Tokamak Plasmas with Density up to 10 Times the Greenwald Limit.

Current-carrying, toroidal laboratory plasmas typically cannot be sustained with an electron density above the empirical Greenwald limit. Presented here are tokamak experiments in the Madison Symmetric Torus with a density up to an unprecedented level about 10 times this limit. This is thought to be made possible in part by a thick, stabilizing, conductive wall, and a high-voltage, feedback-controlled power supply driving the plasma current. The radial profile of the toroidal current flattens around twice the limit, without the edge collapse routinely observed in other experiments.

Dependence of Perpendicular Viscosity on Magnetic Fluctuations in a Stochastic Topology.

In a magnetically confined plasma with a stochastic magnetic field, the dependence of the perpendicular viscosity on the magnetic fluctuation amplitude is measured for the first time. With a controlled, ∼ tenfold variation in the fluctuation amplitude, the viscosity increases ∼100-fold, exhibiting the same fluctuation-amplitude-squared dependence as the predicted rate of stochastic field line diffusion. The absolute value of the viscosity is well predicted by a model based on momentum transport in a stochastic field, the first in-depth test of this model.

Kinetic stress and intrinsic flow in a toroidal plasma.

A new mechanism for intrinsic plasma flow has been experimentally identified in a toroidal plasma. For reversed field pinch plasmas with a few percent ß (ratio of plasma pressure to magnetic pressure), measurements show that parallel pressure fluctuations correlated with magnetic fluctuations create a kinetic stress that can affect momentum balance and the evolution of intrinsic plasma flow. This implies kinetic effects are important for flow generation and sustainment.

Role of nonlinear coupling and density fluctuations in magnetic-fluctuation-induced particle transport.

Three-wave nonlinear coupling among spatial Fourier modes of density and magnetic fluctuations is directly measured in a magnetically confined toroidal plasma. Density fluctuations are observed to gain (lose) energy from (to) either equilibrium or fluctuating fields depending on the mode number. Experiments indicate that nonlinear interactions alter the phase relation between density and magnetic fluctuations, leading to strong particle transport.

Classical impurity ion confinement in a toroidal magnetized fusion plasma.

High-resolution measurements of impurity ion dynamics provide first-time evidence of classical ion confinement in a toroidal, magnetically confined plasma. The density profile evolution of fully stripped carbon is measured in MST reversed-field pinch plasmas with reduced magnetic turbulence to assess Coulomb-collisional transport without the neoclassical enhancement from particle drift effects. The impurity density profile evolves to a hollow shape, consistent with the temperature screening mechanism of classical transport. Corroborating methane pellet injection experiments expose the sensitivity of the impurity particle confinement time to the residual magnetic fluctuation amplitude.

Anisotropic ion heating and tail generation during tearing mode magnetic reconnection in a high-temperature plasma.

Complementary measurements of ion energy distributions in a magnetically confined high-temperature plasma show that magnetic reconnection results in both anisotropic ion heating and the generation of suprathermal ions. The anisotropy, observed in the C(+6) impurity ions, is such that the temperature perpendicular to the magnetic field is larger than the temperature parallel to the magnetic field. The suprathermal tail appears in the majority ion distribution and is well described by a power law to energies 10 times the thermal energy. These observations may offer insight into the energization process.

Bifurcation to 3D helical magnetic equilibrium in an axisymmetric toroidal device.

We report the first direct measurement of the internal magnetic field structure associated with a 3D helical equilibrium generated spontaneously in the core of an axisymmetric toroidal plasma containment device. Magnetohydrodynamic equilibrium bifurcation occurs in a reversed-field pinch when the innermost resonant magnetic perturbation grows to a large amplitude, reaching up to 8% of the mean field strength. Magnetic topology evolution is determined by measuring the Faraday effect, revealing that, as the perturbation grows, toroidal symmetry is broken and a helical equilibrium is established.

Multi-energy reconstructions, central electron temperature measurements, and early detection of the birth and growth of runaway electrons using a versatile soft x-ray pinhole camera at MST.

