The interaction of the extended, fully-conjugated macrocycle, dipyridyldibenzotetraaza[14]annulene (1), with the square planar palladium(II) and platinum(II) complexes [M(dppp)(triflate)(2)].2H(2)O (M = Pd, Pt) has been investigated in both solution and the solid state. In each case solid products showing a 1:1 ratio of metal complex: 1 were obtained. A 1:1 mixture of and [Pd(dppp)(2)(triflate)(2)].2H(2)O in dichloromethane yielded two major products as evidenced by the presence of two singlets in the (31)P {(1)H}-NMR spectrum of the reaction solution. Similarly, two singlets were evident in the corresponding spectrum obtained on dissolving the 1:1 product in nitrobenzene. The temperature and concentration dependence of the spectra clearly showed that the two species present in each case were in equilibrium. From the temperature dependence, the low field signal was assigned to the smaller of the two species. Broadly parallel behaviour was observed for the corresponding platinum-containing system. The MS-ESI spectrum of the platinum derivative showed the presence of a dinuclear species corresponding to [Pt(dppp)(1)](2)(2+) and an X-ray structure of this product confirmed that a corresponding dinuclear complex exists in the solid state. This product has a geometry in which two curved macrocyclic side units bridge two metal centres to yield an ellipse-shaped structure. Attempts to employ pulsed-field gradient spin-echo (PGSE) (31)P NMR confirmed that the lower-field resonance corresponded to the smaller of the two species in solution. STM (HOPG) imaging of the palladium- and platinum-containing products revealed arrays that appear to be composed of "zipper-like" rows of dimer units, with the dimensions of the latter comparing well with those found in the X-ray structure of [Pt(dppp)(1)](2)(2+).

Oscillating-gradient spin echo (OGSE) diffusion experiments have long been used to measure the short-time apparent diffusion coefficient, D(app)(t), in the presence of restricted diffusion, as well as the spectrum of the slow-motion velocity autocorrelation function. In this work, we focus on two previously unexplored aspects of OGSE experiments: convection compensation and acquisition of pure-phase diffusion spectra in the presence of homonuclear scalar couplings. We demonstrate that convection compensation afforded by single-echo OGSE compares well with that in double-echo convection-compensated PGSE experiments. We also show that, in the presence of homonuclear scalar couplings, setting the OGSE echo time to 1/2J enables acquisition of pure-phase diffusion spectra and yields more reliable D estimates than mixed-phase PGSE or OGSE spectra. Pure-phase OGSE acquisition is also compatible with measurements of the apparent diffusion coefficient at an arbitrary diffusion time. These features of OGSE can be valuable in diffusion measurements of scalar-coupled small-molecule probes in cellular and other heterogeneous systems.

The water residence time and diffusional water permeability in colonic epithelial T84 cancer cells was measured using (1)H NMR spectroscopy; the values estimated were 35.2+/-2.8 ms and (7.4+/-0.6)x10(-3)cms(-1), respectively. Water permeability was inhibited to approximately 10% of its original value by the mercurial diuretic, p-chloromercuribenzenesulfonate (PCMBS; 1mM), and fully restored by dithiothreitol (DTT; 1mM). The permeability was also inhibited reversibly to approximately 55%, by extracellular glibenclamide (1mM), an inhibitor of some ATP-binding cassette (ABC) transporters, including the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IMBX; 0.1-1mM) and the adenylate cyclase activator, forskolin (0.1-1mM) did not alter water permeability. It is concluded that in T84 cells water diffuses through the membrane lipid bilayer and via channels that are inhibited by PCMBS, including the channels that are known to be inhibited by glibenclamide.

31P magic angle spinning NMR (MAS-NMR) spectra were obtained from suspensions of human red blood cells (RBCs) that contained the cell-volume-sensitive probe molecule, dimethyl methylphosphonate (DMMP). A mathematical representation of the spectral-peak shape, including the separation and width-at-half-height in the 31P NMR spectra, as a function of rotor speed, enabled us to explore the extent to which a change in cell volume would be reflected in the spectra if it occurred. We concluded that a fractional volume change in excess of 3% would have been detected by our experiments. Thus, the experiments indicated that the mean cell volume did not change by this amount even at the highest spinning rate of 7 kHz. The mean cell volume and intracellular 31P line-width were independent of the packing density of the cells and of the initial cell volume. The relationship of these conclusions to other non-NMR studies of pressure effects on cells is noted.

