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We report on systematic measurements of the electrical resistance of one- and three-dimensional (1D and 3D) metallic and oxidized granular materials under uni-axial compression. Whatever the dimension of the packing, the resistance follows a power law versus the pressure ([Formula: see text]), with an exponent [Formula: see text] much larger than the ones expected either with elastic or plastic contact between the grains. A simple model based on a statistical description of the micro-contacts between two grains is proposed. It shows that the strong dependence of the resistance on the pressure applied to the granular media is a consequence of large variabilities and heterogeneities present at the contact surface between two grains. Then, the effect of the three-dimensional structure of the packing is investigated using a renormalization process. This allows to reconcile two extreme approaches of a 3D lattice of widely distributed resistances: the effective medium and the percolation theories.
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The Superfluid High REynolds von Kármán experiment facility exploits the capacities of a high cooling power refrigerator (400 W at 1.8 K) for a large dimension von Kármán flow (inner diameter 0.78 m), which can work with gaseous or subcooled liquid (He-I or He-II) from room temperature down to 1.6 K. The flow is produced between two counter-rotating or co-rotating disks. The large size of the experiment allows exploration of ultra high Reynolds numbers based on Taylor microscale and rms velocity [S. B. Pope, Turbulent Flows (Cambridge University Press, 2000)] (Rλ > 10000) or resolution of the dissipative scale for lower Re. This article presents the design and first performance of this apparatus. Measurements carried out in the first runs of the facility address the global flow behavior: calorimetric measurement of the dissipation, torque and velocity measurements on the two turbines. Moreover first local measurements (micro-Pitot, hot wire, ) have been installed and are presented.
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Heat transfer measurements performed by Riedinger et al. (Phys. Fluids, 25, 015117 (2013)) showed that in an inclined channel, heated from below and cooled from above with adiabatic walls, the flow is laminar or intermittent (local bursts can occur in the laminar flow) when the inclination angle is sufficiently high and the applied power sufficiently low. In this case, gravity plays a crucial role in the characteristics of the flow. In this paper, we present velocity measurements, and their derived tensors, obtained with Particle Image Velocimetry inside the channel. We, also, propose a model derived from a jet interpretation of the flow. Comparison between experiment and model shows a fair agreement.
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We present a collection of eight data sets from state-of-the-art experiments and numerical simulations on turbulent velocity statistics along particle trajectories obtained in different flows with Reynolds numbers in the range R{lambda}in[120:740]. Lagrangian structure functions from all data sets are found to collapse onto each other on a wide range of time lags, pointing towards the existence of a universal behavior, within present statistical convergence, and calling for a unified theoretical description. Parisi-Frisch multifractal theory, suitably extended to the dissipative scales and to the Lagrangian domain, is found to capture the intermittency of velocity statistics over the whole three decades of temporal scales investigated here.
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We report on measurements of the electrical conductivity in both a 2D triangular lattice of metallic beads and in a chain of beads. The voltage/current characteristics are qualitatively similar in both experiments. At low applied current, the voltage is found to increase logarithmically in good agreement with a model of widely distributed resistances in series. At high enough current, the voltage saturates due to the local welding of microcontacts between beads. The frequency dependence of the saturation voltage gives an estimate of the size of these welded microcontacts. The DC value of the saturation voltage ( approximately 0.4 V per contact) gives an indirect measure of the number of welded contact carrying the current within the 2D lattice. Also, a new measurement technique provides a map of the current paths within the 2D lattice of beads. For an isotropic compression of the 2D granular medium, the current paths are localized in few discrete linear paths. This quasi-one-dimensional nature of the electrical conductivity thus explains the similarity between the characteristics in the 1D and 2D systems.
Assuntos
Coloides/química , Metais/química , Modelos Químicos , Simulação por Computador , Condutividade Elétrica , Conformação Molecular , PorosidadeRESUMO
We have developed a small, neutrally buoyant, wireless temperature sensor. Using a camera for optical tracking, we obtain simultaneous measurements of position and temperature of the sensor as it is carried along by the flow in Rayleigh-Bénard convection, at Ra approximately 10;{10}. We report on statistics of temperature, velocity, and heat transport in turbulent thermal convection. The motion of the sensor particle exhibits dynamics close to that of Lagrangian tracers in hydrodynamic turbulence. We also quantify heat transport in plumes, revealing self-similarity and extreme variations from plume to plume.
