A cautionary note on the use of stable transformed cells

Gene transfection and ectopic expression is a widely used method in experimental biology. In the present report, we would like to point out that this approach may, in certain circumstances, lead to a modification of the transfected cell phenotype. Indeed, we observed that after transfection of bcl-2 gene in the neuronal PC12 cell line some of the selected clones have lost their neuronal and catecholaminergic characteristics, i.e. TH expression and ability to grow neurites in response to NGF. Thus, the resistance of some PC12-Bcl-2 clones against neurotoxic insults may not necessarily reflect the potential benefit afforded by Bcl-2 expression. We therefore encouraged authors to verify cell phenotype after stable transfection to avoid misinterpretation of their results.     

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of ³¹P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.

Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy

Addition of choline, ethanolamine, or hemicholinium-3 (a choline kinase inhibitor) to the perfusate of human breast cancer cells monitored by 31P NMR spectroscopy resulted in significant changes to phosphomonoester (PME) and phosphodiester (PDE) signals. These results enable us to assign the PMEs to phosphcholine (PC) and phosphoethanolamine (PE), the PDEs to glycerophosphorylcholine and glycerophosphorylethanolamine, and to define the pathways producing them. The PMEs are products of choline and ethanolamine kinases, the first steps in phospholipid synthesis; and the PDEs are substrates of glycerophosphorylcholine phosphodiesterase, the last step in phospholipid catabolism. Furthermore, PC and PE peaks are twice as intense in cells at log phase versus confluency. We also observed these signals in vivo in human colon and breast tumors grown in mice. Since PMEs are low in most nonproliferating tissues, they could form a basis for noninvasive diagnosis. Also, PE and PC are situated between the control enzymes of two major synthetic pathways and will allow noninvasive 31P NMR studies of these pathways in intact cells and in vivo.     

An alternative expeditious analysis of phospholipid composition in human blood plasma by 31P NMR spectroscopy

31P nuclear magnetic resonance spectroscopy has been applied to quantitate phospholipids in human blood plasma and in separate lipoprotein fractions. The addition of suitable detergents to samples produces an excellent chemical shift dispersion and allows the identification and integration of the peaks of the most important phospholipids. Results are in agreement with those obtained with enzymatic colorimetric and TLC methods: our method is characterized by good accuracy and reproducibility.     

Multipoint linkage analysis. A cautionary note.

Multipoint linkage analysis is commonly used to evaluate linkage of a disease to multiple markers in a small region. Multipoint analysis is particularly powerful when the IBD relations of family members at the trait locus are ambiguous. The increased power arises because, unlike single-marker analyses, multipoint analysis uses haplotype information from several markers to infer the IBD relations. We wish to temper this advantage with a cautionary note: multipoint analysis is sensitive to power loss due to misspecification of intermarker distances. Such misspecification is especially problematic when dealing with closely spaced markers. We present computer simulations comparing the power of single-point and multipoint analyses, both when IBD relations are ambiguous, and when the intermarker distances are misspecified. We conclude that when evaluating markers in a small region to confirm or refute previous findings, a situation in which p values of modest statistical significance are important, single marker analyses may provide more reliable measures of the strength of support for linkage than multipoint statistics.     

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. ³¹P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in ³¹P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid ³¹P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible ³¹P NMR spectra of phospholipids.     

A cautionary note on the use of certain restriction endonucleases with methylated substrates

Methylation of adenine and cytosine residues in DNA isolated from common strains of Escherichia coli K-12 can render that DNA resistant to cleavage by certain restriction endonucleases at those sites at which the recognition sequence for such an endonuclease overlaps (but does not include) a sequence recognized by methylases specified by the dam or dcm gene.     

Phosphoglycerides and derivatives in Taenia crassiceps examined by 31P NMR spectroscopy

Examination of the larval stage of the tapeworm, Taenia crassiceps, by 31P NMR spectroscopy revealed the presence of a major phosphoglyceride component. However, using saturation transfer, no exchange between glycerophosphorylcholine and phosphoglyceride or any other NMR-detectable phosphorus metabolites was detected.     

Thermolysin-inhibitor complexes examined by 31P and 113Cd NMR spectroscopy

Complexes between phosphoramidon (N-(alpha-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptoph an) and zinc thermolysin and between phosphoramidon or N-phosphoryl-L-leucineamide and 113Cd-substituted thermolysin have been examined by 31P and 113Cd NMR spectroscopy. 113Cd resonances are observed at 168 and 152 ppm for the phosphoramidon and N-phosphoryl-L-leucineamide complexes, respectively. There are large but different chemical shift anisotropy contributions to the 113Cd line widths for the two complexes, which reflect the known structural differences for the zinc-enzyme complexes. 113Cd-31P spin-spin coupling is also seen and differs for the two cadmium complexes, being larger, 28 Hz, for the bidentate N-phosphoryl-L-leucineamide ligand than for the monodentate phosphoramidon, 16 Hz. Large changes in chemical shift, 7.5-10.9 ppm, are seen for the 31P resonances of the inhibitors upon binding to the enzyme reflecting direct phosphoryl-metal ligation. Chemical shift anisotropy is the dominant relaxation mechanism for the 31P nuclei at 9.4 T, while the dipole-dipole contribution seems to be unaffected by a change of solvent from H2O to D2O.     

