A Lorentz model for weak magnetic field bioeffects: Part II—Secondary transduction mechanisms and measures of reactivity

In Part I it was shown that the thermal component of the motion of a charged particle in an oscillator potential, that is, within a molecular binding site, rotates at the Larmor frequency in an applied magnetic field. It was also shown that the Larmor angular frequency is independent of the thermal noise strength and thus offers a mechanism for the biological detection of weak (µT‐range) magnetic fields. Part II addresses the question of how the Larmor trajectory could affect biological reactivity. The projection of the motion onto a Cartesian axis measures the nonuniformity of the Larmor trajectory in AC and combined AC/DC magnetic fields, suggesting a means of assessing resonances. A physically meaningful measure of reactivity based upon the classical oscillator trajectory is suggested, and the problem of initial conditions is addressed through averaging over AC phases. AC resonance frequencies occur at the Larmor frequency and at other frequencies, and are dependent upon the ratio of AC/DC amplitudes and target kinetics via binding lifetime. The model is compared with experimental data reported for a test of the ion parametric resonance (IPR) model on data from Ca²⁺ flux in membrane vesicles, neurite outgrowth from PC‐12 cells and a cell‐free calmodulin‐dependent myosin phosphorylation system, and suggests Mg²⁺ is the target for these systems. The results do not require multiple‐ion targets, selection of isotopes, or additional curve fitting. The sole fitting parameter is the binding lifetime of the target system and the results shown are consistent with the literature on binding kinetics. Bioelectromagnetics 30:476–488, 2009. © 2009 Wiley‐Liss, Inc.     

Ring-current shifts in the 300 MHz nuclear magnetic resonance spectra of six purified transfer RNA molecules

We present the 300 MHz high-resolution proton nuclear magnetic resonance spectra of the ring NH hydrogen-bonded protons of six purified tRNAs. Good agreement was obtained between the observed spectra and those computed on the assumption of the suitable cloverleaf models. In the computation it is assumed that the hydrogen-bonded ring NH in each type of base pair has an intrinsic position with respect to 2,2-dimethyl-2-silapentane-5-sulfonate, i.e. in A·U it is at ?14·8 parts per million, in G·C at ?13·7 parts per million and in A·Ψat ?13·5 parts per million. The shifts of these resonances from these positions are calculated by including ring current fields from the nearest neighbors. The agreement is very good, adding support to our earlier findings that there is no evidence for additional Watson-Crick base pairs detected beyond those in the cloverleaf. In general, resolved resonances are fitted by the computed spectra to within ±0·2 part per million showing that there is no need for any additional physical mechanism to explain the nuclear magnetic resonance positions. Hence, the nuclear magnetic resonance spectra can be interpreted in terms of the structure of their neighbors and in a few important cases this has been particularly valuable in understanding the structure beyond the end of a helical region. In the tRNA^Glu_E.coli′ for example, the positions of the resonances in A·U no. 7 and A·U no. 49 at the interior ends of the acceptor and -T-Ψ-C- stems, respectively, strongly suggest that these two stems are in a continuous helix. Other structural effects at the ends of the helical regions are also suggested by the nuclear magnetic resonance spectra.     

Transient effect of weak electromagnetic fields on calcium ion concentration in Arabidopsis thaliana

Background  

Weak magnetic and electromagnetic fields can influence physiological processes in animals, plants and microorganisms, but the underlying way of perception is poorly understood. The ion cyclotron resonance is one of the discussed mechanisms, predicting biological effects for definite frequencies and intensities of electromagnetic fields possibly by affecting the physiological availability of small ions. Above all an influence on Calcium, which is crucial for many life processes, is in the focus of interest. We show that in Arabidopsis thaliana, changes in Ca²⁺-concentrations can be induced by combinations of magnetic and electromagnetic fields that match Ca²⁺-ion cyclotron resonance conditions.     

