10,586 research outputs found
Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting
No full textWe have Investigated the kinetics of dissociation of actlnomycin D from DNA by a variation of the foot-printing technique. Complexes of actlnomycin with a radiolabelied DNA fragment (tyrT) were dissociated by addition of a large excess of uniabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37°C, 20°C and 4°C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20°C. In contrast, the dissociation of distamycln was too fast to measure (<5 s) even at 4°C.</p
Dissociation kinetics of echinomycin from CpG binding sites in different sequence environments
No full textWe have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 °C, echinomycin dissociates more slowly from CpG sites flanked by (AT)(n) (t( 1/2 ) ~ .40 min) and (CA)n) · (TG)(n) (t( 1/2 ) ~ 11 min) than by A(n) · T(n) (t( 1/2 ) < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)4CG(TTA)4 also represents a very good binding site (t 1/2 ) ~ 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)10(G/C)4(AT)10, the dissociation from CGGC is slower than that from CCCG or CCGC.</p
Visualising the dissociation of sequence selective ligands from individual binding sites on DNA
No full textAbstractWe have used a modification of the footprinting technique to measure the dissociation of mithramycin, echinomycin and nogalamycin from their binding sites in a natural DNA fragment. Complexes with radiolabelled DNA were dissociated by addition of unlabelled DNA. Samples were removed at various times and subjected to DNase I digestion, and the rate of dissociation from each site was estimated from the time-dependent disappearance of the footprints. For echinomycin the slowest rate of dissociation is from ACGT, while the slowest site for mithramycin contains four contiguous guanines. The dissociation of nogalamycin is extremely slow, even from its weaker sites; the slowest rate was from ACGTA, which took longer than 4 h, even at 37°C
Dissociation kinetics of actinomycin D from individual GpC sites in DNA
No full textWe have examined the kinetics of dissociation of actinomycin from GpC sites in several DNA fragments containing synthetic DNA inserts, by a variation of the footprinting technique. Complexes of the ligand with radiolabelled DNA fragments were dissociated by adding a large excess of unlabelled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the GpC sites located in different sequence environments. Actinomycin dissociates more slowly from GpC sites flanked by (AT)n than An · Tn. Within regions of alternating AT, TGCA represents a better binding site than AGCT, and CGCA is a better binding site than GGCA. GpC sites flanked by (AC)n · (GT)n present good binding sites; in this context, dissociation from CGCG is faster than from TGCA.</p
On the Michael addition of water to C = C bonds
No full text?-Hydroxy carbonyl compounds are an important class of compounds often found as a common structural motif in natural products. Although the molecules themselves look rather simple, their synthesis can be challenging. Water addition to conjugated C = C bonds opens up a straightforward route for the preparation of ?-hydroxy carbonyl compounds. Moreover, water addition to C = C bonds benefits a lot from its simplicity and excellent atom economy. However, the enantioselective addition of water to ?,?-unsaturated carbonyl (Michael) acceptors still represents a chemically very challenging reaction, due to the poor nucleophilicity of water and its small size, which make regio- and stereoinduction difficult. Equally, the often unfavorable equilibrium of water-addition reactions remains to be solved. In contrast, enzymes such as fumarase, malease, citraconase, aconitase, and enoyl-CoA hydratase have been successfully used on industrial scale, and their excellent (enantio-) selectivities are highly valued. Unfortunately, most hydratases are part of the primary metabolism where perfect substrate specificity is required. This very high substrate selectivity severely limits their practical applicability in organic synthesis. Thus, a straightforward approach with broad applicability still had not been described. The aim of the research presented in this thesis was to take up this challenge and dedicated to the search for a Michael hydratase with a more relaxed substrate specificity for the preparation of important ?-hydroxy carbonyl compounds. The stereospecificity of enzyme-catalysed reactions has been a fruitful source of information about the mechanisms of enzyme catalysis and vice versa; the application of stereospecifically labelled substrates allows for studying the course of the reaction. It offers a very promising opportunity to comprehensively understand the precise mechanistic and kinetic details of even the most complex enzymatic reactions. Thus Chapter 1 provides unifying ideas for stereochemistry of the enzymatic water addition to C = C bonds. This enhances our understanding of the chemistry of water addition to C = C bonds, and further allows us to find more hydratases from natural sources or obtained via protein engineering. In Chapter 2, a direct, enantioselective Michael addition of water in water to prepare important ?