On the noninvasive measurement of intracellular free magnesium by 31P NMR spectroscopy

We previously introduced a noninvasive measurement of the concentration of free Mg2+ in intact cells and tissues using 31P NMR. To resolve a controversy in the literature concerning the affinity of Mg2+ for ATP used in our procedure, the apparent dissociation constant of MgATP under simulated intracellular conditions has been determined by three independent magnetic resonance methods, including a newly developed combination procedure for determining this value at intracellular ATP levels. The new combination method, which utilizes 31P NMR to determine the degree of Mg2+ chelation of ATP and the dye antipyrylazo III for optical determination of free Mg2+, yielded a value of (50 +/- 10) microM for this apparent dissociation constant at pH 7.2 in the presence of 0.15 M K+ and 25 degrees C. We further show that hydroxyquinolines are not satisfactory indicators for optical determination of the Mg2+-nucleotide dissociation constant. From our determinations a low value of free Mg2+ (less than 1 mM) is established for all of the tissues studied, including perfused heart muscle, contrary to a recent report in the literature. Saturating human erythrocytes with Mg2+ results in an alpha- and beta-phosphorus resonance separation for intracellular ATP that is indistinguishable from that observed in a noncellular MgATP control under similar conditions, showing that MgATP resonances in this cell are unaffected by the cellular environment.     

Improved technique for investigation of cell metabolism by31P NMR spectroscopy

SIp NMR studies on microorganisms have been carried out with the cells embedded in agarose gel. The novel use of the gel for the NMR studies has advantages over the usual liquid suspensions in terms of improved reproducibility of data and cell viability, with no net loss of spectral quality. Polyphosphate formation in Escherichia coli was monitored continuously for up to 24 h and metabolic changes in yeast for 6 h. Changes of the intracellular pH during glycolysis in yeast were determined from the chemical shift of the internal Pi. NMR titration curves of Pi in the presence of Mg²⁺ indicate uncertainties in internal pH values estimated by this technique.     

Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy

The use of new 1H-detected heteronuclear 1H-31P shift correlation experiments is demonstrated for oligonucleotides of 12 and 40 base pairs. The methods give unambiguous assignments of the 31P resonances and also permit identification of the C4' and C5' sugar protons. Use of the new methods enables one to make sequence-specific resonance assignments without reference to a known or assumed conformation of the DNA fragment.     

Diffusional limitations of immobilized Escherichia coli in hollow-fiber reactors: influence on 31P NMR spectroscopy

Escherichia coli cells were immobilized and grown in hollow-fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using (35)S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 degrees C were shown to be severe, with regions of starved cells occupying up to 80% of the cell-containing region. Phosphorus-31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume-averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur-35 autoradiographs of a reactor operated at 16 degrees C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near-normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.     

Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectroscopy and 31P NMR spectroscopy

The small GTPase Ras plays a key role as a molecular switch in the intercellular signal transduction. On Mg(2+) --> Mn(2+) substituted samples, the first ligand sphere of the metal ion in the inactive, GDP-bound Ras has been studied by continuous wave EPR at 94 GHz (W-band). Via replacement of normal water with (17)O-enriched water, the (17)O--(55)Mn superhyperfine coupling was used to determine the number of water ligands bound to the metal ion. In contrast to EPR data on frozen solutions and X-ray data from single crystals where four direct ligands to the metal ion are found, the wild-type protein has only three water ligands bound in solution at room temperature. The same number of water ligands is found for the mutant Ras(T35S). However, for the alanine mutant in position 35 Ras(T35A) as well as for the oncogenic mutant Ras(G12V), four water ligands can be observed in liquid solution. The EPR studies were supplemented by (31)P NMR studies on the Mg(2+) x GDP complexes of the wild-type protein and the three mutants. Ras(T35A) exists in two conformational states (1 and 2) with an equilibrium constant K(1)(1,2) of approximately 0.49 and rate constants k(1--1) which are much smaller than 40 s(-1) at 298 K. For wild-type Ras and Ras(T35S), the two states can also be observed with equilibrium constants K(1)(1,2) of approximately 0.31 and 0.21, respectively. In Ras(G12V), only one conformational state could be detected.     

31P NMR analysis of the surface homogeneity of mixed sphingomyelin-phosphatidylcholine vesicles

The surface compositional symmetry of a mixed sphingomyelin-phosphatidylcholine vesicle has been studied by ³¹P NMR spectrometry. The molecules on the outer vesicle surface could be distinguished from the molecules on the inner vesicle surface by utilization of the shift reagent, Eu³⁺. This polyvalent cation interacts with the outer surface phosphate groups and, as a result, shifts these molecules upfield. Analysis of the data obtained indicates that a slight excess of sphingomyelin is present on the outer surface of the vesicle, compared to phosphatidylcholine, which appears to have a slight preference for the inner surface.     

31P NMR spin-transfer in the phosphoglyceromutase reaction

The rate of exchange of phosphoryl groups between 2- and 3-phosphoglycerate catalysed by (a) high concentrations (approximately equal to 5.0 mg protein ml-1) of rabbit muscle phosphoglyceromutase and (b) lysed human erythrocytes was measured using saturation and inversion transfer techniques with 31P-NMR spectroscopy. This is the first reported application of these techniques to a study of this particular enzymic reaction either in vitro or in situ in a cell cytosol. Selective irradiation of resonances was achieved by the DANTE pulse sequence which had not previously been used for spin-transfer studies. New equilibrium exchange theory was developed for the simplest model of a two-reactant enzyme-catalysed reaction and this was used to calculate turnover rates for the enzymes. There was a close similarity between the turnover rates calculated from the spin-transfer data obtained from the systems in vitro and in situ and those obtained by conventional enzymic assays, at low enzyme concentrations. This suggested an absence of any homogeneous enzyme-enzyme interactions which modify the kinetics at high protein concentrations either in lysates or in the system in vitro.