Nuclear magnetic resonance studies on oligo-deoxyribonucleotides containing thedam methylation site GATC. Adenine methylation of d(GGATCC) does not change the helix geometry

We have studied the conformation of two hexanucleotides d(GGATCC) and d(GGm⁶ATCC) using proton nuclear magnetic resonance. Nuclear Overhauser effect measurements show that d(GGATCC) assumes a normal right handed B helix. The single and double strand resonances are in fast exchange on a proton nuclear magnetic resonance time scale. For d(GGm⁶ATCC), up to the Tm separate resonances are observed for each state, indicating slow exchange, though above the Tm it becomes more rapid. The orientation of the adenosine methyl-amino group, preferentiallycis to N¹, hinders base pair formation.The connectivities of the resonances of the two states were established by saturation transfer experiments. At 0°C irradiation of the m⁶ A-T imino proton gives an nuclear Overhauser effect to AH² showing that base pairing is Watson-Crick. Intra and interresidue nuclear Overhauser effects starting from the 3′ terminus show that the helix is right handed and in the B-form.The results on the two oligomers demonstrate that adenosine methylation induces little or no change in the conformation of the helix, but reduces the Tm from 45° to 32°C and slows the opening and closing of the m₆A.T base pair by a factor of about 100.     

13C nuclear magnetic resonance of the ferrichrome peptides: Structural and strain contributions to the conformational state

The ¹³C nuclear magnetic resonance spectra of l-ornithine, of the di- and tripeptide linear derivatives, and of the siderophore-occurring, modified residue δ-N-acetyl-δ-N-hydroxy-l-ornithine (Orn) are reported for ²H₂O solutions, at pD 7. The assignment of all the resonances is directly established from the comparative data. This information, together with available literature data, is used to identify the resonances of the metal-free cyclohexapeptides deferriferrichrome, deferriferricrocin, and deferriferrichrysin. The spectra of the analogous peptides at pD 7 in ²H₂O are shown to vary in the pattern of the Orn C_β resonances, suggesting different conformations for deferriferrichrome and the two seryl-containing analogs, in agreement with previously resported ¹H nuclear magnetic resonance data. Co-ordination of the Al³⁺ ion results in extensive changes in both the carbonyl and the aliphatic regions which enhance the overall resolution of the spectra. Most resonances are identified, and many assigned, from the comparative data for the three metal ion co-ordinated analogs. Except for the hydroxamate moiety, directly involved in the complexation event, the drastic chemical shifts induced by metal binding reflect an overall change in the conformational state of the peptides. Differences in the C_a region of the Al³⁺-bound and metal-free peptides are attributed to strain or environmental effects rather than to inductive effects arising from primary structure.     

P NMR Observations on the Effect of the External pH on the Intracellular pH Values in Plant Cell Suspension Cultures

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to monitor the response of oil palm (Elaeis guineensis) and carrot (Daucus carota) cell suspensions to changes in the external pH. An airlift system was used to oxygenate the cells during the NMR measurements and a protocol was developed to enable a constant external pH to be maintained in the suspension when required. Phosphonoacetic acid was used as an external pH marker and the intracellular pH values were measured from the chemical shifts of the cytoplasmic and vacuolar orthophosphate resonances. In contrast to earlier studies the cytoplasmic pH was independent of the external pH over the range 5.5 to 8.0 and it was only below pH 5.5 that the cytoplasmic pH varied, falling at a rate of 0.12 pH unit per external unit. Loss of pH control was observed in response to sudden increases in external pH with the response of the cells depending on the conditions imposed. A notable feature of the recovery from these treatments was the transient acidification of the cytoplasm that occurred in a fraction of the cells and overshoot phenomena of this kind provided direct evidence for the time dependence of the regulatory mechanisms.     