Differences in magnetic susceptibility between various compartments in heterogeneous samples can introduce unanticipated complications to NMR spectra. On the other hand, an understanding of these effects at the level of the underlying physical principles has led to the development of several experimental techniques that provide data on cellular function that are unique to NMR spectroscopy. To illustrate some key features of susceptibility effects we present, among a more general overview, results obtained with red blood cells and a recently described model system involving diethyl phthalate in water. This substance forms a relatively stable emulsion in water and yet it has a significant solubility of 5 mmol/L at room temperature; thus, the NMR spectrum has twice as many resonances as would be expected for a simple solution. What determines the relative intensities of the two families of peaks and can their frequencies be manipulated experimentally in a predictable way? The theory used to interpret the NMR spectra from the model system and cells was first developed in the context of electrostatics nearly a century ago, and yet some of its underlying assumptions now warrant closer scrutiny. While this insight is used in a practical way in this article, the accompanying article deals with the mathematics and physics behind this new analysis.

As part of a programme of comparative measurements of diffusional water permeability (Pd) the red blood cells (RBC) from Little Penguin (Eudyptula minor) were studied. The cell dimensions were measured with light and electron microscopy, and by a newly described non-invasive technique, NMR q-space analysis. In view of its relative novelty for cell biologists, an overview of this technique is presented. The RBC revealed an ellipsoidal shape that is characteristic of avian RBC, with axis lengths ("diameters") estimated to be: a=16.0 microm; b=9.6 microm; c=5.0 microm. The values of P(d)were: 2.0 x 10(-3)cm s(-1)at 5 degrees C, 3.3 x 10(-3)cm s(-1)at 10 degrees C, 4.6 x 10(-3)cm s(-1)at 15 degrees C and approximately 5.4 x 10(-3)cm s(-1)at 20, 25, 30, 37 and 42 degrees C. There was a lack of inhibition of water permeability by p-chloromercuribenzensulfonate (PCMBS), the well-known inhibitor of RBC aquaporin. It was notable that in the temperature range 5-20 degrees C the NMR parameters, and hence the permeability, varied linearly as is found for other species, but at temperatures higher than 20 degrees C there was no temperature-dependence of Pd. Consequently, there was an obvious break at approximately 20 degrees C in the Arrhenius plot, of the mean residence life time of water inside the cells, 1/Te, versus temperature. For temperatures less than 20 degrees C the activation energy E(a,d) was 45.6 +/- 6.6 kJ/mol. For temperatures higher than 25 degrees C E(a,d) was zero. The lack of inhibition of water permeability by PCMBS and the very high value of E(a,d) for diffusive water exchange suggests that the water permeation occurs primarily via the membrane bilayer per se, i.e., there is no aquaporin in Little Penguin RBC. The discontinuity at approximately 20 degrees C in the Arrhenius plot is an interesting finding, not seen before in other species, and we suggest that it reflects a phase transition in the membrane lipids.

The diffusion coefficients are reported of rubbery ternary systems consisting of the polymer, its monomer analogue (i.e., the saturated equivalent of the monomer), and trace quantities of oligomers (dimer, trimer, tetramer and hexamer) for 2-hydroxyethyl methacrylate (HEMA). These have been obtained with pulsed-field-gradient NMR spectroscopy with a polymer weight fraction (f(p)) of 0 less than or equal to fp less than or equal to 0.4. The oligomers are macromonomers synthesized with a cobalt catalytic chain-transfer agent. The diffusion coefficients are about an order of magnitude smaller than those for monomers such as methyl methacrylate; this effect is ascribed to hydrogen bonding in HEMA. The diffusion coefficient D-i of an i-meric oligomer has been fitted with moderate accuracy by an empirical universal scaling relation, D-i(f(p))/D-1(f(p)) approximate to i(-(0.66+2fp)), previously found to provide an adequate fit to corresponding data for styrene and for methyl and butyl methacrylates. The approximate empirical scaling relation seems to hold for a remarkably wide range of types of monomer/polymer systems. These results are of use in modeling rates and molecular weight distributions in free-radical polymerization, particularly for termination (which is chain-length-dependent and is controlled by the diffusion coefficient of chains of the low degrees of polymerization studied here). (C) 2003 Wiley Periodicals, Inc.