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It is argued that the method used by Cleve et al. [Phys. Rev. E 69, 066316 (2004)], to determine the intermittency exponent mu is biased at moderate Reynolds numbers R lambda. Thus, the claimed dependence of mu upon (R lambda) is questionable. Other determinations give a constant value for mu.
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We measure the relation between convective heat flux and temperature gradient in a vertical channel filled with water, the average vertical mass flux being zero. Compared to the classical Rayleigh-Bénard case, this situation has the advantage of avoiding plates and, thus, their neighborhood, in which is usually concentrated most of the temperature gradient. Consequently, inertial processes should control the convection, with poor influence of the viscosity. This idea gives a good account of our observations, if we consider that a natural vertical length, different from the channel width, appears. Our results also suggest that heat fluxes can be deduced from velocity measurements in free convective flows. This confers to our results a wide range of applications.
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The 1/2 power law is reported in a Rayleigh-Bénard experiment: Nu approximately Ra(1/2), where Ra and Nu are the Rayleigh and Nusselt numbers. This observation is coherent with the predictions of the ultimate convection regime, characterized by fully turbulent heat transfers. Ordered rough boundaries are used to cancel the correction due to the thickness variation of the viscous sublayer, and the observation of the asymptotic regime is therefore possible. This result supports the interpretation of a laminar-turbulent boundary-layer transition to account for the observation of Chavanne et al. of a new regime [X. Chavanne et al., Phys. Rev. Lett. 79, 3648 (1997)].
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The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1. Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired with a cytosine, [8-OxoG.C], are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1) nM(-)(1) for the wild-type and the K241R protein, respectively. On the other hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities of yOgg1. In contrast, the K241R and K241Q proteins have conserved wild-type DNA binding properties. K(dapp) values for binding of [8-OxoG.C] are 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respectively. The results also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins. Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand. On the complementary strand, only the cytosine placed opposite Pr was strongly protected. The same footprints were observed with the K241R and K241Q proteins, confirming their wild-type DNA binding properties. These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizable substrates such as 8-OxoG or an AP site.
Assuntos
Arginina/genética , Proteínas de Ligação a DNA/química , Glutamina/genética , Lisina/genética , N-Glicosil Hidrolases/química , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação/efeitos dos fármacos , Catálise , Ciclopentanos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , DNA-Formamidopirimidina Glicosilase , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Furanos/farmacologia , Guanosina/análogos & derivados , Guanosina/metabolismo , Hidrólise , Radical Hidroxila/análise , Radical Hidroxila/metabolismo , Cinética , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Propilenoglicóis/farmacologia , Purinas/metabolismo , Pirimidinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
The homodimeric form alpha(2) of the Escherichia coli DNA-binding protein HU was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals belong to space group I222, with unit-cell parameters a = 31.09, b = 55.34, c = 117. 63 A, and contain one monomer per asymmetric unit. A full diffraction data set was collected to 2.3 A resolution on a conventional X-ray source. The molecular-replacement method, using the HU crystallographic model from Bacillus stearothermophilus as a starting point, gave a reliable solution for the rotation and translation functions.
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Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Cristalização , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Geobacillus stearothermophilus , Difração de Raios XRESUMO
The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM). The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes. The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively). These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand. This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.
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Proteínas de Escherichia coli , N-Glicosil Hidrolases/metabolismo , Ciclopentanos/metabolismo , Pegada de DNA , Reparo do DNA , DNA-Formamidopirimidina Glicosilase , Escherichia coli/enzimologia , Furanos/metabolismo , Radical Hidroxila , Lactococcus lactis/enzimologia , Liases/metabolismo , Propilenoglicóis/metabolismo , Ligação Proteica , Purinas/química , Pirimidinas/química , Especificidade por SubstratoRESUMO
In this study, we have identified a protein in Escherichia coli that specifically binds to double-stranded DNA containing a single-stranded gap of one nucleotide. The gap-DNA binding (GDB) protein was purified to apparent homogeneity. The analysis of the amino-terminal sequencing of the GDB protein shows two closely related sequences we identify as the alpha and beta subunits of the HU protein. Furthermore, the GDB protein is not detected in the crude extract of an E. coli double mutant strain hupA hupB that has no functional HU protein. These results led us to identify the GDB protein as the HU protein. HU binds strongly to double-stranded 30-mer oligonucleotides containing a nick or a single-stranded gap of one or two nucleotides. Apparent dissociation constants were measured for these various DNA duplexes using a gel retardation assay. The KD(app) values were 8 nM for the 30-mer duplex that contains a nick and 4 and 2 nM for those that contain a 1-or a 2-nucleotide gap, respectively. The affinity of HU for these ligands is at least 100-fold higher than for the same 30-mer DNA duplex without nick or gap. Other single-stranded breaks or gaps, which are intermediate products in the repair of abasic sites after incision by the Fpg, Nth, or Nfo proteins, are also preferentially bound by the HU protein. Due to specific binding to DNA strand breaks, HU may play a role in replication, recombination, and repair.