-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. This opens up an entirely new approach for the preparation of important ?-hydroxy carbonyl compounds. Deuterium labelling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry, which belongs to the family members of hydratases: oleate hydratase, fumarase, malease, aconitase and type II dehydroquinase with a preference for the anti-addition; whereas, type I dehydroquinase, enoyl-CoA hydratase and artificial hydratase exclusive prefer for the syn-addition, as discussed in Chapter 1. The biocatalytic reaction system was carefully optimized for gram-scale synthesis, resulting in good conversions and excellent enantioselectivities. Under the optimized conditions, whole cells could be reused for 4 cycles without significant loss of activity while maintaining up to 90% ee. Since whole cells from Rhodococcus strains were used to catalyse the Michael addition of water in water to a series of ?,?-unsaturated carbonyl compounds, and when the work presented in Chapter 2 started, no genomic information of Rhodococcus strains was publically available, we sequenced and annotated the strain R. rhodochrous ATCC 17895. This is described in Chapter 3 together with features of the R. rhodochrous ATCC 17895. It is a Gram-positive aerobic bacterium with a rod-like morphology. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Our study suggests the Michael hydratase has not been described before. In the work presented in Chapter 2, we found that most ?-hydroxy ketones are not commercially available or commercially expensive as we mentioned in the first paragraph, which made the stereoselectivity determination of Michael addition products difficult. Indeed, many seemingly simple molecules have to be prepared via multi-step syntheses, in particular so if they are optically active. Therefore a straightforward approach to enantiomerically enriched (R)- and (S)-3-hydroxycyclopentanone was established by kinetic resolution in Chapter 4. This methodology allows us to prepare more ?-hydroxy carbonyl compounds structurally closely related to 3-hydroxycyclopentanone. The isolated chiral alcohols were used to determine the stereochemistry of the Michael addition of water in Chapter 2, saving us a lot of laboratory work. Moreover, unexpected stereoselective reduction of conjugated C = C bonds was discovered during studies on the enantioselective Michael addition of water. As mentioned in Chapter 2, the whole cells of R. rhodochrous ATCC 17895 reduced ?,?-unsaturated cyclic ketones into the corresponding ketones as initially undesired side reaction for the addition of water to C = C bonds. Therefore, ene-reductase activity was also investigated in Chapter 5. A series of substrates, including activated ketones, aldehydes, amines and nitro-compounds were screened for ene-reductase activity using whole cells of R. rhodochrous ATCC 17895. This showed that R. rhodochrous is a very promising catalyst for the reduction of C = C bonds and harbours ene-reductases. Indeed, looking for the annotated ene reductase from the genome of R. rhodochrous ATCC 17895 as described in Chapter 3, three candidates were observed and were classified as ene-reductases by amino acid sequence alignment with the known Old Yellow Enzymes (OYEs). Thus, the putative ene-reductase genes from R. rhodochrous ATCC 17895 were heterologously overexpressed in Escherichia coli and one of the encoded proteins was purified and characterized for their biocatalytic and biochemical properties. Based on these accomplishments it can be concluded that we have discovered a new Michael hydratase and three new ene reductases from Rhodococcus strains. Genome sequence and annotation of strain R. rhodochrous ATCC 17895 has been done, offering an excellent opportunity for the discovering novel enzymes, for instance, the Michael hydratase and S-selective ene reductase. The important chiral ?-hydroxy carbonyl compounds can be prepared by kinetic resolution of racemic alcohols using lipases or the direct enantioselective Michael addition of water using whole cells of Rhodococcus strains. The isolated products from kinetic resolution were readily used for the stereochemistry determination of Michael addition of water in water, completes the story of water addition to C = C bonds.BiotechnologyApplied Science
Delocalization by disorder in GaAs/AlGaAs heterostructures
No full textAn experimental study of quasiparticle and quasi-one dimensional properties of strongly correlated two-dimensional (2D) electron systems has been carried out. The samples were low disordered
GaAs/AlGaAs heterostructures. Measurements were performed at low temperatures, down to 24 mK, in a dilution refrigerator (MCK 50-100 TOF) equipped with a superconducting magnet.
The goal of our work was to study quasiparticle properties of 2DEG and to investigate the effect of local disorder on the conductance of the sample.