A multi-energy soft x-ray pinhole camera has been designed, built, and deployed at the Madison Symmetric Torus to aid the study of particle and thermal transport, as well as MHD stability physics. This novel imaging diagnostic technique employs a pixelated x-ray detector in which the lower energy threshold for photon detection can be adjusted independently on each pixel. The detector of choice is a PILATUS3 100 K with a 450 µm thick silicon sensor and nearly 100 000 pixels sensitive to photon energies between 1.6 and 30 keV. An ensemble of cubic spline smoothing functions has been applied to the line-integrated data for each time-frame and energy-range, obtaining a reduced standard-deviation when compared to that dominated by photon-noise. The multi-energy local emissivity profiles are obtained from a 1D matrix-based Abel-inversion procedure. Central values of Te can be obtained by modeling the slope of the continuum radiation from ratios of the inverted radial emissivity profiles over multiple energy ranges with no a priori assumptions of plasma profiles, magnetic field reconstruction constraints, high-density limitations, or need of shot-to-shot reproducibility. In tokamak plasmas, a novel application has recently been tested for early detection, 1D imaging, and study of the birth, exponential growth, and saturation of runaway electrons at energies comparable to 100 × Te,0; thus, early results are also presented.

Mass-dependent ion heating during magnetic reconnection in a laboratory plasma.

Noncollisional ion heating in laboratory and astrophysical plasmas and the mechanism of conversion of magnetic energy to ion thermal energy are not well understood. In the Madison Symmetric Torus reversed-field pinch experiment, ions are heated rapidly during impulsive reconnection, attaining temperatures exceeding hundreds of eV, often well in excess of the electron temperature. The energy budget of the ion heating and its mass scaling in hydrogen, deuterium, and helium plasmas were determined by measuring the fraction of the released magnetic energy converted to ion thermal energy. The fraction ranges from about 10%-30% and increases approximately as the square root of the ion mass. A simple model based on stochastic ion heating is proposed that is consistent with the experimental data.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel.

BACKGROUND: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. RESULTS: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. CONCLUSIONS: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions. The influence of in situ field gradients.

The 1H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T2 values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with tau = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25-1.98 G/cm and 0.89-2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.

Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure.

Methylphosphonate in conjunction with 31P-NMR spectroscopy was used for the measurement of transmembrane delta pH in human erythrocytes stored at 4 degrees C for up to 5 weeks in a nutrient medium. Intra- and extracellular pH was determined using calibration curves based on the pH-dependent separation between the NMR resonances of methylphosphonate and orthophosphate (Pi). A comprehensive statistical procedure is presented for the determination of the variance of NMR-based pH estimates. The entry of methylphosphonate into erythrocytes was more rapid at low pH and uptake was fully inhibited by the band 3 reagent, disodium 4,4-diisothiocyano-2,2'-disulphonic acid stilbene. The distribution ratio of methylphosphonate concentration inside and outside the cells was used to calculate the membrane potential; the analysis depends on a consideration of the Donnan equilibrium for an anion with one or two charges. Furthermore, the analysis does not depend on the pH estimates but relies solely on concentration estimates. The chemical shift of methylphosphonate was not subject to the variations associated with specific intracellular binding encountered with many other phosphorus compounds, including Pi. On the other hand, the ionic strength dependence of the chemical shift of methylphosphonate, contrary to earlier reports, is comparable in magnitude (but opposite in sign) to that of Pi.

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system.

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 mumol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [14C]pyruvate to haemoglobin and the rate of efflux of [14C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent Km and Vmax for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.

Glucose transport in human erythrocytes measured using 13C NMR spin transfer.

We present the results of a new NMR-based procedure for measuring the fast transmembrane exchange of D-[1-13C]glucose in human erythrocytes. The method relies on different rates of exchange between the alpha- and beta-anomers of glucose inside and outside the cells; the rate outside the cells is greatly increased by the addition of mutarotase to the suspension. Theory is developed to describe nuclear-spin transfer in the present system and is used to analyse the data to yield estimates of transmembrane-exchange rate constants and their statistical uncertainties. For a total glucose concentration of 25.5 mmol/l at 40 degrees C the first order efflux rate constants for the alpha- and beta-anomers were 1.20 +/- 0.40 s-1 and 0.71 +/- 0.30 s-1, respectively.

Evidence of red cell alignment in the magnetic field of an NMR spectrometer based on the diffusion tensor of water.