The nature of the Ln3+ —angiotensin II complex. A 13C nmr study of the binding of Yb3+ to angiotensin ii

The nature of the Yb³⁺-angiotensin II complex is examined by ^13C nuclear magnetic resonance. The ytterbium-induced shifts of most resonances are observed to be strongly dependent on pD, while a few are observed to be largely independent of pD. These observations are shown to be consistent with stepwise binding of the lanthanide ion to the carboxylates of aspartic acid and the C-terminus.     

Letters to the editor: Nuclear magnetic resonance studies of protein-RNA interactions. I. The elongation factor Tu-GTP aminoacyl-tRNA complex

The 300 MHz high-resolution nuclear magnetic resonance spectra of hydrogen-bonded protons in Escherichia coli tRNA^Glu and yeast tRNA^Phe have previously been reported and the resolved resonances assigned to specific base-pairs. Here we show that in complexes of these two tRNAs with elongation factor Tu there is no discernible loss of base-paired protons. Within the experimental accuracy this means that no helical arms open upon complex formation.     

Letter: Nuclear magnetic resonance of protein-nucleic acid interactions. II. The E. coli tRNA-Glu complex with glutamyl-tRNA synthetase

We have observed the 300 MHz high-resolution proton nuclear magnetic resonance spectrum of the Escherichia coli tRNA^Glu complex with the glutamyl-tRNA synthetase. The observations are fitted very well by a computer-simulated spectrum of the E. coli tRNA^Glu itself, with the individual resonances broadened from 45 Hz to 150 Hz because of the increased molecular weight. This indicates that no helical arms open upon complex formation, nor is there evidence for any additional Watson-Crick base-pairs in the complex.     

Use of Na-Nuclear Magnetic Resonance To Follow Sodium Uptake and Efflux in NaCl-Adapted and Nonadapted Millet (Panicum miliaceum) Suspensions

Cellular Na⁺ transport was followed in vivo by ²³Na nuclear magnetic resonance (NMR) using anionic dysprosium-based shift reagents to resolve internal and external ²³Na⁺ resonances. Proso millet (Panicum miliaceum) cell suspensions adapted for rapid growth on 130 mm NaCl had biphasic ²³Na efflux kinetics when shifted to low Na⁺ medium, while nonadapted cells had little measurable Na⁺ efflux after preloading with ²³NaCl. Uptake of ²³Na was also observed using ²³Na NMR. The resonance frequency of the external Na⁺-dysprosium (III) triphosphate, relative to that of the ²³Na in the cells, was sensitive to pH, permitting the pH of the external medium to be followed during the course of in vivo experiments.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

Fluorinated Vitamin B12 Analogs Are Cofactors of Corrinoid-Dependent Enzymes: a 19F-Labeled Nuclear Magnetic Resonance Probe for Identifying Corrinoid-Protein Interactions

The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B₁₂ analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B₁₂. Hence, it is possible to use ¹⁹F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.     

Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in Rhodococcus opacus 1cp

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and ¹⁹F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The ¹⁹F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.     

A P Nuclear Magnetic Resonance Study of Intracellular pH of Plant Cells Cultivated in Liquid Medium

³¹P nuclear magnetic resonance has been used to study the vacuolar and cytoplasmic pH of Acer pseudoplatanus, Catharanthus roseus, and Glycine max cells grown as cell suspensions. The adaptation of this technique to plant cells grown in liquid medium is described with emphasis on the removal of Mn²⁺ and phosphate from the extracellular medium and on providing the O₂ supply of the cells in the nuclear magnetic resonance tube and the various problems of calibration. Aerobic and anaerobic cells show large differences in their glucose-6-phosphate, their cytoplasmic inorganic phosphate pools, and their cytoplasmic pH. Differences in the relative sizes of the cytoplasmic and vacuolar inorganic phosphate pools have been observed for the three cell strains studied.     

