of the D+/- production asymmetry in 7 TeV pp collisions
No full textThe asymmetry in the production cross-section \sigma of D+/- mesons, A_P = (\sigma(D+) - \sigma(D-))/(\sigma(D+) + \sigma(D-)), is measured in bins of pseudorapidity \eta and transverse momentum p_T within the acceptance of the LHCb detector. The result is obtained with a sample of D+ -> K_S pi+ decays corresponding to an integrated luminosity of 1.0 fb^-1, collected in pp collisions at a centre of mass energy of 7 TeV at the Large Hadron Collider. When integrated over the kinematic range 2.0 K_S pi+ decay is negligible. No significant dependence on \eta or p_T is observed
D* (D)over-bar* molecule interpretation of Z(c)(4025)
No full textWe have used QCD sum rules to study the newly observed charged state Z(c)(4025) as a hidden-charm D*(D) over bar* molecular state with the quantum numbers I-G(J(P)) =1(+)(1(+)). Using a D*(D) over bar* molecular interpolating current, we have calculated the two-point correlation function and the spectral density up to dimension eight at leading order in alpha(s). The extracted mass is m(X) = (4.04 +/- 0.24) GeV. This result is compatible with the observed mass of Z(c)(4025) within the errors, which implies a possible molecule interpretation of this new resonance. We also predict the mass of the corresponding hidden-bottom B*(B) over bar* molecular state: m(Zb) = (9.98 +/- 0.21) GeV.Physics, Particles & FieldsSCI(E)[email protected]; [email protected]; [email protected]; [email protected]
Prompt charm production in pp collisions at √<span style="text-decoration:overline">s</span>=7 TeV
No full textCharm production at the LHC in pp collisions at s√=7 TeV is studied with the LHCb detector. The decays D0→K−π+, D+→K−π+π+, D⁎+→D0(K−π+)π+, D+s→ϕ(K−K+)π+, Λ+c→pK−π+, and their charge conjugates are analysed in a data set corresponding to an integrated luminosity of 15 nb−1. Differential cross-sections dσ/dpT are measured for prompt production of the five charmed hadron species in bins of transverse momentum and rapidity in the region 0<pT<8 GeV/c and 2.0<y<4.5. Theoretical predictions are compared to the measured differential cross-sections. The integrated cross-sections of the charm hadrons are computed in the above pT-y range, and their ratios are reported. A combination of the five integrated cross-section measurements gives
σ(cc¯)pT<8 GeV/c,2.0<y<4.5=1419±12(stat)±116(syst)±65(frag) μb,
where the uncertainties are statistical, systematic, and due to the fragmentation functions
Z(c)(3900) as a (D)over-barD* molecule from the pole counting rule
No full textA comprehensive study on the nature of the Zc(3900) resonant structure is carried out in this work. By constructing the pertinent effective Lagrangians and considering the important final-state-interaction effects, we first give a unified description to all the relevant experimental data available, including the J/psi pi and pi invariant mass distributions from the e(+)e(-) -> J/psi pi process, the h(c)pi distribution from e(+)e(-) -> h(c)pi pi, and also the D (D) over bar* spectrum in the e(+)e(-) -> D (D) over bar*pi process. After fitting the unknown parameters to the previous data, we search the pole in the complex energy plane and find only one pole in the nearby energy region in different Riemann sheets. Therefore, we conclude that Z(c)(3900) is of D (D) over bar* molecular nature, according to the pole counting rule method [Nucl. Phys. A543, 632 (1992); Phys. Rev. D 35, 1633 (1987)]. We emphasize that the conclusion based upon the pole counting method is not trivial, since both the D (D) over bar* contact interactions and the explicit Z(c) exchanges are introduced in our analyses andNational Nature Science Foundations of China (NSFC) [10925522, 11021092, 11575052, 11105038]; Natural Science Foundation of Hebei Province [A2015205205]; inoGerman Collaborative Research Center "Symmetries and the Emergence of Structure in QCD" [CRC 110]; DFG; NSFCSCI(E)ARTICLE119
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