The general anesthetic 2,6-diisopropylphenol (propofol) is very poorly soluble in water and is normally administered in the form of an emulsion. We demonstrated that several commercially available nonionic surfactants (Tween 80, Cremophor EL, Poloxamer 188, Poloxamer 407, Solutol HS15, and Vitamin E TPGS) render propofol soluble with a specific solubilization capacity of at least 0.1 g/g. The room-temperature stability of the solutions appeared to be limited only by the chemical stability of the compounds involved. The association between propofol and the surfactants was investigated by various NMR approaches, including measurements of diffusion coefficients, H-1 longitudinal relaxation times, and the magnitude of intermolecular nuclear Overhauser effects. The results were consistent with the micellar solubilization mechanism of propofol by the surfactants (unimer solubilization in the case of Poloxamer 188). The H-1 longitudinal relaxation and diffusion behavior of propofol were monoexponential in each case. Solubilization caused a considerable shortening of propofol's proton T-1's. The values of the diffusion coefficient of propofol were several percent higher than those of surfactants. This was explained by the partitioning of propofol between swollen micelles and the aqueous solution. Diffusion measurements also revealed the presence of a rapidly diffusing ethylene oxide population in surfactant solutions, which is consistent with free poly(ethylene oxide) (PEO) known to be present in commercially produced surfactants. The free PEO blocks exhibited molecular association with the extramicellar propofol.

N-Methylacetamide (NMA) is known to self-associate in solution through the formation of hydrogen bonds. This behavior, and the presence in the molecule of an amide bond, make this an interesting model for study since the oligomer can be considered to be a protein analogue. The aims of the present study were to estimate the thermodynamic parameters of the self-association process, through the measurement of the diffusion coefficient of NMA in carbon tetrachloride (CCl4), and to predict the population distribution of oligomers. as a function of the concentration of NMA. Diffusion coefficients were measured using pulsed field gradient spin-echo (PGSE) NMR spectroscopy. A computer model based on the Kirkwood-Riseman theory of macromolecular diffusion and an attenuative model of self-association were used to fit the experimental data and to derive estimates of the thermodynamic association constants and bond length, and these values were, in turn, used to estimate the oligomer distribution. In addition, the temperature dependence of the diffusion coefficient was measured at three different concentrations in order to calculate the apparent Arrhenius activation energies so as to provide additional insight into the self-association process. Finally, the concentration dependence of the viscosity of NMA in CCl4 was measured to characterize further the hydrodynamic behavior of the polydisperse system. Copyright (C) 2002 John Wiley Sons, Ltd.

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.

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.

: The diffusional water permeability ( P d ) of red blood cells (RBCs) from horses introduced to Australia and from European horses was measured by a Mn 2+ doping nuclear magnetic resonance (NMR) technique. The values of P d were ∼3.5 × 10 −3 cm/s at 25°C, 4.1 × 10 −3 cm/s at 30°C, 5.6 × 10 −3 cm/s at 37°C and 6.3 × 10 −3 cm/s at 42°C with no significant differences between the two strains of horse. Systematic studies on the effect of p-chloromercuribenzene sulphonate (PCMBS) on water diffusion indicated that the maximal inhibition (60%) was reached in 60 min at 22°C with 1 m m PCMBS. The basal permeability to water was estimated at 1.2 × 10 −3 cm/s at 25°C, 1.7 × 10 −3 cm/s at 30°C, 2.0 × 10 −3 cm/s at 37°C and 2.7 × 10 −3 cm/s at 42°C. The activation energy ( E a,d ) of water diffusion was ∼25 kJ/mol and increased to ∼39 kJ/mol after incubation with PCMBS, in conditions of maximal inhibition of water diffusion. The membrane protein electrophoretic pattern of horse RBCs was compared with its human counterpart. The horse RBC membranes had lower amounts of the proteins migrating as bands 4.1 and 4.2 and higher amounts of the protein migrating as band 4.9, and band 6 (glyceraldehyde-3-phosphate dehydrogenase) was undetectable. A noteworthy feature was the appearance of considerable differences in protein migration distances in the region of bands 7 and 9, between horse and human membranes.

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 amidotransfer 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.