We have performed independent measurements of effective electron mass m*, transport and quantum scattering time at three different temperatures and effective g-factor. We used Shubnokov de Haas
effect , the oscillations in the longitudinal resistance in Hall effect to study the effective mass, scattering lifetimes and effective g factor. We found out that effective mass is unaffected and agrees well with
the typical value of GaAs/AlGaAs system i.e. 0.067 me, where me is the electron mass. The quantum scattering time was studied at three different temperatures, base temperature (24mK), 200 mK and
400 mK as function of electron density. We found out that the quantum scattering time at 24 mK is independent of electron density while at higher temperatures it decreases with decrease in density.
The ratio of two lifetimes τ_t and τ_q is more than 10 for all temperatures. It means that remote Coulomb centres play a dominating role in the scattering mechanisms of our sample.
More insight is needed to study the quantum scattering time at higher temperatures. The effective g-factor experiments were done with and without in-plane magnetic field. In both cases, the g-factor shows
dependence on the magnetic field.
The main purpose of this thesis work is to investigate possible breakdown of the Anderson localization in presence of local disorder. To implement the local disorder and create delocalization
we have used fine surface gates which tuned the potential barriers in the 2DEG. This was done in two different types of samples. In one sample the finger gates and top surface gate are isolated by
an insulating layer of SiO2 and in the other sample, the two gates are intercalated. The spacing in between the finger gates is determined by the mean free path of the system. The experiments are done in
absence of magnetic field. We found out that the sample with SiO2shows the effect of disorder with decrease in the resistance. But due to charge trapped in the SiO2layer, the effect was not repeated.
In the intercalated samples with two different finger gate spacings, the effect was not visible. The delocalization was not set in these sample even at high temperatures. More study is needed to prove this
effect in GaAs/AlGaAs heterostructures. A low mobility wafer can be considered as the future candidate for this experiment
Clinical pharmacology in the UK, c. 1950–2000: industry and regulation
No full textClinical pharmacology in the UK in the 1950s and 1960s was an exciting profession. Many important new drugs were developed and brought to market and a more systematic knowledge of drug effects in humans was needed, as well as ensuring the safety and efficacy of new and existing drugs, especially following the unexpected problems arising from the use of thalidomide. This Witness Seminar followed an earlier meeting on the history of the general development of clinical pharmacology and focuses on the development of clinical pharmacology in pharmaceutical companies and drug regulation. Professor Rod Flower chaired the meeting of clinical pharmacologists and others who shaped the discipline, which discussed the main centres of influence; the attraction to clinical pharmacologists of working in industry; whether the decline in the number of academic clinical pharmacologists was paralleled in drug companies; what drove drug regulation; and the relationships between companies and regulatory authorities.
Participants included Dr Jeffrey Aronson, Professor Nigel Baber, Sir Alasdair Breckenridge, Sir Iain Chalmers, Professor Joe Collier, Professor Donald Davies, Dr Peter Fletcher, Dr Arthur Fowle, Professor Sir Charles George, Professor David Grahame-Smith, Professor John Griffin, Dr Andrew Herxheimer, Professor Ray Hill, Dr Peter Lewis, Dr Tim Mant, Professor Denis McDevitt, Professor Michael Orme, Dr Anthony Peck, Professor Brian Prichard, Professor Sir Michael Rawlins, Professor John Reid, Professor Philip Routledge, Dr Julian Shelley, Dr Robert Smith, Professor Cameron Swift, Professor Tilli Tansey, Dr Duncan Vere, and the late Professor Owen Wade
Church’s Thesis, Turing’s Limits, and Deutsch’s Principle
No full textIn the search for a precise definition of the notion of effective calculability, Turing’s computability has proven to be more convincing than other definitions proposed almost simultaneously. Turing does not define a particular class of “calculable functions”, but which processes can be carried out in computing. His conceptual analysis brings about an intimate and inescapable link between computability and “measurability”, hence between mechanical procedures and physical processes. The full significance of this link is captured in Deutsch’s physical version of the Church-Turing thesis, namely, his claim that every physical system can be simulated by a universal quantum Turing machine. Quantum physics supports more efficient forms of computation, even though quantum computation remains within Turing’s limits. Why? This chapter explores how logical principles and physical processes coalesce into the notion of computation
Michael Connelly Interview: 9/11, City of Bones, and Lost Light
No full textAn interview with American crime author Michael Connelly, exploring the role of crime narratives as outlets for critical engagement with the politics of the war on terror and the associated neoconservative narrativising of 9/11. The discussion principally delves into Connelly’s first two post 9/11 Harry Bosch novels; City of Bones (2002) and Lost Light (2003)
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