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Proteínas de Bactérias/metabolismo , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Reparo do DNA , Proteínas de Ligação a DNA/isolamento & purificação , Ligantes , Dados de Sequência Molecular , Ligação ProteicaRESUMO
A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E. coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.
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DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/química , Guanina/análogos & derivados , N-Glicosil Hidrolases/metabolismo , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Cisteína/genética , DNA/análise , DNA-Formamidopirimidina Glicosilase , Glicina/genética , Guanina/metabolismo , Cinética , Dados de Sequência Molecular , Mutagênese , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Especificidade por Substrato , Dedos de ZincoRESUMO
The Fpg protein of Escherichia coli is a DNA repair enzyme with DNA glycosylase, abasic site nicking, and deoxyribose excising activities. Analysis of the amino acid sequence of this protein suggests that the Fpg protein is a zinc finger protein with a Cys-X2-Cys-X16-Cys-X2-Cys motif. Competition experiments show that the Fpg protein substitutes Cu(II), Cd(II), and Hg(II), metal ions classically associated with substitutions in zinc finger proteins. The Fpg protein activities are inhibited following the reaction with a Cys-specific reagent at low protein:reagent ratios, suggesting that these residues are important for the enzymatic activities. Site-directed mutagenesis was used to produce 6 mutant Fpg proteins with Cys-->Gly mutations. Substitution of the zinc in these proteins by 65Zn(II) indicates that all the proteins bind zinc, but the Zn(II) is not retained as strongly in the zinc finger mutants. The mutations in the Fpg protein outside the zinc finger consensus sequence do not eliminate the Fapy-DNA glycosylase and abasic site nicking. One of the Fpg mutant proteins outside the zinc finger has a reduced capacity to release deoxyribose from abasic sites. Cys-->Gly mutations in the zinc finger consensus sequence reduce all three aforementioned activities substantially. The purified Fpg proteins with Cys-->Gly mutations in the zinc finger consensus sequence do not incise DNA at abasic sites with the same efficiency nor mechanism as the native Fpg protein. The wild type Fpg protein and the Fpg proteins mutated outside the zinc finger sequence bind an oligonucleotide with a unique chemically reduced abasic site in a defined sequence as assayed by retention on nitrocellulose filters, whereas the mutant Fpg proteins within the zinc finger sequence do not bind to the same oligonucleotide. Therefore, the disruption of zinc coordination in the zinc finger of the Fpg protein is associated with decreased binding capacity to DNA as well as decreased enzymatic activities.
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Cisteína/química , Proteínas de Escherichia coli , N-Glicosil Hidrolases/química , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Sequência Consenso , Cisteína/metabolismo , DNA Bacteriano/metabolismo , DNA-Formamidopirimidina Glicosilase , Eletroforese em Gel de Poliacrilamida , Glicosilação , Cinética , Metais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Compostos de Fenilmercúrio/farmacologia , Conformação ProteicaRESUMO
The E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.
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Ácido Apurínico/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , N-Glicosil Hidrolases/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Sequência de Bases , Boroidretos , DNA-Formamidopirimidina Glicosilase , Eletroforese , Cinética , Dados de Sequência Molecular , OxirreduçãoRESUMO
We have purified the product of the NAM2 gene, the mitochondrial leucyl-tRNA synthetase, from yeast mitochondria. The purified protein cross-reacts with antibodies raised against the product of a LacZ/NAM2 gene fusion and antibodies raised against the purified Escherichia coli leucyl-tRNA synthetase. The mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 100 kDa, consistent with the size predicted by the gene sequence (102 kDa). The N-terminal sequence of the protein has been determined and shows that the first nine amino acids predicted by the gene sequence have been removed, probably during transport into the mitochondria.