The diffusional water permeability ( P d ) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p -chloromercuribenzene sulphonate (PCMBS). The values of P d were, in the case of alpaca RBC≈4.6×10 −3 cm/s at 25°C, 5.4×10 −3 cm/s at 30°C, 6.6×10 −3 cm/s at 37°C and 7.7×10 −3 cm/s at 42°C. In case of camel RBC the values of P d where ≈4.2×10 −3 cm/s and 9.0×10 −3 cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10 −3 cm/s at 25°C, 1.7×10 −3 cm/s at 30°C and of camel RBC as 1.8×10 −3 cm/s at 25°C and 3.0×10 −3 cm/s at 37°C. The values of the activation energy of water diffusion ( E a, d ) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.

The diffusive motion of water in wood pulps has been studied as a function of time between gradient pulses (Delta) using the pulsed field gradient stimulated echo NMR method while varying the gradient magnitude (g). The basis of this experiment is the attenuation of the echo signal by thermal or diffusive motion of water. Two types of liquid water in a pulp wet to below saturation can be identified on the basis of the attenuation of this signal, water which has its diffusional motion restricted by the cell lumen and water which exists outside the fibres for which the attenuation is less pronounced. The echo amplitude is plotted versus the spatial wave number vector, q = Vg delta/2 Pi, where Y is the magnetogyric ratio of the nucleus under consideration, g is the field gradient vector and delta is the gradient pulse duration. The Fourier transform of the echo amplitude in inverse space (q-space) gives the probability distribution of molecules being displaced a distance R during the time between the gradient pulses (Delta). From this representation an average pore size of 50 mu m for pulp fibre lumens over the entire sample is measured from a Gaussian peak-width at half height at a diffusion time of 1200 ms. This result is discussed in terms of the orientation of fibre lumens.

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.

The glutamine metabolism was studied in glucose-starved and glucose-sufficient hybridoma and Sp2/0-Ag14 myeloma cells. Glucose starvation was attained by cultivating the hybridoma cells with fructose instead of glucose, and the myeloma cells with a low initial glucose concentration which was rapidly exhausted. Glutamine used in the experiments was labeled with 15N, either in the amine or in the amide position. The fate of the label was monitored by 1H/15N NMR analysis of released 15NH+4 and 15N-alanine. Thus, NH+4 formed via glutaminase (GLNase) could be distinguished from NH+4 formed via glutamate dehydrogenase (GDH). In the glucose-sufficient cells a small but measurable amount of 15NH+4 released by GDH could be detected in both cell lines (0.75 and 0.31 micromole/10(6) cells for hybridoma and myeloma cells, respectively). The uptake of glutamine and the total production of NH+4 was significantly increased in both fructose-grown hybridoma and glucose-starved myeloma cells, as compared to the glucose-sufficient cells. The increased NH+4 production was due to an increased throughput via GLNase (1.6 -1.9-fold in the hybridoma, and 2.7-fold in the myeloma cell line) and an even further increased metabolism via GDH (4.8-7.9-fold in the hybridoma cells, and 3.1-fold in the myeloma cells). The data indicate that both GLNase and GDH are down-regulated when glucose is in excess, but up-regulated in glucose-starved cells. It was calculated that the maximum potential ATP production from glutamine could increase by 35-40 % in the fructose-grown hybridoma cells, mainly due to the increased metabolism via GDH.

The utility of 1H nuclear magnetic resonance (NMR) diffusion-diffraction of water as a tool for characterising red cell shape was investigated. Experiments were conducted on various cell suspensions which contained different shapes/forms of erythrocytes prepared by manipulating the conditions of the suspension medium, such as osmolality, and altering metabolism to affect the adenosine triphosphate concentration. Abnormal red cells from patients with hereditary stomatocytosis and megaloblastic anemia were also studied in order to assess the practical application of this “new” technique. The results clearly show that NMR diffusion-diffraction is sensitive to very small changes in mean cell dimensions and that a “characteristic” q-space plot/profile can be ascribed to each erythrocyte form. It was also found that the homogeneity of the cell shape and/or size is an important factor that affects the intensity of the diffusion-diffraction peaks. This study demonstrates the potential of the NMR diffusion-diffraction technique as a diagnostic tool in hematology.

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 3(10) 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-l, 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.