The alignment of human erythrocytes in aqueous suspensions in the magnetic field B(0) (called the z-direction) of an NMR spectrometer was shown by calculating the diffusion tensor for water in the sample. The diffusion was measured using a pulsed-field-gradient spin-echo NMR method. The extent of diffusion anisotropy for water was exemplified by the values of the apparent diffusion coefficients with erythrocytes of normal shape and volume: for a typical experiment the values for the x-, y-, and z-directions were (6.88 +/- 0.17) x 10(-10), (7.07 +/- 0.17) x 10(-10), and (10.20 +/- 0.17) x 10(-10) m(2) s(-1), respectively. Cells in hypo- and hyperosmotic media were also studied and they too showed the anisotropy of the apparent diffusion coefficients but the extents were different. A new method of data analysis was developed using the Standard Add-On Packages in a Mathematica program. The experimental findings support evidence of erythrocyte alignment that was previously obtained with a high-field-gradient q-space method.

Assignment of coherence features in NMR q-space plots to particular diffusion modes in erythrocyte suspensions.

NMR q-space plots derived from water diffusing inside and around erythrocytes in a suspension display reproducible and characteristic coherence features. The aim of the present work was to determine which water population gives rise to the respective features. The central experimental strategy was to use choline and choline phosphate which are virtually membrane impermeant on the time scale of the experiment; the former was incorporated into erythrocytes by a lysis-resealing method and the latter was simply added to the suspensions. Dimethyl sulfoxide, which readily but more slowly exchanges across the cell membranes than water, also yielded q-space plots which were similar to those of water, but the differences were able to be accounted for on the basis of its slower transmembrane exchange rate. Random walk simulations using a Monte Carlo procedure, together with a model of an array of biconcave discocytes, helped verify the interpretations of the assignment of the features of the plots to molecules diffusing in the two regions. In addition, the simulations revealed how the presence or absence of transmembrane exchange affects the form of q-space plots.

Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay.

This is the second of two papers [Drews, M., Doverskog, M., Ohman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., Häggström, L., 2000. Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR. J. Biotechnol. 78, 23-37]. where the general goal has been to determine and characterise the glutamine metabolism in Sf9 cells. The presence of glutamate synthase (GOGAT) activity was investigated in cell-free extracts of S. frugiperda (Sf9) insect cells by modified 1H/15N spin-echo and gradient enhanced multiple quantum coherence NMR spectroscopy techniques. Cell-free extracts were prepared from cells cultured in a serum-free medium. The assay conditions were based on conventional spectrophotometric and chromatographic methods. NMR data showed that nitrogen from [5-15N] glutamine was selectively incorporated into 2-oxoglutarate forming [2-15N] glutamate with a specific activity of 4.15 +/- 0.21 nmol [2-15N] glutamate min -1 (mg total protein)-1 in the cell-free extracts. The enzyme activity was exclusively dependent on NADH as coenzyme and was completely inhibited by 1 mM azaserine. From the results obtained, we conclude that Sf9 cells possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glutamate dehydrogenase.

Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR.

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.

Mutarotase equilibrium exchange kinetics studied by 13C-NMR.

The rates of exchange between the alpha- and beta-anomers of D-[1-13C]glucose, at equilibrium catalyzed by porcine kidney mutarotase (EC 5.1.3.3), were measured using 13C-NMR spin-transfer procedures. This entailed inversion-transfer and saturation-transfer experiments, and two-dimensional exchange spectroscopy (2D EXSY). The concentration and temperature dependences of the fluxes were studied; equilibrium exchange Michaelis constants, and the activation energy of the catalyzed reaction were thereby measured.

Strong and weak binding of water to proteins studied by NMR triple-quantum filtered relaxation spectroscopy of (17)O-water.

The triple-quantum filtered (TQF) spin-echo signal of (17)O-water, in the presence of proteins, was analysed to yield estimates of the number of weakly, and strongly bound water molecules. The analysis used a constrained direct iterative regression procedure with a three-state model of fast-exchange. Thus, the population size of free, weakly, and strongly bound water were determined simultaneously. The two fractions of the bound water were estimated by using correlation time(s) estimated in other studies. Bovine serum albumin (BSA), basic pancreatic trypsin inhibitor (BPTI), lysozyme and oxyhaemoglobin were studied. Of the four proteins, BSA contained the largest number of strongly and weakly bound water molecules, there being approximately 30 of the former and approximately 3000 of the latter under conditions of high protein concentration. The correlation time of the proteins increases with their concentration in solution, and when this was taken into account for BSA the estimated number of strongly bound water molecules did not change significantly. This NMR technique, and data analysis, will probably also be useful in studies of water binding and mobility in various systems including hydrogels, protein networks, membranes, cells and tissues.