Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 ? resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 ? and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.     

Design and optimization of modified simplex methods

Four test functions are used to evaluate different modifications of the modified simplex method and to optimize the contraction, reflection and expansion parameters. The evaluation shows that some of the modifications and the optimization of the parameters result in a simplex method that has gained in convergence ability, with some improvement in speed.     

Collective behavior of Franck-Condon excited states and energy transfer in DNA double helices

Absorption of UV radiation by DNA bases is known to induce carcinogenic mutations. The lesion distribution depends on the sequence around the hotspots, suggesting cooperativity between bases. Here we show that such cooperativity may intervene at the very first step of a cascade of events by formation of Franck-Condon states delocalized over several bases and subsequent energy transfer faster than 100 fs. Our study focuses on the double helix poly(dA).poly(dT), whose fluorescence, induced by femtosecond pulses at 267 nm, is probed by the upconversion technique and time-correlated single photon counting, over a large time domain (100 fs to 100 ns). The time-resolved fluorescence decays and fluorescence anisotropy decays are discussed in relation with the steady-state absorption and fluorescence spectra in the frame of exciton theory.     

Superporous agarose beads as a hydrophobic interaction chromatography support

Superporous agarose beads were used as a support for hydrophobic interaction chromatography. These beads have large connecting flow pores in addition to their normal diffusion pores. The flow pores, which are approximately one fifth of the overall diameter of the superporous agarose beads, were earlier shown to give the beads improved mass transfer properties relative to homogeneous agarose beads (Gustavsson and Larsson, J. Chromatogr. A, 734 (1996) 231–240). Superporous agarose beads and homogeneous agarose beads of the same particle size range (106–180 μm) were derivatized with phenyl groups. The properties of the superporous beads were then compared with the homogeneous beads in the separation of a mixture of three model proteins (ribonuclease A, lysozyme and bovine serum albumin) at various superficial flow velocities from 30 to 600 cm/h. The superporous beads gave satisfactory separation at flow velocities five times higher than was possible for homogeneous beads. The performance of the two types of beads was also compared in the purification of lactate dehydrogenase from a beef heart extract at a superficial flow velocity of 150 cm/h. The superporous beads performed considerably better, leading to twice the purification factor and twice the concentration of the desired product. The results were interpreted using the theoretical treatment given by Carta and Rodrigues (Carta and Rodrigues, Chem. Eng. Sci., 48 (1993) 3927).     

Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.     

Control of interaction-induced dephasing of Bloch oscillations

We report on the control of interaction-induced dephasing of Bloch oscillations for an atomic Bose-Einstein condensate in an optical lattice. We quantify the dephasing in terms of the width of the quasimomentum distribution and measure its dependence on time for different interaction strengths which we control by means of a Feshbach resonance. For minimal interaction, the dephasing time is increased from a few to more than 20 thousand Bloch oscillation periods, allowing us to realize a BEC-based atom interferometer in the noninteracting limit.     

Variable-range projection model for turbulence-driven collisions

We discuss the relative speeds DeltaV of inertial particles suspended in a highly turbulent gas when the Stokes number, a dimensionless measure of their inertia, is large. We identify a mechanism giving rise to the distribution P(DeltaV) approximately exp(-C|DeltaV|(4/3)) (for some constant C). Our conclusions are supported by numerical simulations, and by the analytical solution of a model equation of motion. The results determine the rate of collisions between suspended particles. They are relevant to the hypothesized mechanism for formation of planets by aggregation of dust particles in circumstellar nebula.     

A microcomputer-based system for potentiometric measurements with ion-selective electrodes

A comparison, on completely equal terms, of a microcomputer-based system with manual measurements is described in detail. The main features compared are the precision and accuracy of the results and the number of determinations per time unit. The example used is a standard addition measurement of fluoride in toothpaste. The microcomputer system and its interfacing to the peripheral units are also described. The microcomputer system for control, supervision and evaluation of the measurements results in a significant gain in precision and speed. Accuracy requires the use of a non-theoretical value of the Nernstian slope. The memory effect of the ion-selective electrode is large when the type of sample is changed.     

Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide

We use agarose gel electrophoresis to characterize how the monovalent catioinic surfactant cetyltrimethylammonium bromide (CTAB) compacts double-stranded DNA, which is detected as a reduction in electrophoretic DNA velocity. The velocity reaches a plateau at a ratio R = 1.8 of CTAB to DNA-phosphate charges, i.e., above the neutralization point, and the complexes retain a net negative charge at least up to R = 200. Condensation experiments on a mixture of two DNA sizes show that the complexes formed contain only one condensed DNA molecule each. These CTAB-DNA globules were further characterized by time-resolved measurements of their velocity inside the gel, which showed that CTAB does not dissociate during the migration but possibly upon entry into the gel. Using the Ogston-model for electrophoresis of spherical particles, the measured in-gel velocity of the globule is quantitatively consistent with CTAB having two opposite effects, reduction of both the electrophoretic charge and DNA coil size. In the case of CTAB the two effects nearly cancel, which can explain why opposite velocity shifts (globule faster than uncomplexed DNA) have been observed with some catioinic condensation agents. Dissociation of the complexes by addition of anionic surfactants was also studied. The DNA release from the globule was complete at a mixing ratio between anionic and cationic surfactants equal to 1, in agreement with equilibrium studies. Circular DNA retained its supercoiling, and this demonstrates a lack of DNA nicking in the compaction-release cycle which is important in DNA transfection and purification applications.