2-(4-Fluorophenyl)-quinazolin-4(3H)-one as a novel tyrosinase inhibitor: Synthesis,inhibitory activity,and mechanism

2-(4-Fluorophenyl)-quinazolin-4(3H)-one (FQ) was synthesized, and its structure was identified with ¹H nuclear magnetic resonance (¹H NMR), ¹³C nuclear magnetic resonance (¹³C NMR), fourier transform infrared spectroscopy (FTIR), and high resolution mass spectrometry (HRMS). From the enzyme analysis, the results showed that it could inhibit the diphenolase activity of tyrosinase (IC₅₀ = 120 ± 2 μM). Furthermore, the results of kinetic studies showed that the compound was a reversible mixed-type inhibitor, and that the inhibition constants were determined to be 703.2 (K_I) and 222.1 μM (K_IS). The results of fluorescence quenching experiment showed that the compound could interact with tyrosinase and the substrates (tyrosine and l-DOPA). Molecular docking analysis revealed that the mass transfer rate was affected by FQ blocking the enzyme catalytic center. In brief, current study identified a novel tyrosinase inhibitor which deserved further study for hyperpigmentation drugs.     

Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers

Summary A three-dimensional ¹H chemical shift/¹H-¹⁵N dipolar coupling/¹⁵N chemical shift correlation spectrum was obtained on a sample of specifically ¹⁵N-labeled magainin peptides oriented in lipid bilayers between glass plates in a flat-coil probe. The spectrum showed complete resolution of the resonances from two labeled amide sites in all three dimensions. The three orientationally dependent frequencies associated with each resonance enabled the orientation of the peptide planes to be determined relative to the direction of the applied magnetic field. These results demonstrate the feasibility of multiple-pulse spectroscopy in a flat-coil probe, the ability to measure three spectral parameters from each site in a single experiment, and the potential for resolving among many labeled sites in oriented membrane proteins.     

Nuclear Magnetic Resonance Studies of Sodium and Potassium in Etiolated Pea Stem

Based on nuclear magnetic resonance (NMR) spectroscopy and other evidence, it has been argued that tissues accumulate, and retain, ions in a binding process by a highly structured water-protoplasm system; thus active membrane transport need not be involved. Recent evidence has accounted for the loss of resonance intensity usually found when investigating quadrupolar ions in animal tissue. Using continuous wave NMR spectroscopy, we have examined two quadrupolar ions, Na⁺ and K⁺, in pea stem cells where about 90% of the ion content is in the largely aqueous vacuoles having a membrane barrier. The NMR resonances from these ions correspond to almost 100% of that expected from independent measurements of total ion content. This indicates that the ions are retained as free ions after accumulation. The small fraction which is NMR invisible may represent ions in an ordered, anisotropic environment, such as that in the wall or cytoplasm.     

Calcium ion cyclotron resonance in dissipative water structures

Weak magnetic and electromagnetic fields affect physiological processes in animals, plants, and microorganisms. Ion cyclotron resonance (ICR) is discussed as one of the sensitive mechanisms, which enable perception of the geomagnetic field and its orientation. Numerous biological effects are observed involving several small ions, showing windows of predicted frequencies and intensities. The pioneering work of Guiliano Preparata and Emilio Del Giudice using quantum electrodynamics showed that spontaneously originating coherent regions in water facilitate ICR effects at incoherent water phase boundaries. Here we examine the ICR response of the calcium ion (Ca²⁺), crucial for many life processes. We use an aqueous solution containing the biologically ubiquitous membrane lipid L-α-phosphatidylcholine that serves as a biomimetic proxy for dynamic light scattering (DLS) and nonlinear dielectric spectroscopy (NLDS) measurements. One notable result is that this system approaches a new equilibrium upon addition of calcium by means of the oscillatory Belousov–Zhabotinsky chemical reaction, oscillations are significantly reduced under Ca²⁺ ICR application. Secondly an “oscillator” of calcium ions appears to be able to itself couple coherently and predictably to large-scale coherent regions in water. This system appears able to regulate ion fluxes in response to very weak environmental electromagnetic fields.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.